CN102370611A - Temperature-sensitive hydrogel containing exendin-4 and injection thereof - Google Patents
Temperature-sensitive hydrogel containing exendin-4 and injection thereof Download PDFInfo
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Abstract
The invention relates to a temperature-sensitive hydrogel containing exendin-4, comprising exendin-4 as an effective ingredient and an amphiphilic copolymer as a medicine slow release carrier, wherein, the amphiphilic copolymer comprises polyester and polyethylene glycol, preferred PLGA-PEG-PLGA. The temperature-sensitive hydrogel is a liquid at room temperature, and is a solid water insoluble gel at the temperature of 37 DEG C, can be used for treating diabetes, and has good drug release property, environmental protection, little irritation, convenient application, and obvious effect. Compared with preparations with a large dose of exendin-4, the hydrogel disclosed herein has the advantages of in vivo formation of gel, easy operation, and continuous release property, and improves bioavailability, reduces administration times, and enhances patient compliance.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, particularly, the present invention relates to comprise the temperature sensitive type water gel and the injection thereof of insulin secretion accelerating element (exendin-4).
Background technology
Diabetes are diseases of a kind of serious threat human health and life, and whole world diabetics surpasses 1.5 hundred million, and wherein the type 2 diabetes mellitus patient accounts for 90%.China type 2 diabetes mellitus patient nearly 4,000 ten thousand, along with growth in the living standard, sickness rate is also increasingly high, and the caused a series of complication of diabetes are having a strong impact on patient's quality of life especially.
Plain-1 (the glucagon-like peptide-1 of insulin secretion accelerating; GLP-1) have blood sugar lowering, promote insulin secretion, promote beta Cell of islet growth, propagation and differentiation and suppress effect such as its apoptosis; Once once be used as the hope place of effective treatment type 2 diabetes mellitus; (Murielle M J.Current Opinion inPharmacology 2006 6:598-605), has limited clinical practice but because of its half-life is extremely lacked (only 2 minutes).Exendin-4 is the conspicuous a kind of natural polypeptides separated the Monster salivary gland that draws from the Southwestern United Stares nefud; Belong to the GLP-1 analog; Contain 39 amino acid peptides, belong to incretin secretions, have 53% homology with the aminoacid sequence of mammiferous GLP-1; The ability of its blood sugar regulation is similar with GLP-1; And in several animal models, shown similar antidiabetic effect with GLP-1, but its half-life obviously prolong, reach (.Pharmacology&Therapeutics 2007 (113) 546-593 such as M á ire E) about 4 hours.Exendin-4 reduces the speed of gastric emptying through stimulating the propagation and the differentiation of beta Cell of islet, and mechanism such as promotion satietion reach the effect of reduction, blood sugar control, are having broad prospects aspect the treatment type 2 diabetes mellitus.
Exendin-4 (for example can also utilize the gene engineering method preparation; Referring to ZL200410052039.4): through the DNA sequence of synthetic exendin-4; Then sequence is inserted into expression plasmid pET32a (+); Transformed into escherichia coli DH5 α makes up and forms again, obtains end product through a series of biotechnology such as expression, purification, fermentations again.It is stable that the product that makes is like this deposited at low temperatures (2～8 ℃).Exendin-4 is as first class national new drug in reorganization, is mainly used in treatment of diabetes clinically, as all protein drugs; Exendin-4 is eliminated in blood circulation fast; In order to keep treating effective blood drug level, usually want heavy dose or frequent drug administration, this method is inconvenient and have a potential side effect (.Biomacromolecules such as Chung Y M; 2002,3 (3): 511-516.).
The aminoacid sequence of Exendin-4 is:
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?GluGlu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly?Pro?SerSer?Gly?Ala?Pro?Pro?Pro?Ser
Be abbreviated as: HGEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPS
Controlled drug delivery system is exactly that medicine is processed certain dosage form, and the control medicine is at the intravital release degree of people, make medicine according to the dosage of design, in the time range that requires, slowly discharge in vivo, to reach the purpose of treatment disease with certain speed.This drug delivery system comprise injectable oil, emulsion, suspension, liposome, microgranule (microcapsule or microsphere), implantation or gel systems etc. (.Plenmu such as Baker R W, NewYork, 1974,15-71.).Amylin and the Alkermes company research and development of cooperation exendin-4 microsphere sustained-release dosage form (exenatide LAR) only need the administration in January 1 time, can obviously improve clinical compliance (US20090239796).
