CN105982845A - Thermotropic hydrogel hypoglycemic drug preparation and preparing method thereof - Google Patents
Thermotropic hydrogel hypoglycemic drug preparation and preparing method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of high polymer materials and medical treatment, and particularly relates to a thermotropic hydrogel hypoglycemic drug preparation and a preparing method thereof. The thermotropic hydrogel hypoglycemic drug preparation comprises hypoglycemic peptide and a thermotropic gelatinization polymer; the preparing method includes the following steps that according to the raw material formula, drugs are directly dissolved into polymer thermotropic gel existing in a solution mode, polypeptide is directly packaged with gel, slow releasing of polypeptide drugs is achieved through the joint-assembling action between the polypeptide and the polymer, and the long-acting releasing preparation capable of reducing blood glucose is prepared.
Description
Technical field
The invention belongs to macromolecular material and field of medical technology, be specifically related to a kind of hydrogel with thermic plastic characteristic
Hypoglycemic medicine preparation and preparation method thereof.
Background technology
It is a kind of by h and E factor interaction for prior art discloses diabetes (diabetes mellitus, DM)
And the metabolic syndrome caused.Owing to hypoinsulinism or insulin sensitivity reduce, the diabetics one is caused to be
Row metabolism disorder, mainly shows as hyperglycemia, and long can cause multiple tract impaired, and causes serious
Complication etc., this illness has become as the worldwide public health problem of serious threat human health.Diabetes are broadly divided into
I type and type ii diabetes, wherein the sickness rate of type ii diabetes accounts for more than the 90% of diabetics sum, its machine of falling ill
Reason is due to beta Cell of islet defect of insulin secretion and insulin resistant.Diabetes are brought sternly to family and the society of patient
The burden of weight.In recent decades, along with the progress of human sciences's technology and developing rapidly of medical level, treating diabetes
The research and development of medicine achieve many progress.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (Liraglutide) is a kind of pancreas researched and developed by Novo Nordisk Co., Ltd of Denmark (Novo Nordisk Inc.)
Glucagon-like peptide-1 (GLP-1) analog, in June, 2009 and in January, 2010 respectively by EMA and FDA
Approval listing, for the treatment of adult's type ii diabetes.The molecular formula of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is C172H265N43O51, molecule
Amount is 3751.20.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and endogenous people's GLP-1 main distinction on the 34th by human body in natural
The arginine of GLP-1 replaces with lysine, by glutamic acid and the fat of 16 carbon on the lysine of the 26th
Acid is connected, and the existence of C16 aliphatic chain considerably increases the hydrophobicity of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], thus improves the stability of medicine,
C16 aliphatic chain is believed to strengthen Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and albuminous non-covalent bond combines, thus is greatly prolonged Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Half-life in vivo.Similar to GLP-1, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] acts on GLP-1 receptor, and exciting this receptor stimulates islets of langerhans
Element discharges according to concentration of glucose dependency mode, reduces plasma glucagon level, and can be done directly on β cell
Promote its propagation and differentiation.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] has delay gastric emptying, increases satiety, reduces appetite, loses weight, fall
The effects such as lower shrinkage pressure.Owing to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and endogenous people GLP-1 have 97% amino acid sequence homology, thus
Incidence rate and the antibody horizontal of its antibody tormation are relatively low, have the highest safety and less side effect.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Commodity injection promise and power() on October 9th, 2011 is formal in Discussion on Chinese Listed.Its specification is 3 milliliters:
18 milligrams of prefilled injection pen, it is adaptable to after metformin alone or the treatment of sulfonylurea drugs maximum tolerable dose, blood glucose is still
Control the best patient, can be with metformin or sulfonylurea drugs use in conjunction.The 3mg of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fills injectionAlso the medicine as treatment chronic fatty is ratified by FDA, as diet control and body in December, 2014
Educate supplementing of exercise.Also it is the currently the only fat-reducing injection ratified by FDA.Other GLP-1 analog
In, such as albiglutide, Li Sina peptide, Du Lalu peptide, Suo Malu peptide etc. is widely studied the most equally, the biggest portion
Divide and gone through listing or be in clinical investigation phase.
But, need 1 subcutaneous injection every day to rise for the Polypeptide-k of injection-type every day, such as Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] injection
To blood sugar reducing function, for patients, this drug administration by injection mode still seems excessively frequent, the most complicated to prescription
Case brings bigger misery and inconvenience to patient.Therefore the long-acting slow-release preparation of the Polypeptide-ks such as Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is prepared to increasing
Adding the bioavailability of medicine, reduce the frequency of drug administration by injection, the compliance improving patient medication has huge clinical valency
Value and meaning.The long-acting slow-release preparation of the Polypeptide-ks such as current Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] there is not yet report.
