CN101658496A - Exenatide release microsphere preparation, preparation method and application thereof - Google Patents
Exenatide release microsphere preparation, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an Exenatide release microsphere preparation mainly comprising the following components in percentage by weight: 0.5-10 percent of Exenatide and 85-99 percent of polylactic acid-glycolic acid copolymer. The invention also discloses a preparation method and an application of the Exenatide release microsphere preparation. The Exenatide release microsphere preparation not onlycan prolong the action time of the Exenatide in the body and reduce the medicine application frequency, but also can maintain the effective blood-medicine concentration of the Exenatide and improve the treatment effect.
Description
Technical field
The present invention relates to Exenatide pharmaceutical preparation, relate in particular to a kind of Exenatide release microsphere preparation and its production and application.
Background technology
Exendin (exendin-4) is Monster (the Gila monster from being distributed in the west and south, North America, Helodermasuspectum) the natural glucagon-like-peptide-1 of separating in the salivary gland (glucagon-like peptide-1, analog GLP-1).Exenatide (exenatide) is the Exendin (exendin-4) of synthetic, is made of 39 amino acid residues, and its aminoacid sequence is as follows:
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Me t-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-As n-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (Eng etc., J.Biol.Chem., 267:7402-05,1992).
Exenatide and mammiferous GLP-1 have 53% homology, with people's glucagon (glucagon) 48% homology is arranged, (GLP-1R) has high affinity to the GLP-1 receptor, itself and the bonded ability of GLP-1R are 2.4 times of (Goke etc. of GLP-1, J Biol Chem, 268:19650-55,1993).
The known pharmacological action of Exenatide comprises: (1) increases glucose dependency insulin secretion accelerating, does not stimulate secretion of insulin in blood sugar level during normal or hypoglycemia, only stimulates insulin secretion when blood sugar level is higher; (2) suppress the secretion of type ii diabetes patient glucagon, and when hyperglycemia, reduce glucagon concentration in the serum, but do not weaken normal glucagon hypoglycemic reaction; (3) suppress digestive tract power and secretory function after the meal, postpone gastric emptying, thereby help the control of post-prandial glycemia; (4) reduce appetite, reduce the absorption of food; (5) stimulate the β apoptosis, thereby increase the quantity of β cell; Phase reaction when (6) recovering the insulin the first of type ii diabetes patient forfeiture significantly increases at first o'clock and reaches second o'clock phase secretion of insulin mutually, improves type ii diabetes patient's empty stomach and level of postprandial blood sugar.
The Exenatide injection obtains U.S. food and drugs administration approved listing in April, 2005, and commodity are called Byetta.Said preparation be proved improve glycemic control and lose weight aspect have good result.But because the half-life of Exenatide only is 2.4 hours (a Byetta description), be steady blood sugar control, need subcutaneous injection every day is administered twice, and frequent injection makes patient's compliance relatively poor, particularly also needs the patient of insulin injection more to be difficult to accept.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of can effectively prolong Exenatide Exenatide release microsphere preparation of action time in vivo, has reduced the administration frequency of Exenatide.
In addition, also need to provide a kind of preparation method of Exenatide release microsphere preparation, and application and Exenatide release microsphere preparation the application in preparation slimming medicine of Exenatide release microsphere preparation in the medicine of preparation treatment type ii diabetes.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of Exenatide release microsphere preparation, mainly comprised Exenatide, polylactic acid-glycolic guanidine-acetic acid copolymer, its mass percent is: Exenatide 0.5%～10%, polylactic acid-glycolic guanidine-acetic acid copolymer 85%～99%.Preferably, it is 0.5%～5% protective agent that described Exenatide release microsphere preparation also comprises mass percent, and this protective agent is selected from a kind of or its mixture in zinc carbonate, human serum albumin, gelatin, trehalose, sucrose or the mannitol.Protective agent can not degraded in the process of preparation microsphere by the protected protein polypeptides matter.
Polylactic acid-glycolic guanidine-acetic acid [poly (lactide-co-glycolide), PLGA] be by lactic acid (LA) and hydroxyacetic acid (GA) a kind of macromolecular material of forming of block copolymerization in varing proportions, have excellent biological compatibility and degradability, its final catabolite is carbon dioxide and water.Preferably, polylactic acid-glycolic guanidine-acetic acid copolymer of the present invention is a polylactic acid: hydroxyacetic acid is 25: 75～75: 25 a copolymer, and its molecular weight is 5000～20000 dalton.