Compare with microsphere sustained-release preparation, the gel sustained release system that grew up in recent years is for reducing drug dose, control drug effect zone, and improving bioavailability of medicament and reducing its toxic and side effects etc. all is a kind of extraordinary administering modes.Gel sustained release medicine has following advantage and characteristics: 1) aqueous solutions of polymers at room temperature is liquid, and viscosity is little, has flowability, can filtration sterilization; 2) various medicines can load with simple method of mixing, and drug loading is high, and not with an organic solvent, helps the maintenance of pharmaceutically active; 3) adopt the mode administration of injecting, reduced administration number of times, patient compliance is strong; The mixture of medicine and polymer solution injects in the body with liquid form, can adapt to cavity shape different in the body, does not have the solution migration, and the fixed point that helps medicine discharges; 4) biodegradable hydrogel can slowly be degraded into the micromolecule product and be absorbed in human body, need not take out through operation behind the injection human body.
Natural and the half-natural injectable macromolecule hydrogel that is used for slow releasing carrier of medication is because characteristics such as its excellent biological compatibility, natural degradable property, wide material sources, cheap price and extensively being paid close attention to.At present, common natural and semi-natural syringeability macromolecule hydrogel material has chitosan class, cellulose family, hyalomitome acids, alginates etc.; The synthetic injectable macromolecule hydrogel that is used for slow releasing carrier of medication can be divided into two big types according to its degradability: one type is non-degradable synthetic high polymer hydrogel, comprises poly-N-isopropyl acrylamide, PEO and PPOX block copolymer; One type is the degradable synthesized polymer hydrogel, comprise Polyethylene Glycol (PEG)/polyester degradable block copolymer with gather organophosphor eyeball etc. (.Plenmu such as Baker RW, NewYork, 1974,15-71.).
Therefore injectable Polyethylene Glycol (PEG)/polyester copolymer has degradability owing to contain aliphatic polyester.Polyester mainly comprises Acetic acid, hydroxy-, bimol. cyclic ester (GA), caprolactone (CL), lactide (LA) etc.The research of this respect is the most outstanding with the Jeong B seminar and the Lee DS seminar of the Kim S W of U.S. group, Korea S.Injectable Polyethylene Glycol (PEG)/polyester copolymer hydrogel does not have physiology toxicity, no antigen and immunogenicity; Has excellent biological compatibility with tissue and blood; Degradable, the gel longer duration, thereby become one of injection aquagel material that has much application prospect (.Drug Discov.Today 2005 such as Veronese F.M; 10,1451-1458).Qiao etc. (Mingxi Qiao.Pharmazie.2008,63 (1): 27-30.) utilizing PLGA-PEG-PLGA copolymer (PLGA is the copolymer of polyglycolic acid and hydroxyacetic acid, and PEG is a Polyethylene Glycol) is biological slow-released carrier; Process IL-2 temperature sensitive type water gel preparation, the dispose procedure of monitoring IL-2, the result finds; The release in vitro of IL-2 was above 20 days; Its release rate reduces along with the rising of copolymer concentration, and in whole dispose procedure, IL-2 can keep the biological activity of 57-90%; In anticancer animal experiment, the temperature sensitive sensitive hydrogel preparation of IL-2 shows good anticancer effect.
Summary of the invention
The temperature sensitive type water gel that the purpose of this invention is to provide a kind of exendin-4 of comprising.Said temperature sensitive type water gel comprises: as the exendin-4 of effective ingredient with as the amphipathic copolymer of slow-released carrier, the block copolymer that wherein amphipathic copolymer is made up of polyester and Polyethylene Glycol.Wherein said exendin-4 is dispersed in the said amphipathic copolymer, and said hydrogel is liquid down in room temperature (term " room temperature " is meant 10 ℃-30 ℃, referring to " 2005 editions note on the use parts of Chinese pharmacopoeia), is the insoluble gel of solid water at 37 ℃.