The selection of long-acting injection drug administration system carrier typically has following three kinds: (1) oily solution or suspension type ejection preparation；
(2) non-biodegradation implants；(3) biodegradable injection.Along with developing rapidly of polymeric materials science,
Biodegradable material is that the drug-supplying system of skeleton progressively becomes the mainstream carrier that long-acting injection is administered, wherein with polyesters material
The application of material is the most ripe.Polyester biodegradable material can gradually be degraded to lactic acid, ethanol in vivo
The internal intrinsic small organic molecules such as acid, then discharge through internal tricarboxylic acid cycle.Wherein, with third hand over fat (D, L-lactide,
LA), glycolide (glycolide, GA) and polymer that 6-caprolactone (ε-caprolactone, CL) is monomer or altogether
Polymers is to use most biodegradable polymers in polyester material.
Nearly ten years, domestic and international researcher is by introducing degradable polyester block or poly-ammonia in PEG hydrophilic block
Base acid block, is prepared for various PEG/ polyester (polyamino acid) amphipathic nature block polymer, and it was found that
When having suitable overall hydrophobe balance, synthesized PEG/ polyester (polyamino acid) block copolymer can be low
Temperature time be dissolved in water, simultaneously because material is amphipathic, block copolymer can by being self-assembly of micelle, along with
Temperature raises, and these micelles will be assembled further, forms the network of " micelle-gel ".This can be with temperature
The polymer water system that degree raises and occurs sol-gel to change is referred to as thermic hydrogel (thermogel).
Compared with other injection delivery systems, PEG/ degradable polyester (polyamino acid) thermic hydrogel has a plurality of advantages:
(1) solution state under low temperature makes it can be able to be implanted by ordinary syringe injection, easy to operate, with
Time the good biological degradability of material and biocompatibility, it is to avoid follow-up operation is taken out；(2) by body temperature from stimulating food
Reason is cross-linked to form gel, gelation and embedding process and is all difficult to make the active substance of embedding to inactivate；(3) " cryogenic fluid,
High temperature gel " feature facilitate the preparation of preparation, only polymer solution need to be simply mixed with medicine, and whole
Individual preparation process relates to any organic solvent hardly；(4) can directly filtration sterilization under cryogenic fluid state, it is not necessary to penetrate
The processing procedure that line, autoclaving etc. are complicated, it is ensured that Drug safety and stability；(5) medicament slow release
Behavior can regulate and control by simply changing material composition, structural property etc..Therefore PEG/ polyester (polyamino acid)
Thermic hydrogel is one of ideal carrier of polypeptide, the research of albumen long-acting injection drug-supplying system.
But, during polypeptide drug aquogel system prepares medicament slow release preparation, owing to polypeptide drug is good
Water solublity so that it is often face that early stage is prominent to be released and the later stage discharges incomplete problem.Early stage is prominent to be released so that its initial stage discharges
Substantial amounts of medicine, does not reaches the purpose of slow release, and later stage release not exclusively causes high amount of drug to remain in gel, makes
Become later stage dose not enough, it is impossible to reach the effect being steadily administered continuously.One of solution currently for described problem is
Adding various adjuvant in pharmaceutical preparation and carry out the release behavior of regulating drug, e.g., Chinese Patent Application No. is
201210305356.7 patent disclose a kind of thermosensitive hydrogel pharmaceutical preparation and preparation method thereof.This thermosensitive hydrogel medicine system
Agent composition of raw materials contains medicine, thermo-sensitive gel composition, and adjuvant C；Described adjuvant C is slaine, saccharide thing
One or more in the hydrophilic polymer of matter and injection；Described slaine is pharmaceutically acceptable and water
Solution dissociates the slaine of divalent metal；Described medicine is the material existed with anionic form in aqueous solution
A, described substance A is one or more in pharmaceutical grade protein, polypeptide drug and glycopeptide antibiotics.This patent
Synergism by multi-medicament adjuvant, it is possible to effectively improve pharmaceutical grade protein, polypeptide drug and glycopeptide class antibiosis
The prominent problem of releasing of element, but it prepares scheme complexity, and the introducing of one or more pharmaceutic adjuvants simultaneously also can strong influence
The syringeability of the prepared preparation of the character of the gel of thermo-sensitive gel composition own, even impact.
So far by medicine and hydrogel material at the controlled water of the mutual self assembly effect regulating drug of molecular level
Gel sustained-release preparation is seldom seen in report.The problem existed in view of prior art, present inventor intends providing a kind of tool
Long-acting gel blood sugar lowering slow releasing preparation that can subcutaneously or intramuscularly inject having thermic plastic characteristic and preparation method thereof, to solve water
The common initial stage during gel delivery polypeptide, protein drug dashes forward and releases problem incomplete with later stage release.
Summary of the invention
It is an object of the invention to for prior art exist problem, it is provided that a kind of have thermic plastic characteristic can subcutaneous or
Long-acting gel blood sugar lowering sustained release pharmaceutical formulation of intramuscular injection and preparation method thereof.