In another aspect of this invention, provide a kind of preparation method of Exenatide release microsphere preparation, may further comprise the steps:
Polylactic acid-glycolic guanidine-acetic acid copolymer is dissolved in organic solvent, and making described polylactic acid-glycolic guanidine-acetic acid copolymer concentration is the oil phase of 100～400mg/ml;
Exenatide is water-soluble, form the interior water that described Exenatide concentration is 200～600mg/ml, water and oil phase volume ratio are 1: 50～1: 100 in this;
Interior water mixed forming colostrum with described oil phase, and this colostrum joined in the outer water aqueous solution that contains surfactant form emulsion, the volume ratio of this outer water aqueous solution and colostrum is 10: 1～20: 1;
After treating the organic solvent volatilization, obtain being encapsulated with the microsphere of Exenatide.
This method adopts W1/O/W2 (W/O/W) method to make Exenatide release microsphere.
Described surfactant is selected from polyvinyl alcohol (polyvinyl alcohol, PVA), a kind of or its mixture in enuatrol, sodium stearate, dodecyl sodium sulfate, carboxymethyl cellulose, lecithin, gelatin, hyaluronic acid, tween or the span, preferably, described surfactant is polyvinyl alcohol (PVA).Preferred, described colostrum joined contain that mixing forms emulsion in the outer water aqueous solution that mass volume ratio is 1%～3% polyvinyl alcohol.
Above-mentioned preparation method also further comprises: with the emulsion mass volume ratio is that 0.5%～1% polyvinyl alcohol water solution dilutes, and then by stirring or other modes, impels the organic solvent volatilization.
In another aspect of this invention, also provide a kind of preparation method of Exenatide release microsphere preparation, may further comprise the steps:
Polylactic acid-glycolic guanidine-acetic acid copolymer is dissolved in organic solvent, and making described polylactic acid-glycolic guanidine-acetic acid copolymer concentration is the oil phase of 50～200mg/ml;
Exenatide is water-soluble, form the interior water that described Exenatide concentration is 50～100mg/ml, water and oil phase volume ratio are 1: 10～1: 50 in this;
Interior water is mixed the formation colostrum with described oil phase, and the non-solvent organic substance is joined this Ruzhong just, the mass ratio of described non-solvent organic substance and oil phase organic solvent is 1: 1～2: 1;
After treating the organic solvent volatilization, obtain being encapsulated with the microsphere of Exenatide.
This method adopts W/O1/O2 (oil bag Water-In-Oil) method to make Exenatide release microsphere.
Described non-solvent organic substance is selected from a kind of or its mixture in dimethicone, liquid paraffin or the petroleum ether.
This method also comprises step: it is that with solidified microsphere, this mixed solvent cumulative volume is 10: 1～20: 1 with oil phase volume of organic solvent ratio in heptane/alcohol mixed solvent of 3～5 ℃ that microsphere is further transferred to temperature.
In another aspect of this invention, also provide a kind of preparation method of Exenatide release microsphere preparation, may further comprise the steps:
Polylactic acid-glycolic guanidine-acetic acid copolymer is dissolved in organic solvent, and making described polylactic acid-glycolic guanidine-acetic acid copolymer concentration is the oil phase of 50～400mg/ml;
Exenatide and Polyethylene Glycol is water-soluble, remove Polyethylene Glycol after the lyophilization, obtain the Exenatide micropowder;
The Exenatide micropowder is dispersed in the described oil phase, makes suspension through homogenize;
Spray into described suspension in the organic solvent of surface coverage liquid nitrogen with spray pattern with spray dryer;
After treating organic solvent volatilization or extraction, obtain being encapsulated with the microsphere of Exenatide.
This method is to make the Exenatide release microsphere of the solid type of oil bag by the mode that spraying is congealed.
Preferably, the mol ratio of described Exenatide and Polyethylene Glycol is 1: 8.