Preferably, the concentration of said exendin-4 in said hydrogel is 0.1-100mg/ml, and more preferably about 0.5-50mg/ml most preferably is about 1-10mg/ml; The concentration of said amphipathic copolymer in said hydrogel is 100-400mg/ml, more preferably 150-250mg/ml.
The number-average molecular weight of said amphipathic copolymer can be 500-28000, is preferably 600-16000, more preferably 1000-8000.The number-average molecular weight of wherein said polyester can be 500-30000, is preferably 800-20000, more preferably 1000-10000; The number-average molecular weight of Polyethylene Glycol can be 200-20000, is preferably 300-10000, more preferably 500-3000.
Preferably, in the said slow-released carrier of said temperature sensitive type water gel, also be dispersed with in buffer agent, stabilizing agent, the antiseptic one or more.
Said exendin-4 is exendin-4 polypeptide or its isoform, mutant, fusion rotein, functional deriv, its active fragment.
In said amphipathic copolymer, polyester and Polyethylene Glycol block configuration are preferably polyester-polyethylene glycol-ester or polyethylene glycol-ester-Polyethylene Glycol, more preferably polyester-polyethylene glycol-ester.Wherein the weight ratio of polyester and Polyethylene Glycol can be 9-6: 1-4 is preferably 9-7: 1-3, more preferably 9-8: 1-2.
Said polyester includes but not limited to copolymer (PLGA), the poly-dl-lactide (D of polylactic acid (PLA), polyglycolic acid and hydroxyacetic acid; L-PLA), raceme polyglycolic acid and co-glycolic acid (D; L-PLGA), any one in end carboxyl polylactic acid (PLA-COOH), end carboxyl polyglycolic acid and the co-glycolic acid (PLGA-COOH) or multiple copolymer; Be preferably PLA and PLGA, more preferably PLGA.
When polyester was selected PLGA for use, the weight ratio of polyglycolic acid and hydroxyacetic acid can be 15-1: 1, be preferably 9-1: and 1,5-1 more preferably: 1.
Said amphipathic copolymer includes but not limited to PLA-PEG-PLA, PLGA-PEG-PLGA, PEG-PLA-PEG, PGE-PLGA-PEG; Be preferably PLGA-PEG-PLGA and PEG-PLGA-PEG, more preferably PLGA-PEG-PLGA.
Preferably, the concentration of said PLGA-PEG-PLGA copolymer in said hydrogel is 100-400mg/ml, more preferably 150-250mg/ml.
Said buffer agent includes but not limited to phosphate buffer, Tris-HCl buffer, citrate buffer, acetate buffer, is preferably phosphate buffer; The concentration of said buffer agent in said hydrogel is 5-100mM, is preferably 20mM; Its pH value scope is 5.0-8.0, is preferably 5.5-6.6.
Said stabilizing agent comprises but is not limited in mannitol, glycine, sorbitol, the trehalose one or more; Its concentration in said hydrogel is 1-5mg/ml.Said stabilizing agent is preferably the glycine of 3mg/ml, and wherein said glycine can make exendin-4 in required period of storage (for example 6 to 12 months), keep stable.Said trehalose can also increase the biological activity of protein and peptide class medicine.
Said antiseptic includes but not limited to one or more in Potassium Benzoate, metacresol, benzoic acid, the phenol; Its concentration in said hydrogel is 0.1-0.2mg/ml.Said antiseptic is preferably the Potassium Benzoate of 0.1mg/ml.
Preferably, the temperature sensitive type water gel of the exendin-4 of comprising according to the invention is made up of following component:
Exendin-4 1mg/ml
PLGA-PEG-PLGA copolymer 1 50mg/ml
PH6.0 phosphate buffer 20mM
Glycine 3mg/ml
Potassium Benzoate 0.1mg/ml
Preferably, the temperature sensitive type water gel of the exendin-4 of comprising according to the invention is made up of following component:
Exendin-4 1mg/ml
PLGA-PEG-PLGA copolymer 200mg/ml
PH6.0 phosphate buffer 20mM
Glycine 3mg/ml
Potassium Benzoate 0.1mg/ml
Preferably, the temperature sensitive type water gel of the exendin-4 of comprising according to the invention is made up of following component:
Exendin-4 1mg/ml
PLGA-PEG-PLGA copolymer 250mg/ml
PH6.0 phosphate buffer 20mM
Glycine 3mg/ml
Potassium Benzoate 0.1mg/ml
The temperature sensitive type water gel of the exendin-4 of comprising of the present invention can prepare through following method: get the PLGA-PEG-PLGA copolymer in glass container, add buffer, stir dissolving down, be mixed with the PLGA-PEG-PLGA copolymer of variable concentrations; Other gets buffer solution and the polymer buffer solution mixing of exendin-4.