The present invention utilizes thermic hydrogel material bag to carry polypeptide drug, prepares the long-acting gel that can subcutaneously or intramuscularly inject and delays
Release formulation, by medicine with hydrogel material in the slow release behavior of the mutual self assembly effect regulating drug of molecular level, solves
The common initial stage during certainly hydrogel delivers polypeptide, protein drug dashes forward and releases problem incomplete with later stage release.The present invention
Slow releasing preparation compared to GLP-1, the structural modification of the Polypeptide-k such as Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the particularly introducing of hydrophobic components,
The stability of medicine can be significantly improved, strengthen medicine and albuminous non-covalent bond combines, it is thus possible to significantly extend peptide drug
The thing half-life in vivo；Meanwhile, the structural modification in slow releasing preparation of the present invention, can be itself and PEG/ polyester (poly-ammonia
Base acid) realize effective intermolecular interaction between amphipathic thermic hydrogel material and provide possibility.
Concrete, the invention discloses a kind of thermic hydrogel hypoglycemic medicine preparation, it contains Polypeptide-k and heat-induced gel
The polymer changed, is prepared by following method: by composition of raw materials, and medicine is directly dissolved in exist in the form of a solution poly-
In compound heat-induced gel, use gel directly to wrap up polypeptide, realize many by the common assembly behavior between polypeptide and polymer
The slow release of peptide medicine, prepares and can drop hypoglycemic long-acting slow-release preparation.
More specifically, the thermic hydrogel hypoglycemic medicine preparation of the present invention, it is characterised in that it is by medicine and injectable
Polymer thermic hydrogel forms；Described medicine is Polypeptide-k, and described thermic hydrogel is by amphipathic block polymerization
Thing and solvent composition, this gel rubber system is in solution state under room temperature or low temperature, can Spontaneous conversion under human body temperature
For gel, wherein the hydrophilic block of amphipathic nature block polymer is Polyethylene Glycol (PEG), and hydrophobic block is degradable poly
Ester or polyamino acid.
In the thermic pharmaceutical hydrogel preparation of the present invention, common assembling between medicine and polymer carrier materials, can be occurred to make
With.
In the thermic pharmaceutical hydrogel preparation of the present invention, its medicine carrying gel rubber system has heat-induced gel performance, pharmaceutical preparation
Under room temperature or low temperature, it is in solution state, can Spontaneous conversion be gel under human body temperature.
In the present invention, the sol-gel transition temperature of thermic its polymer water system of pharmaceutical hydrogel preparation is between 4-40 DEG C
Between, in one embodiment of the present of invention, the sol-gel transition temperature of preferred polymers aqueous systems between 20-37 DEG C,
In an alternative embodiment of the invention, more preferably the sol-gel transition temperature of polymer water system is between 30-37 DEG C.
In the block copolymer of the heat-induced gel pharmaceutical preparation of the present invention:
1) mean molecule quantity of described hydrophilic Polyethylene Glycol block A is 400-8000, and content is 10-90wt%；
2) mean molecule quantity of described hydrophobic polyester block or polyamino acid B block is 500-40000, and content is
90-10wt%；Wherein, hydrophobic polyester block is selected from poly DL-lactide, poly-D-lactide, PLLA, poly-second
Lactide, poe, poly-epsilon-caprolactone, poly-ε-alkyl replaces caprolactone, poly-δ-valerolactone, poly-Isosorbide-5-Nitrae, 8-trioxa spiral shell
[4.6]-9-hendecanone, poly-para-dioxanone, polyesteramide, Merlon, polyacrylate, polyether ester or above-mentioned
The copolymer that two or more polyester in each polyester is formed；
Wherein, hydrophobic polyamino acid block selected from polyalanine, polyphenylalanine, poly-leucine, polylysine,
The copolymer that two or more polyamino acid in polyglutamic acid, poly-aspartate or above-mentioned each polyamino acid is formed；
3) described block copolymer can be two block copolymerizations of the triblock copolymer of ABA or BAB type, AB type
Thing, the graft copolymer of A-g-B or B-g-A type, (A-B)nOr (B-A)nStar block copolymer and A (BA)nOr
B(AB)nThe segmented copolymer of configuration, wherein n is the integer of 2 to 10；
4) the part or all of end of described block polymer is connected to function end group, and end group is hydrophilic hydroxyl, amino, carboxylic
Any one of base, imidazole radicals, aldehyde radical, cyano group, nitro；Or hydrophobic alkyl, sterin, alkoxyl, virtue
Any one of perfume base, aromatic heterocyclic, amide ester group, halogen atom, trichloromethyl, ester group, sulfydryl.
In the thermic pharmaceutical hydrogel preparation of the present invention, its polymer can be single amphipathic nature block polymer, it is possible to
Compositions for the amphipathic nature block polymer of two or more different molecular structures.
The medicine wrapping load in the heat-induced gel pharmaceutical preparation of the present invention is Polypeptide-k, including Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], albiglutide,
One or more in Li Sina peptide, Du Lalu peptide, Suo Malu peptide.
In the present invention, the content of medicine accounts for the 0.01-5% of heat-induced gel pharmaceutical preparation, is preferably 0.1-2%.
Heat-induced gel pharmaceutical preparation in the present invention, polymer content in the formulation is 3-50wt%.
Heat-induced gel pharmaceutical preparation in the present invention, the solvent in polymer solution be pure water, normal saline, buffer solution,
The body fluid of tissue culture medium, cell culture fluid, animals and plants or human body or for other aqueous solution with organic solvent be not
The medium of main body.