In the above-mentioned three kinds of preparation methoies of the present invention, preferred, when described Exenatide is water-soluble, also in this aqueous solution, add protective agent, do not degrade in the process of preparation microsphere with the protection Exenatide; The organic solvent of described dissolving polylactic acid-glycolic guanidine-acetic acid copolymer is not for dissolving the organic solvent of encapsulated medicine, be selected from a kind of or its mixture in dichloromethane, chloroform, acetonitrile, ethyl acetate or the acetone, after treating the organic solvent volatilization, copolymer is separated out, and obtains being encapsulated with the microsphere of Exenatide.
In another aspect of this invention, also provide the application of a kind of above-mentioned Exenatide release microsphere preparation in the medicine of preparation treatment type ii diabetes.
In another aspect of this invention, also provide the application of a kind of above-mentioned Exenatide release microsphere preparation in the slimming medicine of preparation.
In the present invention, term " sustained-release micro-spheres " is the microgranule that forms with behind the polymeric encapsulate active substance.Usually, the sphere that microsphere is not necessarily strict, the form of microsphere may be very irregular, according to the technology difference, its porose or atresia in surface.The particle diameter of microsphere is 0.5～1000 μ m, preferred 4～200 μ m, more preferably 10～150 μ m, because microsphere is generally used for drug administration by injection, particle diameter too senior general causes using the more injection needle of large size, patient's pain is stronger, and particle diameter too the young pathbreaker cause well packaging medicine of copolymer, do not reach the good slow release effect.
Exenatide release microsphere preparation of the present invention can not only prolong Exenatide action time in vivo, reduces administration frequency, and can keep the effective blood drug level of Exenatide, improves therapeutic effect.Extracorporeal releasing experiment is the result show, Exenatide release microsphere preparation slow release effect of the present invention is obvious, and cumulative release reached more than 80% in 7 days, and promptly the slow release cycle is 7 days, discharges to meet approximate zero mode, is suitable for treating type ii diabetes and controlling body weight.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is the cumulative in vitro release profiles of the prepared Exenatide release microsphere preparation of the embodiment of the invention 1;
Fig. 2 is the cumulative in vitro release profiles of the prepared Exenatide release microsphere preparation of the embodiment of the invention 4;
Fig. 3 is the cumulative in vitro release profiles of the prepared Exenatide release microsphere preparation of the embodiment of the invention 7;
Fig. 4 is the 1st day blood sugar concentration-time plot after the prepared Exenatide release microsphere preparation administration of the embodiment of the invention 1;
Fig. 5 is the 3rd day blood sugar concentration-time plot after the prepared Exenatide release microsphere preparation administration of the embodiment of the invention 1;
Fig. 6 is the 5th day blood sugar concentration-time plot after the prepared Exenatide release microsphere preparation administration of the embodiment of the invention 1;
Fig. 7 is the 7th day blood sugar concentration-time plot after the prepared Exenatide release microsphere preparation administration of the embodiment of the invention 1.
The specific embodiment
For overcoming short, the frequent shortcoming of medication number of times of Exenatide half-life, after experience is repeatedly tested repeatedly, finally develop Exenatide release microsphere preparation of the present invention, mainly comprise Exenatide, polylactic acid-glycolic guanidine-acetic acid copolymer, its mass percent is: Exenatide 0.5%～10%, polylactic acid-glycolic guanidine-acetic acid copolymer 85%～99%.
Preferably, it is 0.5%～5% protective agent that the Exenatide release microsphere preparation also comprises mass percent, and this protective agent is selected from a kind of or its mixture in zinc carbonate, human serum albumin, gelatin, trehalose, sucrose or the mannitol.Protective agent can not degraded in the process of preparation microsphere by the protected protein polypeptides matter.
Preferably, polylactic acid-glycolic guanidine-acetic acid copolymer is a polylactic acid: hydroxyacetic acid is 25: 75～75: 25 a copolymer, and its molecular weight is 5000～20000 dalton.
Exenatide release microsphere preparation of the present invention can be by following three kinds of methods preparation.
(1.W1/O/W2 W/O/W) solvent evaporation method:
(1) preparation oil phase
Host material PLGA is dissolved in organic solvent dichloromethane makes oil phase, concentration is 100～400mg/ml, and this organic solvent also can be selected chloroform, acetonitrile, ethyl acetate or acetone etc. for use, and other can not dissolve the organic solvent of encapsulated medicine.