In addition; Also can the temperature sensitive type water gel of the exendin-4 of comprising of the present invention further be passed through the filtration step of aseptic condition, for example (as: 4 ℃) carry out filtration sterilization with 0.22 micron membranes under the low viscous temperature in that the PLGA-PEG-PLGA hydrogel is maintained.The solution that obtains is installed in bottle, ampoule, the syringe, process injection.
Hydrogel of the present invention is meant and can self forms the crosslinking net thing of the hydrophilic polymer of the three dimensional structure that contains large quantity of moisture.It at room temperature is an aqueous solution, can change solid gel in vivo, solid gel can not only prolong drug release time, also can keep higher drug level, and can increase the sensitivity of medicine.
The PLGA-PEG-PLGA copolymer can form micelle in aqueous solution; When higher concentration and/or raising temperature, the PLGA-PEG-PLGA copolymer experiences gelling (solution is to gel conversion) process because of intermicellar interaction makes micelle combine to form liquid crystal state (gel); When higher temperature, gel melts again then.The phase transition temperature of PLGA-PEG-PLGA copolymer depends on its concentration in water.Usually, when the copolymer concentration scope when 10% (g/ml) changes to 40% (g/ml), solution takes place when 5-40 ℃ of temperature to gel conversion, change to solution and gel takes place in the time of temperature 40-70 ℃.Therefore, PLGA-PEG-PLGA copolymer of the present invention has the performance that under human body temperature, shows gelling (solution is to gel conversion).PLGA-PEG-PLGA does not have physiology toxicity, antigenicity and immunogenicity, has excellent biological compatibility with tissue and blood, degradable, and the gel longer duration, convenient drug administration can improve patient's compliance greatly, can be widely used in clinical.
The temperature sensitive type water gel of the exendin-4 of comprising of the present invention is used to treat diabetes, not only has good release property, and environmental protection, zest are little, convenient in application, effect are obvious.Compare with heavy dose of exendin-4 preparation, the advantage of hydrogel of the present invention is to form gel in vivo, and is simple to operate, demonstrates lasting release characteristic, improves bioavailability, reduces administration number of times, strengthens patient's compliance.
The standard dose scope of Exendin-4 administration is 10-20 μ g (microgram) for each person every day.Therefore; When using the temperature sensitive type water gel that comprises exendin-4 of the present invention to treat type 2 diabetes mellitus; Can be administered once weekly or per two weeks are administered once; Preferred dosage is everyone weekly 1-140 μ g exendin-4, more preferably everyone weekly 10-100 μ gexendin-4.
Hydrogel of the present invention can be made into injection, through intramuscular and subcutaneous route injection.
Description of drawings
Fig. 1 Exendin-4 from the temperature sensitive type water gel of the PLGA-PEG-PLGA that contains 150mg/ml, 200mg/ml, 250mg/ml, discharge 7 days the drug release curve ratio.
The whole drug release curve that Fig. 2 Exendin-4 discharges from the temperature sensitive type water gel that contains 200mg/ml PLGA-PEG-PLGA.
37 ℃ of each cycle biological activity dose response diagrams of Fig. 3 Exendin-4 hydrogel.
37 ℃ of each cycle biological activitys of Fig. 4 Exendin-4 hydrogel change diagram.
The situation of change of db/db mouse blood sugar in Fig. 5 .Exendin-4 temperature sensitive type water gel pharmacodynamics test.
The specific embodiment
Below description and combination figure through the specific embodiment the present invention is described further; But this is not to be limitation of the present invention; Those skilled in the art are according to basic thought of the present invention; Can make various modifications or improvement, but only otherwise break away from basic thought of the present invention, all within scope of the present invention.