The present invention prepares heat-induced gel preparation by following method: first at dissolution in low temperature polymer in aqueous solution, then
At-20 DEG C or following store for future use, redissolve before using and be directly added into described Polypeptide-k, after mixing, becoming solution note
Penetrate agent.
The above low temperature refers to the solution-gel transition temperature less than polymer；Prepared heat-induced gel pharmaceutical preparation exists
Thermal reversion hydrogel can be formed when temperature is higher than solution-gel transition temperature.
Heat-induced gel pharmaceutical preparation in the present invention, described heat-induced gel pharmaceutical preparation can be subcutaneously or intramuscularly by entry needle
Inject in tissue.
In said method, block copolymer is by PEG macromole and degradable polyester monomer or the open loop of polyamino acid monomer
Polymerization obtains.
The heat-induced gel material of the present invention has good biocompatibility and degradability, and this material can be within a certain period of time
Degradable for nontoxic α-ol acid and other corresponding monomers.
The heat-induced gel pharmaceutical preparation of the present invention can control Polypeptide-k and discharge the most constantly.
The heat-induced gel pharmaceutical preparation of the present invention does not only have long-acting blood sugar reducing function, is alternatively arranged as long-acting slimming medicine preparation.
It is an advantage of the current invention that: utilize thermic hydrogel material directly to wrap up polypeptide drug, without various medicinal
Adjuvant or in the case of being chemically modified, makees in the mutual self assembly of molecular level with hydrogel material only by medicine
With the slow release behavior of regulating drug, solve hydrogel and deliver the common initial stage during polypeptide, protein drug and prominent release and the later stage
The incomplete problem of release.
The present invention has good reference for utilizing polypeptide drug-material interaction to prepare prolonged drug slow releasing preparation
It is worth and Practical significance.
Accompanying drawing explanation
Fig. 1 is the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous solution of embodiment 25 preparation, Copolymer-1 and Copolymer-1/ Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Mixture aqueous solution dynamic light scattering figure.
Fig. 2 is the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous solution of embodiment 26 preparation, Copolymer-4 and Copolymer-4/ Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Mixture aqueous solution dynamic light scattering figure.
Fig. 3 is Copolymer-1 thermic hydrogel and the thermic hydrogel medicine of embodiment 18 preparation of embodiment 10 preparation
The storage modulus of thing preparation and viscosity with temperature variation diagram.
Fig. 4 is Copolymer-4 thermic hydrogel and the thermic hydrogel medicine of embodiment 19 preparation of embodiment 11 preparation
The storage modulus of thing preparation and viscosity with temperature variation diagram.
Fig. 5 is the Copolymer-1 thermic pharmaceutical hydrogel agent in vitro releasing curve diagram of embodiment 18 preparation.
Fig. 6 is the Copolymer-4 thermic pharmaceutical hydrogel agent in vitro releasing curve diagram of embodiment 19 preparation.
Fig. 7 is the Copolymer-5 thermic pharmaceutical hydrogel agent in vitro releasing curve diagram of embodiment 20 preparation.
Fig. 8 is thermic pharmaceutical hydrogel preparation and the single medicine aqueous solution shadow to ICR mouse blood sugar of embodiment 18 preparation
Ring graph of a relation.
Fig. 9 is that subcutaneous injection of thermic pharmaceutical hydrogel preparation of embodiment 18 preparation is to blood glucose during ICR mice the 0th day
Affect graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 10 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 1st day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 11 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 2nd day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 12 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 3rd day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 13 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 4th day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 14 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 5th day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 15 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 6th day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 16 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 7th day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 17 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 8th day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 18 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation is to the shadow of blood glucose during ICR mice the 9th day
Ring graph of a relation；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Figure 19 is that a thermic pharmaceutical hydrogel preparation shot for embodiment 18 preparation affects relation to the change of ICR Mouse Weight
Figure；" * ": p ＜ 0.05, " * * ": p ＜ 0.01, " * * * ": p ＜ 0.001vs NaCl.
Detailed description of the invention
Further describe the present invention below by example, but the most therefore limit the present invention in these embodiments.
Embodiment 1
Taking 30g PEG (1500) in 250mL there-necked flask, 150 DEG C of lower evacuation 3h of stirring are to remove PEG
The moisture of middle residual.Then, 65.6g 6-caprolactone (CL), 7.4g Acetic acid, hydroxy-, bimol. cyclic ester (GA) and 0.1wt% are added pungent
Acid stannous, below 100 DEG C, evacuation 30min will be will wherein remove by toluene.It is warming up to 160 DEG C, treats that monomer is whole
After Rong Rong, react 12h under an argon atmosphere.After reaction terminates, vacuum filtration 3h is to remove unreacted monomer and low
Boiling product, pours out product while hot, washed several times with water postlyophilization, obtains triblock copolymer, and productivity is about
80%.Described BAB block copolymerization is measured by chromatograph of gel permeation (GPC) (using polystyrene as standard specimen)
The number of thing (PCGA-PEG-PCGA, Copolymer-1) all with weight average molecular weight (Mn, Mw) it is respectively 7530 Hes
10010, molecular weight distribution index (Mw/Mn) it is 1.33.The aqueous systems of this copolymer has the characteristic of heat-induced gel.