(2) water in the preparation
Get water in the water-soluble formation of an amount of Exenatide and protective agent, Exenatide concentration is 200～600mg/ml, and protective agent is selected from gelatin, trehalose, sucrose and mannitol etc., and its concentration is 200～300mg/ml; Interior water and oil phase volume ratio are 1: 50～1: 100, preferable range 1: 60～1: 80.
(3) preparation microsphere
Interior water is added above-mentioned oil phase mix the formation colostrum, adopt ultrasound wave, machinery, magnetic force concussion or other modes to strengthen this mixed process during mixing usually.Then, colostrum dripped rapidly in mass volume ratio is 1%～3% polyvinyl alcohol water solution form emulsion, the volume ratio of this polyvinyl alcohol water solution and colostrum is 10: 1～20: 1, and surfactants such as the also available enuatrol of this polyvinyl alcohol, sodium stearate, dodecyl sodium sulfate, carboxymethyl cellulose, lecithin, gelatin or hyaluronic acid substitute.(mixing speed is 1000～1800rpm), and the polyvinyl alcohol water solution of reuse 0.5%～1% dilutes, and (mixing speed is 300～600rpm), and washing is collected, and lyophilization gets final product to continue stirring at low speed under the room temperature 4 hours through the abundant homogenize of mechanical agitation.
(2.W/O1/O2 oil bag Water-In-Oil) solvent-nonsolvent method:
(1) preparation oil phase
Host material PLGA is dissolved in dichloromethane makes oil phase, concentration is 50～200mg/ml, and this organic solvent also can be selected chloroform, acetonitrile, ethyl acetate or acetone etc. for use, and other can not dissolve the organic solvent of encapsulated medicine.
(2) water in the preparation
Get water in the water-soluble formation of an amount of Exenatide and protective agent, Exenatide concentration is 50～100mg/ml, and protective agent is selected from trehalose, sucrose and mannitol etc., and its concentration is 10～25mg/ml; Interior water and oil phase volume ratio are 1: 10～1: 50, preferable range 1: 15～1: 25.
(3) preparation microsphere
Interior water is added above-mentioned oil phase mix the formation colostrum, adopt ultrasound wave, machinery, magnetic force concussion or other modes to strengthen this mixed process during mixing usually.Then, dimethicone is slowly joined Ruzhong just, this dimethicone and dichloromethane mass ratio 1: 1～2: 1, elementary microsphere that must be softer, this dimethicone also can select for use other non-solvent organic substances such as liquid paraffin or petroleum ether to substitute.
(4) microsphere solidifies and collects
It is in 3～5 ℃ heptane and the ethanol mixed solvent (heptane/ethanol mol ratio 4: 1) that elementary microsphere is transferred to temperature rapidly, mixed solvent cumulative volume and methylene chloride volume were than 10: 1～20: 1, (mixing speed was 300～600rpm) to 3 ℃ of following stirring at low speed, solidified microsphere in 1 hour.Abandon solvent then, add fresh normal heptane rinsing filter cake, 3～5 ℃ of dryings are 6 hours under the vacuum condition, are warming up to 41 ℃ gradually then in 6 hours, continue dry 84 hours again, collect to get final product.
3. congealing spray.
(1) preparation Exenatide micropowder
An amount of polyethylene glycol 6000 and Exenatide and protective agent (its mol ratio is 8: 1: 2) are scattered in the water; after the lyophilization,, remove polyethylene glycol 6000 with washed with dichloromethane, centrifugal; obtain the Exenatide micropowder, protective agent is selected from trehalose, sucrose and mannitol etc.
(2) preparation microsphere
With the dichloromethane solution of Exenatide micropowder adding PLGA, the concentration of PLGA is 50～400mg/ml, and suspension is made in homogenize, and dichloromethane also can be selected chloroform, acetonitrile, ethyl acetate or acetone etc. for use, and other can not dissolve the organic solvent of encapsulated medicine;
Spray-dried device sprays in the cold ethanol of surface coverage liquid nitrogen with spray pattern;
The ethanol volatilization is with the dichloromethane extraction in the PLGA drop;
Fling to liquid nitrogen under the freezing conditions, lyophilization obtains microsphere.