Exendin-4 produces for self-control among the embodiment, and its preparation technology is referring to Chinese patent 200410052039.4; All the other reagent are and are purchased:
PLGA-PEG-PLGA (MW=6000) purchases the organic chemistry institute in Chengdu, lot number 20081124;
NaH
2PO
42H
2O purchases in Jinhuada Chemical Agent Co., Ltd., Guangzhou City, lot number 20061020;
Na
2HPO
412H
2O purchases in Jinhuada Chemical Agent Co., Ltd., Guangzhou City, lot number 20050425;
Glycine is purchased the consor thing Science and Technology Ltd. in the source, Shanghai, lot number 20040726.
Benzoic acid is purchased the chemical reagent company limited in Guangzhou, lot number 20050816.
Embodiment 1:
Take by weighing 0.88g Na
2HPO
412H
2O, 2.74g NaH
2PO
42H
2O, the 1.0g Potassium Benzoate, the 30.0g glycine, with the dissolving of 800ml ultra-pure water, transferring pH is 6.0, is settled to 1000ml, uses the 0.25mM filtering with microporous membrane, is prepared into the phosphate buffer of 200mM, places 4 ℃ of refrigerators to preserve.
Placing the PA tube of ice bath; 6g PLGA-PEG-PLGA (MW=6000) copolymer with weighing; Slowly join in refrigerative (0-4 ℃) 20ml ultra-pure water, under magnetic agitation (400-600rpm), dissolve fully, its abundant swelling is disperseed up to polymer; Being mixed with concentration is the PLGA-PEG-PLGA copolymer of 300mg/ml, places 4 ℃ of refrigerators for use.
Embodiment 2:
Get the 300mg/ml PLGA-PEG-PLGA of 1.5ml embodiment 1 preparation, place centrifuge tube, add the exendin-4 aqueous solution 300 μ l of 10mg/ml then respectively; The 200mM phosphate buffer 300 μ l that add the pH6.0 of embodiment 1 preparation simultaneously are with ice bath ultra-pure water polishing 3ml, in the interpolation process; Centrifuge tube places on the agitator always, and frequency of oscillation is 100-200rpm, makes its mix homogeneously; Be distributed into parallel 3 pipes, place 4 ℃ of refrigerator overnight.The concentration of final PLGA-PEG-PLGA copolymer is 150mg/ml, and the concentration of exendin-4 is 1.0mg/ml.
Embodiment 3:
Identical with embodiment 2 operations, get the 300mg/mlPLGA-PEG-PLGA of 2ml embodiment 1 preparation, place centrifuge tube; The exendin-4 aqueous solution 300 μ l that add 10mg/ml then respectively add the 200mM phosphate buffer 300 μ l of the pH6.0 of EXAMPLE l preparation, simultaneously with ice bath ultra-pure water polishing 3ml; In the interpolation process, centrifuge tube places on the agitator always, and frequency of oscillation is 100-200rpm; Make its mix homogeneously, be distributed into parallel 3 pipes, place 4 ℃ of refrigerator overnight.The concentration of final PLGA-PEG-PLGA copolymer is 200mg/ml, and the concentration of exendin-4 is 1.0mg/ml.
Embodiment 4:
Identical with embodiment 2 operations, get the 300mg/mlPLGA-PEG-PLGA of 2.5ml embodiment 1 preparation, place centrifuge tube; The exendin-4 aqueous solution 300 μ l that add 10mg/ml then respectively add the 200mM phosphate buffer 300 μ l of the pH6.0 of embodiment 1 preparation, simultaneously in the interpolation process; Centrifuge tube places on the agitator always, and frequency of oscillation is 100-200rpm, makes its mix homogeneously; Be distributed into parallel 3 pipes, place 4 ℃ of refrigerator overnight.The concentration of final PLGA-PEG-PLGA copolymer is 250mg/ml, and the concentration of exendin-4 is 1.0mg/ml.