Embodiment 2
Taking 30g PEG (1500) in 250mL there-necked flask, 150 DEG C of lower evacuation 3h of stirring are to remove PEG
The moisture of middle residual.Then, 36.7g DL-lactide (LA), 34.5g Acetic acid, hydroxy-, bimol. cyclic ester (GA) and 0.1wt% are added
Stannous octoate, 150 DEG C, react 12h under argon gas atmosphere.After reaction terminates, vacuum filtration 3h is to remove unreacted
Monomer and low-boiling products.Crude product being dissolved in dichloromethane solution, ether sedimentation, productivity is about 85%.Pass through
GPC measures the number of described BAB block copolymer (PLGA-PEG-PLGA, Copolymer-4) all and Weight-average molecular
Amount (Mn, Mw) it is respectively 6650 and 8050, molecular weight distribution index (Mw/Mn) it is 1.21.The water of this copolymer
System has the characteristic of heat-induced gel.
Embodiment 3
Taking 30g PEG (1500) in 250mL there-necked flask, 150 DEG C of lower evacuation 3h of stirring are to remove wherein
The moisture of residual.Then, 75.6g 6-caprolactone (CL) and 0.1wt% stannous octoate are added, at 150 DEG C, argon
12h is reacted under gas atmosphere.After reaction terminates, vacuum filtration 3h is to remove unreacted monomer and low-boiling products.Take advantage of
Product is poured out by heat, and washed several times with water postlyophilization obtains triblock copolymer, and productivity is about 80%.By solidifying
Glue penetration chromatograph (GPC) (using polystyrene as standard specimen) measures described BAB block copolymer
The number of (PCL-PEG-PCL, Copolymer-5) all with weight average molecular weight (Mn, Mw) it is respectively 8750 and 12090,
Molecular weight distribution index (Mw/Mn) it is 1.38.The aqueous systems of this copolymer has the characteristic of heat-induced gel.
Embodiment 4
Take 22.5g mono methoxy end-blocking Polyethylene Glycol (MPEG 750) in 250mL there-necked flask, 130 DEG C
The lower evacuation 3h of stirring is to remove the moisture of residual in MPEG.Then, add 45g DL-LA, 6g GA and
0.1wt% stannous octoate, below 100 DEG C, evacuation 30min will be will wherein remove by toluene.It is warming up to 150 DEG C, treats
After monomer all melts, react 12h under an argon atmosphere.After reaction terminates, vacuum filtration 3h is unreacted to remove
Monomer and low-boiling products, pour out product while hot, washed several times with water postlyophilization, obtains di-block copolymer material
Material, productivity is about 75%.Described BA di-block copolymer (MPEG-PLGA, Copolymer-6) is measured by GPC
Number all with weight average molecular weight (Mn, Mw) it is respectively 3580 and 4760, molecular weight distribution index (Mw/Mn) it is 1.33.
The aqueous systems of this copolymer has the characteristic of heat-induced gel.
Embodiment 5
Taking 11.0g MPEG (550) and be dissolved in 80mL toluene, distillation has removed residual moisture in polymer to 30mL.
Adding 21.0g CL and 0.3wt% stannous octoate, reflux at 120 DEG C 24h.It is subsequently added HDMI 1.62mL,
7h is reacted at 60 DEG C.Then adding ether in above-mentioned solution, precipitation obtains head product.Gained head product is dissolved in 30
ML dichloromethane, is slowly added to ether so that its precipitation, and in product, residual solvent is removed by evacuation, obtains
PECE material, productivity is about 65%.Described ABA block copolymerization is measured by GPC
The number of thing (PEG-PCL-PEG, Copolymer-10) all with weight average molecular weight (Mn, Mw) it is respectively 5260 and 6260,
Molecular weight distribution index (Mw/Mn) it is 1.19.The aqueous systems of this copolymer has the characteristic of heat-induced gel.
Embodiment 6
Taking 8.5g PEG (1000) in 250mL there-necked flask, 130 DEG C of lower evacuation 3h of stirring are to remove PEG
The moisture of middle residual.Then, add CL 16.9g, trimethylene carbonate 7.6g and 0.1wt% stannous octoate,
120 DEG C, react 24h under argon gas atmosphere.After reaction terminates, vacuum filtration 3h is to remove unreacted monomer and low
Boiling product.Head product being dissolved in dichloromethane solution, ether sedimentation, productivity is about 85%.Measured by GPC
The number of described BAB block copolymer (PCTC-PEG-PCTC, Copolymer-11) all with weight average molecular weight (Mn,
Mw) it is respectively 4940 and 6520, molecular weight distribution index (Mw/Mn) it is 1.32.The aqueous systems of this copolymer has
The characteristic of heat-induced gel.
Embodiment 7
Take 8.8g MPEG (550) and be dissolved in 80mL toluene, distill to 30mL to remove remaining moisture content in polymer.