With PLGA (RG502H, LA: GA=50: 50, Mw=10000) 800mg is dissolved in the 3.0ml dichloromethane and makes oil phase, Exenatide 20mg is dissolved in water in (including the 12.5mg gelatin) forms in the double distilled water of 0.05ml, it is added above-mentioned oil phase, ultrasonic emulsification, form the colostrum of W1/O, will contain 2%PVA solution 50ml and place stirred vessel, with colostrum abundant homogenize of aqueous phase outside high-speed stirred (1200rpm) adds down fast, after three minutes, rotating speed is adjusted downward to 400rpm, adds 0.75% poly-vinyl alcohol solution, stirred 4 hours under the room temperature, microsphere sclerosis back centrifugalize and washing, lyophilization gets final product.The envelop rate of Exenatide microsphere is 90%, particle diameter＜100 μ m.
With PLGA (RG502H, LA: GA=25: 75, Mw=5000) 250mg is dissolved in the 2.5ml dichloromethane and makes oil phase, Exenatide 10mg is dissolved in water in (including the 10mg gelatin) forms in the double distilled water of 0.05ml, it is added above-mentioned oil phase, ultrasonic emulsification, form the colostrum of W1/O, will contain 1%PVA solution 50ml and place stirred vessel, with colostrum abundant homogenize of aqueous phase outside high-speed stirred (1200rpm) adds down fast, after three minutes, rotating speed is adjusted downward to 400rpm, adds 0.5% poly-vinyl alcohol solution, stirred 4 hours under the room temperature, microsphere sclerosis back centrifugalize and washing, lyophilization gets final product.The envelop rate of Exenatide microsphere is 85%, particle diameter＜90 μ m.
With PLGA (RG502H, LA: GA=75: 25, Mw=20000) 2000mg is dissolved in the 5.0ml dichloromethane and makes oil phase, Exenatide 30mg is dissolved in water in (including the 15mg gelatin) forms in the double distilled water of 0.05ml, it is added above-mentioned oil phase, ultrasonic emulsification, form the colostrum of W1/O, will contain 3%PVA solution 50ml and place stirred vessel, with colostrum abundant homogenize of aqueous phase outside high-speed stirred (1200rpm) adds down fast, after three minutes, rotating speed is adjusted downward to 400rpm, adds 1% poly-vinyl alcohol solution, stirred 4 hours under the room temperature, microsphere sclerosis back centrifugalize and washing, lyophilization gets final product.The envelop rate of Exenatide microsphere is 97%, particle diameter＜120 μ m.
Embodiment 4 W/O1/O2 solvent-nonsolvent legal systems are equipped with the Exenatide release microsphere preparation
With PLGA (RG502H, PLA: PGA=50: 50, Mw=10000) 930mg is dissolved in the 10.8ml dichloromethane and makes oil phase, Exenatide 50mg is dissolved in water in (including 10.8mg sucrose) forms in the double distilled water of 0.6ml, it is added above-mentioned oil phase, ultrasonic emulsification, form the colostrum of W/O1, the 16.2ml dimethicone is slowly joined Ruzhong just, and elementary microsphere that must be softer is transferred to elementary microsphere rapidly in 173.0ml normal heptane/alcohol mixed solvent of 3 ℃ then, 3 ℃ of following 400rpm stirred after 1 hour, discard solvent, add fresh normal heptane 9.0ml rinsing filter cake, the following 3 ℃ of dryings of vacuum condition 6 hours, in 6 hours, be warming up to 41 ℃ gradually then, continued dry 84 hours again, and collected, the sealing packing gets final product.The envelop rate of Exenatide microsphere is 92%, particle diameter＜120 μ m.