7 days release tests of embodiment 5:Exendin-4
Get the centrifuge tube that exendin-4 and PLGA-PEG-PLGA copolymer mixed liquor are housed of embodiment 2-4 preparation respectively, be vertically placed in the centrifuge tube shelf, then centrifuge tube shelf is placed 37 ℃ of shaking table hybridization instruments.After treating that solution is completed into milky gel (about 30 minutes), slowly add and be preheated to 37 ℃ slow release medium (phosphate buffer of 20mM) 2ml, (test method is referring to Wei Wang, et al.Acceleration of diabetic wound healing withchitosan-crosslinked collagen sponge containing recombinant humanacidic fibroblast growth factor in healing-impaired STZ diabetic rats.Life Sciences in 37 ℃ of shaking table hybridization instruments (shake speed and be 75V/min), to carry out release test; 82 (2008), 190-204), respectively at 1h; 2h, 4h, 6h; 8h, 1d, 2d; 3d, 4d, 5d; 6d, 7d take out whole slow release media, blank slow release medium (phosphate buffer of the 20mM) 2ml of restock equivalent equality of temperature.With on be set forth in sample that different time points obtains respectively through 0.22 μ m filtering with microporous membrane and centrifugal; With exendin-4 content in HPLC (HPLC) working sample; Calculate the cumulative release percentage rate of exendin-4; With the cumulative release percentage rate time is mapped, obtain the exendin-4 release profiles.Test repetition 3 times is averaged.Exendin-4 discharges 7 days drug release curve ratio and sees Fig. 1 from the temperature sensitive type water gel of the PLGA-PEG-PLGA that contains 150mg/ml, 200mg/ml, 250mg/ml.The result shows that along with polymer concentration increases, rate of release reduces.Exendin-4 is that the 7th day average accumulated discharges percentage amounts and reaches 76%, 61%, 49% in the copolymer of 150mg/ml, 200mg/ml, 250mg/ml in concentration.
24 days release tests of embodiment 6:Exendin-4
Identical with embodiment 5 operations, get the centrifuge tube that exendin-4 and polymer (200mg/ml PLGA-PEG-PLGA) mixed liquor are housed of embodiment 3 preparations, be vertically placed in the centrifuge tube shelf, then centrifuge tube shelf is placed 37 ℃ of shaking table hybridization instruments.After treating that solution is completed into milky gel (about 30 minutes), slowly add and be preheated to 37 ℃ slow release medium (PBS) 2ml, (test method is referring to Wei Wang, et al.Acceleration of diabetic woundhealing with chitosan-crosslinked collagen sponge containingrecombinant human acidic fibroblast growth factor in healing-impairedSTZ diabetic rats.Life Sciences, 82 (2008) in 37 ℃ of shaking table hybridization instruments (shake speed and be 75V/min), to carry out release test; 190-204), respectively at 1h, 2h, 4h; 6h, 8h, 1d, 2d; 3d, 4d, 5d, 6d; 7d, 8d, 9d, 10d; 13d, 17d, 24d take out whole slow release media, blank slow release medium (phosphate buffer of the 20mM) 2ml of restock equivalent equality of temperature.With on be set forth in sample that different time points obtains respectively through 0.22 μ m filtering with microporous membrane and centrifugal; With exendin-4 content in HPLC (HPLC) working sample; Calculate the cumulative release percentage rate of exendin-4; With the cumulative release percentage rate time is mapped, obtain the exendin-4 release profiles.Test repetition 3 times is averaged.The whole release profiles that Exendin-4 discharges from the temperature sensitive type water gel that contains 200mg/ml PLGA-PEG-PLGA is seen Fig. 2.The result shows; Exendin-4 discharges in hydrogel, and the individual rapid release phase is arranged at first, and reason is that part exendin-4 is not and due to hydrogel combines fully; After 1 day; Exendin-4 is curved release in hydrogel, surpasses 90% to about the 17th day accumulative total release rate, has the effect of long-acting slow-release.