Adding 48g trimethylene carbonate and 0.1wt% stannous octoate, reflux at 120 DEG C 24h.Then above-mentioned
Adding ether in solution, precipitation obtains head product.Gained head product is dissolved in 30mL dichloromethane, be slowly added to ether with
Making it precipitate, in product, residual solvent is removed by evacuation, obtains MPEG-PTMC di-block copolymer material,
Productivity is about 65%.Described BA di-block copolymer (MPEG-PTMC, Copolymer-12) is measured by GPC
Number all with weight average molecular weight (Mn, Mw) it is respectively 5610 and 7850, molecular weight distribution index (Mw/Mn) it is 1.40.
The aqueous systems of this copolymer has the characteristic of heat-induced gel.
Embodiment 8
Take 5g one end be methoxyl group other end be that the PEG (1000) of amino is dissolved in 80mL toluene, distill to 15mL
Remove remaining moisture content in polymer.Add anhydrous chloroform and DMF solvent 50mL and 4.2g L-that volume ratio is 2/1
Alanine-N-carboxylic acid anhydrides, reacts 24h at 40 DEG C.Then adding ether in above-mentioned solution, precipitation obtains primiparity
Thing.Gained head product is dissolved in 30mL dichloromethane, is slowly added to ether so that its precipitation, and in product, residual solvent passes through
Evacuation removes, and obtains MPEG-L-PA di-block copolymer material, and productivity is about 70%.Institute is measured by GPC
State the number of BA di-block copolymer (MPEG-L-PA, Copolymer-16) all with weight average molecular weight (Mn, Mw) point
It is not 1100 and 1320, molecular weight distribution index (Mw/Mn) it is 1.20.The aqueous systems of this copolymer has thermic and coagulates
The characteristic of gel.
Embodiment 9
The basic step be given according to embodiment 1, synthesizes other by the PEG of different molecular weight and different monomers various embedding
Section copolymer, its characterization result as shown in Table 1 and Table 2:
Table 1
In above-mentioned table 1, block copolymer is respectively provided with the performance of heat-induced gel, and copolymer is made into certain density aqueous solution,
It is solution state when temperature is less than gel transition temperature, rises with temperature and gradually forms gel, and this process is reversible
Process；
Table 2
In above-mentioned table 2, block copolymer does not the most have the performance of heat-induced gel, all can only be dissolved in the water；
Table 3
In above-mentioned table 3, block copolymer does not the most have the performance of heat-induced gel, and copolymer all can not be dissolved in the water or only
Can be partially dissolved in water；
The present embodiment can take one or more block copolymers shown in table 1 further and mix by a certain percentage, in temperature
Less than being dissolved in water below gel transition temperature, when temperature is higher than gel transition temperature, the solution shape of polymeric blends
Become gel；Or, take one or more block copolymers shown in table 1 and/or one or more block copolymers in table 2
And/or one or more block copolymers in table 3 mix by a certain percentage, it is less than below gel transition temperature in temperature molten
Xie Yushui, when temperature is higher than gel transition temperature, the solution of polymeric blends forms gel；Or, take in table 2 one
Plant or multiple block copolymer mixes by a certain percentage with one or more block copolymers in table 3, in temperature less than solidifying
Being dissolved in the water below glue transition temperature, when temperature is higher than gel transition temperature, the solution of polymeric blends is formed solidifying
Glue.
Embodiment 10
Weigh appropriate block copolymer C opolymer-1, add a certain amount of phosphate buffered solution, prepare to obtain 25wt%
Solution, this solution has heat-induced gel characteristic, when temperature higher than sol-gel phase transition temperature time, it is possible to spontaneous
Form gel, measure its sol-gel phase transition temperature by test tube anastrophe, the test tube of sample will be loaded with in water-bath
180 ° of inversions after middle balance 10min, substantially flow if not observing sample in 30s, are then judged as gel state, knot
Fruit shows that its gel transition temperature is 32 DEG C.
Embodiment 11
Weigh appropriate block copolymer C opolymer-4, add a certain amount of normal saline, prepare to obtain the solution of 25wt%,
This solution has heat-induced gel characteristic, when temperature is higher than sol-gel phase transition temperature, it is possible to spontaneously form gel,
Measuring its sol-gel phase transition temperature by test tube anastrophe, result shows that its gel transition temperature is 34 DEG C.
Embodiment 12
Weigh appropriate block copolymer C opolymer-6, add a certain amount of deionized water, prepare to obtain the solution of 15wt%,
This solution has heat-induced gel characteristic, when temperature is higher than sol-gel phase transition temperature, it is possible to spontaneously form gel,
Measuring its sol-gel phase transition temperature by test tube anastrophe, result shows that its gel transition temperature is 40 DEG C.
Embodiment 13
Block copolymer C opolymer-1 and the Copolymer-6 of quality such as weigh, add a certain amount of deionized water,
Preparing to obtain the solution of 30wt%, this solution has heat-induced gel characteristic, when temperature is higher than sol-gel phase transition temperature
Time, it is possible to spontaneously forming gel, measure its sol-gel phase transition temperature by test tube anastrophe, result shows that it coagulates
Glue transition temperature is 36 DEG C.