Embodiment 5 W/O1/O2 solvent-nonsolvent legal systems are equipped with the Exenatide release microsphere preparation
With PLGA (RG502H, PLA: PGA=25: 75, Mw=8000) 300mg is dissolved in the 6ml dichloromethane and makes oil phase, Exenatide 30mg is dissolved in water in (including 6mg sucrose) forms in the double distilled water of 0.6ml, it is added above-mentioned oil phase, ultrasonic emulsification, form the colostrum of W/O1, the 12.5ml dimethicone is slowly joined Ruzhong just, and elementary microsphere that must be softer is transferred to elementary microsphere rapidly in 60.0ml normal heptane/alcohol mixed solvent of 5 ℃ then, 5 ℃ of following 400rpm stirred after 1 hour, discard solvent, add fresh normal heptane 9.0ml rinsing filter cake, the following 5 ℃ of dryings of vacuum condition 6 hours, in 6 hours, be warming up to 41 ℃ gradually then, continued dry 84 hours again, and collected, the sealing packing gets final product.The envelop rate of Exenatide microsphere is 90%, particle diameter＜120 μ m.
Embodiment 6 W/O1/O2 solvent-nonsolvent legal systems are equipped with the Exenatide release microsphere preparation
With PLGA (RG502H, PLA: PGA=75: 25, Mw=15000) 6000mg is dissolved in the 30ml dichloromethane and makes oil phase, Exenatide 60mg is dissolved in water in (including 15mg sucrose) forms in the double distilled water of 0.6ml, it is added above-mentioned oil phase, ultrasonic emulsification, form the colostrum of W/O1, the 83.1ml dimethicone is slowly joined Ruzhong just, and elementary microsphere that must be softer is transferred to elementary microsphere rapidly in 600ml normal heptane/alcohol mixed solvent of 4 ℃ then, 4 ℃ of following 400rpm stirred after 1 hour, discard solvent, add fresh normal heptane 9.0ml rinsing filter cake, the following 4 ℃ of dryings of vacuum condition 6 hours, in 6 hours, be warming up to 41 ℃ gradually then, continued dry 84 hours again, and collected, the sealing packing gets final product.The envelop rate of Exenatide microsphere is 95%, particle diameter＜140 μ m.
96mg polyethylene glycol 6000 and Exenatide 8mg and trehalose 1.6mg are scattered in the 1ml double distilled water, and vortex mixed about 3 minutes, after the lyophilization, with washed with dichloromethane, centrifugal, removed Polyethylene Glycol, obtained the Exenatide micropowder.With PLGA (RG502H, LA: GA=50: 50, Mw=10000) 150.4mg is dissolved in dichloromethane 2ml and makes oil phase, and micropowder is added ultra-sonic dispersion in the oil phase, spray-dried device sprays in the cold ethanol of surface coverage liquid nitrogen with spray pattern; The ethanol volatilization extracts the dichloromethane in the PLGA drop; Fling to liquid nitrogen under the freezing conditions, lyophilization obtains microsphere; The envelop rate of Exenatide microsphere is 91%, particle diameter＜80 μ m.
Embodiment 8 congealing spraies prepare the Exenatide release microsphere preparation
96mg polyethylene glycol 6000 and Exenatide 8mg and trehalose 1.6mg are scattered in the 1ml double distilled water, and vortex mixed about 3 minutes, after the lyophilization, with washed with dichloromethane, centrifugal, removed Polyethylene Glycol, obtained the Exenatide micropowder.With PLGA (RG502H, LA: GA=25: 75, Mw=5000) 100mg is dissolved in dichloromethane 2ml and makes oil phase, and micropowder is added ultra-sonic dispersion in the oil phase, spray-dried device sprays in the cold ethanol of surface coverage liquid nitrogen with spray pattern; The ethanol volatilization extracts the dichloromethane in the PLGA drop; Fling to liquid nitrogen under the freezing conditions, lyophilization obtains microsphere; The envelop rate of Exenatide microsphere is 82%, particle diameter＜80 μ m.
Embodiment 9 congealing spraies prepare the Exenatide release microsphere preparation
96mg polyethylene glycol 6000 and Exenatide 8mg and trehalose 1.6mg are scattered in the 1ml double distilled water, and vortex mixed about 3 minutes, after the lyophilization, with washed with dichloromethane, centrifugal, removed Polyethylene Glycol, obtained the Exenatide micropowder.With PLGA (RG502H, LA: GA=75: 25, Mw=20000) 800mg is dissolved in dichloromethane 2ml and makes oil phase, and micropowder is added ultra-sonic dispersion in the oil phase, spray-dried device sprays in the cold ethanol of surface coverage liquid nitrogen with spray pattern; The ethanol volatilization extracts the dichloromethane in the PLGA drop; Fling to liquid nitrogen under the freezing conditions, lyophilization obtains microsphere; The envelop rate of Exenatide microsphere is 95%, particle diameter＜80 μ m.