Embodiment 7:Exendin-4 determination of activity
Identical with embodiment 5 operations, get the centrifuge tube that exendin-4 and polymer (200mg/ml PLGA-PEG-PLGA) mixed liquor are housed of embodiment 3 preparations, be vertically placed in the centrifuge tube shelf, then centrifuge tube shelf is placed 37 ℃ of shaking tables.After treating that solution is completed into milky gel (about 30 minutes); Slowly add and be preheated to 37 ℃ slow release medium (PBS) 2ml; (test method is referring to Wei Wang in 37 ℃ of shaking table hybridization instruments (shake speed and be 75V/min), to carry out release test; Et al.Acceleration of diabetic wound healing withchitosan-crosslinked collagen sponge containing recombinant humanacidic fibroblast growth factor in healing-impaired STZ diabetic rats.Life Sciences; 82 (2008); 190-204), respectively at taking out whole slow release media on the the 1st, 3,7 and 14 day, blank slow release medium (phosphate buffer of the 20mM) 2ml of restock equivalent equality of temperature.With on be set forth in sample that different time points obtains respectively through 0.22 μ m filtering with microporous membrane and centrifugal; The amount of GLP-1 cAMP that receptors bind produces through detecting exendin-4 and cell surface; Activity to exendin-4 is measured (assay method is referring to Chinese patent 200610076398.2), detects its burst size through HPLC simultaneously.The result sees Fig. 3, Fig. 4.Wherein the result of Fig. 3 shows: the activity of exendin-4 has dose-dependence, and sample did not have significant difference in the 1st, 3,7 days, has significant difference, and explains that exendin-4 began inactivation at 7-14 days in the 14th day and 1,3,7 days; The result of Fig. 4 shows: the 1st, 3, the exendin-4 activity of sampling in 7 days does not have significant difference, explains that the exendin-4 activity still remains intact in 7 days; And 14 days exendin-4 activity slightly descend, and explain that exendin-4 began inactivation in the time of 7-14 days.
The pharmacodynamic experiment of embodiment 8:Exendin-4 hydrogel
The PLGA-PEG-PLGA copolymer concentration of experimental drug: embodiment 3 preparations is 250mg/ml, and exendin-4 concentration is the hydrogel of 1.0mg/ml, uses the water for injection dilution.
Laboratory animal: the db/db mice in 6 ages in week, totally 30.
Experiment is divided into groups: 30 mices are divided into 5 groups at random, that is:
The exendin-4 matched group (exendin-4 dosage 0.2 μ g/kg, once a day; Be called for short matched group);
PLGA-PEG-PLGA gel group (no exendin-4, weekly; Be called for short the gel group);
The heavy dose of group of exendin-4 hydrogel (exendin-4 dosage 4 μ g/kg, weekly; Be called for short heavy dose of group);
Dose groups in the exendin-4 hydrogel (exendin-4 dosage 2 μ g/kg, weekly; Dose groups in the abbreviation);
Exendin-4 hydrogel small dose group (exendin-4 dosage 1 μ g/kg, weekly; Be called for short small dose group).
Experimental technique: with laboratory animal at 25 ℃ of temperature, relative humidity 60%, freely drink water, raise a week at regular time and quantity the environment, subcutaneous injection administration then, fasting is 4 hours before each administration.Get mouse tail vein blood in 0h, 1h, 2h, 4h, 8h, 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 10d, 12d, 14d after the administration and carry out rapid blood sugar mensuration (used blood glucose meter and blood sugar test paper are Johson & Johnson and produce).
Experimental result: experimental result is seen Fig. 5, wherein the equal obvious blood sugar lowering of exendin-4 hydrogel group; Compare with matched group, exendin-4 hydrogel group blood sugar lowering is slow slightly, but can reach the effect of remarkable blood sugar lowering about 8 hours, and blood glucose maintains normal level always after 12 hours, does not have significant difference between three dose groups; The animal blood glucose of gel group does not have significant change.
Claims (10)
1. temperature sensitive type water gel comprises:
(1) as the exendin-4 of effective ingredient, its concentration in said hydrogel is 0.1-100mg/ml;
(2) as the amphipathic copolymer of slow-released carrier, the block copolymer that this amphipathic copolymer is made up of polyester and Polyethylene Glycol, its number-average molecular weight is 500-28000, its concentration in said hydrogel is 100-400mg/ml;
Wherein said exendin-4 is dispersed in the said amphipathic copolymer; And said hydrogel at room temperature is a liquid, is the insoluble gel of solid water at 37 ℃.