Embodiment 14
Weigh block copolymer C opolymer-16 and Copolymer-19 that mass ratio is 3/1, add a certain amount of cell
Culture fluid, prepares to obtain the solution of 10wt%, and this solution has heat-induced gel characteristic, when temperature is higher than sol-gel phase
During transition temperature, it is possible to spontaneously form gel, measure its sol-gel phase transition temperature, result by test tube anastrophe
Show that its gel transition temperature is 25 DEG C.
Embodiment 15
Weigh block copolymer C opolymer-4 and Copolymer-23 that mass ratio is 2/1, add a certain amount of phosphoric acid
Salt buffer, prepares to obtain the solution of 20wt%, and this solution has heat-induced gel characteristic, when temperature is higher than sol-gel phase
During transition temperature, it is possible to spontaneously form gel, measure its sol-gel phase transition temperature, result by test tube anastrophe
Show that its gel transition temperature is 28 DEG C.
Embodiment 16
Weigh appropriate block copolymer C opolymer-19 and Copolymer-23 in mass ratio 1/1 mixing, add certain
The normal saline of amount, prepares to obtain the solution of 25wt%, and this solution has heat-induced gel characteristic, when temperature higher than colloidal sol-
During gel phase transition temperature, it is possible to spontaneously form gel, measure its sol-gel phase transition temperature by test tube anastrophe,
Result shows that its gel transition temperature is 30 DEG C.
Embodiment 17
Weigh appropriate block copolymer C opolymer-4, Copolymer-19 and Copolymer-23 in mass ratio 1/1/1
Mixing, adds a certain amount of deionized water, prepares to obtain the solution of 25wt%, and this solution has heat-induced gel characteristic,
When temperature higher than sol-gel phase transition temperature time, it is possible to spontaneously form gel, by test tube anastrophe measure its colloidal sol-
Gel phase transition temperature, result shows that its gel transition temperature is 33 DEG C.
Embodiment 18
Take appropriate polymer Coplymer-1 to be dissolved in phosphate buffered solution, be configured to the aqueous solution of 25wt%, mistake
0.22 μm filter membrane sterilizing is standby, and precision weighs Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] dry powder 10mg, is dissolved in the above-mentioned polymer solution of 5mL,
Stirring makes medicine dissolution, prepares thermic pharmaceutical hydrogel preparation.
Embodiment 19
Take appropriate polymer Coplymer-4 to be dissolved in phosphate buffered solution, be configured to the aqueous solution of 25wt%, mistake
0.22 μm filter membrane sterilizing is standby, and precision weighs Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] dry powder 15mg, is dissolved in the above-mentioned polymer solution of 7.5mL
In, stirring makes medicine dissolution, is prepared into thermic pharmaceutical hydrogel preparation.
Embodiment 20
Take appropriate polymer Coplymer-5 to be dissolved in phosphate buffered solution, be configured to the aqueous solution of 25wt%, go out
After bacterium standby, precision weighs Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] dry powder 20mg, is dissolved in the above-mentioned polymer solution of 10mL, stirring make medicine
Thing dissolves, and is prepared into thermic pharmaceutical hydrogel preparation.
Embodiment 21
Take appropriate polymer Coplymer-1 to be dissolved in phosphate buffered solution, be configured to the aqueous solution of 23wt%, mistake
0.22 μm filter membrane sterilizing is standby, and precision weighs Du Lalu peptide dry powder 15mg, is dissolved in the above-mentioned polymer solution of 5mL,
Stirring makes medicine dissolution, is prepared into thermic pharmaceutical hydrogel preparation.
Embodiment 22
Take block copolymer C opolymer-4 that mass ratio is 2/1 and Copolymer-23 is dissolved in phosphate buffered solution
In, it being configured to the aqueous solution of 20wt%, 0.22 μm filter membrane sterilizing excessively is standby, and precision weighs albiglutide dry powder 100mg,
Being dissolved in the above-mentioned polymer solution of 5mL, stirring makes medicine dissolution, is prepared into thermic pharmaceutical hydrogel preparation.
Embodiment 23
Take block copolymer C opolymer-16 that mass ratio is 3/1 and Copolymer-19 be dissolved in cell culture fluid,
Being configured to the aqueous solution of 10wt%, 0.22 μm filter membrane sterilizing excessively is standby, and precision weighs Li Sina peptide dry powder 2.5mg, molten
Solution is in the above-mentioned polymer solution of 5mL, and stirring makes medicine dissolution, is prepared into thermic pharmaceutical hydrogel preparation.
Embodiment 24
Block copolymer C opolymer-19 and the Copolymer-23 that take mass ratio 1/1 are dissolved in phosphate buffered solution
In, it is configured to the aqueous solution of 25wt%, crosses 0.22 μm filter membrane sterilizing standby.Precision weighs Suo Malu peptide dry powder 50mg,
Being dissolved in the above-mentioned polymer solution of 5mL, stirring makes medicine dissolution, is prepared into thermic pharmaceutical hydrogel preparation.