The Exenatide release microsphere preparation of the foregoing description preparation is carried out the prominent mensuration of releasing with release profiles of microsphere, assay method is: precision takes by weighing pastille microsphere 50mg and puts in the 10ml centrifuge tube, with pH is that 7.4 phosphate buffer (contains 0.02% Hydrazoic acid,sodium salt as antibacterial, 0.02%F-68 is as wetting agent) be release medium, place the water bath with thermostatic control shaking table, under hunting speed 100rpm, 37 ℃ ± 0.5 ℃ condition of temperature, carry out the release in vitro degree of microsphere and measure.Take out the release medium that whole release medium also more renew, high effective liquid chromatography for measuring Exenatide content at 1d, 2d, 4d and 7d respectively.Fig. 1,2,3 is respectively the cumulative in vitro release profiles of the prepared Exenatide release microsphere preparation of embodiment 1,4,7.Can be drawn by Fig. 1～3, the Exenatide release microsphere preparation that three kinds of preparation methoies of the present invention make all has the good slow release effect, and prominent in 1 day released and be about 23%, 7 day cumulative release and reach more than 85%.
Embodiment 11 animal experiments
Choose 16 of adult Wistar rats, about body weight 200g, male and female half and half, be divided into administration group and blank group at random, the an amount of microsphere of administration group subcutaneous injection (make the 2.0mg microsphere according to embodiment 1, contain the about 4.5 μ g of Exenatide), blank group subcutaneous injection is with the normal saline of volume.Respectively at the 1st, 3,5,7 day the same time after the administration, give every rats by intraperitoneal injection 18mmol/kg glucose, every Mus is adopted blank blood sample earlier before the injection, gets blood at 5,10,15,20,30 and 50 minutes eye sockets then, measures the blood glucose before and after injecting.Blood sugar detection carries out with reference to the description of glucose assays test kit (glucose oxidase method, Beijing Chemical Plant).Make blood sugar concentration and the curve chart of time, Fig. 4,5,6,7 is respectively after the prepared Exenatide release microsphere preparation administration of embodiment 1 blood sugar concentration-time plot of the 1st, 3,5,7 day, by Fig. 4～7 as seen, blank group lumbar injection glucose, blood glucose obviously raises after 5 minutes, fall after rise slowly, just reached the ground state blood sugar concentration in 240 minutes gradually.The administration group can be observed the obvious functions of blood sugar effect at the 1st, 3,5,7 day, promptly fall back to ground state concentration in 30 minutes, illustrate that Exenatide microsphere of the present invention has tangible slow release and hypoglycemic activity in vivo, the slow release formulation that can be used as Exenatide is used for the treatment of type ii diabetes or controlling body weight.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (17)
1. an Exenatide release microsphere preparation is characterized in that, mainly comprises Exenatide, polylactic acid-glycolic guanidine-acetic acid copolymer, and its mass percent is: Exenatide 0.5%～10%, polylactic acid-glycolic guanidine-acetic acid copolymer 85%～99%.
2. Exenatide release microsphere preparation according to claim 1; it is characterized in that; also comprise mass percent and be 0.5%～5% protective agent, this protective agent is selected from a kind of or its mixture in zinc carbonate, human serum albumin, gelatin, trehalose, sucrose or the mannitol.
3. Exenatide release microsphere preparation according to claim 1 is characterized in that, described polylactic acid-glycolic guanidine-acetic acid copolymer is a polylactic acid: hydroxyacetic acid is 25: 75～75: 25 a copolymer, and its molecular weight is 5000～20000 dalton.