2. temperature sensitive type water gel according to claim 1 wherein also is dispersed with in buffer agent, stabilizing agent, the antiseptic one or more in said slow-released carrier.
3. temperature sensitive type water gel according to claim 1, wherein said exendin-4 are exendin-4 polypeptide or its isoform, mutant, fusion rotein, functional deriv, active fragment.
4. temperature sensitive type water gel according to claim 1; Wherein said polyester is selected from copolymer (PLGA), the poly-dl-lactide (D of polylactic acid (PLA), polyglycolic acid and hydroxyacetic acid; L-PLA), raceme polyglycolic acid and co-glycolic acid (D, L-PLGA), end carboxyl polylactic acid (PLA-COOH), end carboxyl polyglycolic acid and co-glycolic acid (PLGA-COOH).
5. temperature sensitive type water gel according to claim 4, wherein said amphipathic copolymer is PLGA-PEG-PLGA.
6. temperature sensitive type water gel according to claim 2, wherein said buffer agent are selected from phosphate buffer, Tris-HCl buffer, citrate buffer, acetate buffer; And the concentration of said buffer agent in said hydrogel is 5-100mM.
7. temperature sensitive type water gel according to claim 2, wherein said stabilizing agent is selected from one or more in mannitol, glycine, sorbitol, the trehalose; And the concentration of said stabilizing agent in said hydrogel is 1-5mg/ml.
8. temperature sensitive type water gel according to claim 2, wherein said antiseptic is selected from one or more in Potassium Benzoate, metacresol, benzoic acid, the phenol; And the concentration of said antiseptic in said hydrogel is 0.1-0.2mg/ml.
9. temperature sensitive type water gel according to claim 2, it is made up of following component:
Exendin-4 1mg/ml
PLGA-PEG-PLGA copolymer 1 50 or 200 or 250mg/ml
PH6.0 phosphate buffer 20mM
Glycine 3mg/ml
Potassium Benzoate 0.1mg/ml
10. injection that comprises each described temperature sensitive type water gel among the claim 1-9.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103622902A (en) * | 2012-08-24 | 2014-03-12 | 上海现代药物制剂工程研究中心有限公司 | A temperature-sensitive gel medicine preparation and a preparation method thereof |
| CN103637978A (en) * | 2013-12-13 | 2014-03-19 | 北京诺康达医药科技有限公司 | Stable gel containing bromelain |
| CN111297799A (en) * | 2019-04-12 | 2020-06-19 | 浙江大学 | Sustained-release composition preparation of polypeptide protein medicine and preparation method thereof |
| CN111743855A (en) * | 2020-03-25 | 2020-10-09 | 天津大学 | Method for synthesizing gel preparation for treating rheumatoid arthritis |
| CN113248723A (en) * | 2021-04-13 | 2021-08-13 | 康汉医药(广州)有限公司 | Preparation method of protein drug sustained-release preparation |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103622902A (en) * | 2012-08-24 | 2014-03-12 | 上海现代药物制剂工程研究中心有限公司 | A temperature-sensitive gel medicine preparation and a preparation method thereof |
| CN103622902B (en) * | 2012-08-24 | 2016-12-21 | 上海现代药物制剂工程研究中心有限公司 | A kind of thermosensitive hydrogel pharmaceutical preparation and preparation method thereof |
| CN103637978A (en) * | 2013-12-13 | 2014-03-19 | 北京诺康达医药科技有限公司 | Stable gel containing bromelain |
| CN111297799A (en) * | 2019-04-12 | 2020-06-19 | 浙江大学 | Sustained-release composition preparation of polypeptide protein medicine and preparation method thereof |
| CN111297799B (en) * | 2019-04-12 | 2021-12-03 | 浙江大学 | Sustained-release composition preparation of polypeptide protein medicine and preparation method thereof |
| CN111743855A (en) * | 2020-03-25 | 2020-10-09 | 天津大学 | Method for synthesizing gel preparation for treating rheumatoid arthritis |
| CN113248723A (en) * | 2021-04-13 | 2021-08-13 | 康汉医药(广州)有限公司 | Preparation method of protein drug sustained-release preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102370611B (en) | 2013-09-18 |
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