4. the preparation method of an Exenatide release microsphere preparation is characterized in that, may further comprise the steps:
Polylactic acid-glycolic guanidine-acetic acid copolymer is dissolved in organic solvent, and making described polylactic acid-glycolic guanidine-acetic acid copolymer concentration is the oil phase of 100～400mg/ml;
Exenatide is water-soluble, form the interior water that described Exenatide concentration is 200～600mg/ml, water and oil phase volume ratio are 1: 50～1: 100 in this;
Interior water mixed forming colostrum with described oil phase, and this colostrum joined in the outer water aqueous solution that contains surfactant form emulsion, the volume ratio of this outer water aqueous solution and colostrum is 10: 1～20: 1;
After treating the organic solvent volatilization, obtain being encapsulated with the microsphere of Exenatide.
5. preparation method according to claim 4, it is characterized in that described surfactant is selected from a kind of or its mixture in polyvinyl alcohol, enuatrol, sodium stearate, dodecyl sodium sulfate, carboxymethyl cellulose, lecithin, gelatin, hyaluronic acid, tween or the span.
6. according to claim 4 or 5 described preparation methoies, it is characterized in that described surfactant is a polyvinyl alcohol.
7. preparation method according to claim 6 is characterized in that, described colostrum is joined contain that mixing forms emulsion in the outer water aqueous solution that mass volume ratio is 1%～3% polyvinyl alcohol.
8. preparation method according to claim 6 is characterized in that, also further comprises: with the emulsion mass volume ratio is that 0.5%～1% polyvinyl alcohol water solution dilutes.
9. the preparation method of an Exenatide release microsphere preparation is characterized in that, may further comprise the steps:
Polylactic acid-glycolic guanidine-acetic acid copolymer is dissolved in organic solvent, and making described polylactic acid-glycolic guanidine-acetic acid copolymer concentration is the oil phase of 50～200mg/ml;
Exenatide is water-soluble, form the interior water that described Exenatide concentration is 50～100mg/ml, water and oil phase volume ratio are 1: 10～1: 50 in this;
Interior water is mixed the formation colostrum with described oil phase, and the non-solvent organic substance is joined this Ruzhong just, the mass ratio of described non-solvent organic substance and oil phase organic solvent is 1: 1～2: 1;
After treating the organic solvent volatilization, obtain being encapsulated with the microsphere of Exenatide.
10. preparation method according to claim 9 is characterized in that, described non-solvent organic substance is selected from a kind of or its mixture in dimethicone, liquid paraffin or the petroleum ether.
11. according to claim 9 or 10 described preparation methoies, it is characterized in that, also comprise step: it is that with solidified microsphere, this mixed solvent cumulative volume is 10: 1～20: 1 with oil phase volume of organic solvent ratio in heptane/alcohol mixed solvent of 3～5 ℃ that microsphere is further transferred to temperature.
12. the preparation method of an Exenatide release microsphere preparation is characterized in that, may further comprise the steps:
Polylactic acid-glycolic guanidine-acetic acid copolymer is dissolved in organic solvent, and making described polylactic acid-glycolic guanidine-acetic acid copolymer concentration is the oil phase of 50～400mg/ml;
Exenatide and Polyethylene Glycol is water-soluble, remove Polyethylene Glycol after the lyophilization, obtain the Exenatide micropowder;
The Exenatide micropowder is dispersed in the described oil phase, makes suspension through homogenize;
Spray into described suspension in the organic solvent of surface coverage liquid nitrogen with spray pattern with spray dryer;
After treating organic solvent volatilization or extraction, obtain being encapsulated with the microsphere of Exenatide.
13. preparation method according to claim 12 is characterized in that, the mol ratio of described Exenatide and Polyethylene Glycol is 1: 8.
14. according to claim 4,9 or 12 described preparation methoies; it is characterized in that; when described Exenatide is water-soluble, also add protective agent in this aqueous solution, this protective agent is selected from a kind of or its mixture in zinc carbonate, human serum albumin, gelatin, trehalose, sucrose or the mannitol.
15., it is characterized in that the organic solvent of described dissolving polylactic acid-glycolic guanidine-acetic acid copolymer is selected from a kind of or its mixture in dichloromethane, chloroform, acetonitrile, ethyl acetate or the acetone according to claim 4,9 or 12 described preparation methoies.
16. the application of the described Exenatide release microsphere preparation of claim 1 in the medicine of preparation treatment type ii diabetes.
17. the application of the described Exenatide release microsphere preparation of claim 1 in the slimming medicine of preparation.
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