Embodiment 25
Weigh 10mg Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] dry powder, be dissolved in phosphate buffered solution, be configured to 500 μ g/mL solution, adopt
Measure the hydrodynamic size of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] with dynamic light scattering, weigh appropriate block copolymer C opolymer-1 and profit
Draw Shandong peptide dry powder, add a certain amount of phosphate buffered solution, be configured to a series of polymer concentration and be fixed as 0.5wt%,
Drug level is respectively the solution of 0,40,500,1000 μ g/mL, uses dynamic light scattering to measure above-mentioned system
Hydrodynamic size, result shows (as shown in Figure 1), and Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] presents two hydrodynamic size in aqueous
Distribution, the size of respectively about 7nm and 200nm, correspond respectively to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] strand and Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] self assembly
The micelle formed, and Copolymer-1 can be self-assembly of micelle in aqueous, micelle size is about 40nm,
After Copolymer-1 mixes with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], both there occurs common assembling, shows as under the addition of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37],
The intensity peak of Copolymer-1 there occurs that change, division are about two peaks of 25nm and 150nm size, and along with profit
Drawing Shandong peptide concentration to increase to 1000 μ g/mL from 40 μ g/mL, there is not notable change in hydrodynamic size distribution；Dynamic
State light scattering result shows that Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and Copolymer-1 hydrogel can occur assembly behavior altogether under aqueous solution state.
Embodiment 26
Weigh 10mg Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] dry powder, be dissolved in phosphate buffered solution, be configured to 500 μ g/mL solution, adopt
The hydrodynamic size of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is measured with dynamic light scattering；Weigh appropriate block copolymer C opolymer-4 and profit
Draw Shandong peptide dry powder, add a certain amount of phosphate buffered solution, be configured to a series of polymer concentration and be fixed as 0.5wt%,
Drug level is respectively the solution of 0,40,500,1000 μ g/mL, uses dynamic light scattering to measure above-mentioned system
Hydrodynamic size, result shows (as shown in Figure 2), and Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] presents two hydrodynamic size in aqueous
Distribution, the size of respectively about 7nm and 200nm, correspond respectively to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] strand and Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] self assembly
The micelle formed, and Copolymer-4 can be self-assembly of micelle in aqueous, micelle size is about 50nm,
After Copolymer-4 mixes with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], both there occurs common assembling, show as Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] add after with
Copolymer-4 merge become one unimodal, along with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] concentration increases to 1000 μ g/mL from 40 μ g/mL, stream
There is not notable change in hydrodynamic size distribution；Dynamic light scattering result shows Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and Copolymer-4 water-setting
Glue can occur assembly behavior altogether under aqueous solution state.
Embodiment 27
Take the block copolymer solution of appropriate embodiment 10 preparation, use rotational rheometer to measure the mould of polymer water system
The rheological property variation with temperature such as amount, viscosity, under fixed shear frequency (w=10rad/s), with 0.5 DEG C/min
Heating rate carry out temperature scanning, take appropriate embodiment 18 preparation thermic pharmaceutical hydrogel preparation, use rotating flow
Become instrument and measure the rheological property variation with temperature such as the modulus of polymer water system, viscosity, at fixed shear frequency (w=
Under 10rad/s), carry out temperature scanning with the heating rate of 0.5 DEG C/min；Result shows (as shown in Figure 3), wherein,
Fig. 3 (a) shows, the Copolymer-1 hydrogel solution at room temperature storage modulus of 25wt% is less, and system has very
Good mobility, and storage modulus steeply rises near 30 DEG C, the phase transition point of solution-gel is 32 DEG C, works as addition
After Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the maximum storage modulus after system phase in version does not occurs significantly to change, and phase transition temperature also becomes
Change；Fig. 3 (b) shows, the Copolymer-1 hydrogel solution of 25wt% at room temperature has less viscosity, in phase
Near transition point, system viscosity steeply rises, and after adding Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the viscosity after system phase in version occurs significantly
Increase；Rheological property result of study shows, the addition of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] causes Copolymer-1 heat-induced gel broad perspectives
Change in matter, also demonstrates on molecular level, and Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and polymer there occurs common assembly behavior.
Embodiment 28
Take the block copolymer solution of appropriate embodiment 11 preparation, use rotational rheometer to measure the mould of polymer water system
The rheological property variation with temperature such as amount, viscosity, under fixed shear frequency (w=10rad/s), with 0.5 DEG C/min
Heating rate carry out temperature scanning, take appropriate embodiment 19 preparation thermic pharmaceutical hydrogel preparation, use rotating flow
Become instrument and measure the rheological property variation with temperature such as the modulus of polymer water system, viscosity, at fixed shear frequency (w=
Under 10rad/s), carry out temperature scanning with the heating rate of 0.5 DEG C/min.Result shows (as shown in Figure 4), wherein,
Fig. 4 (a) shows, the Copolymer-4 hydrogel solution at room temperature storage modulus of 25wt% is less, and system has very
Good mobility, and storage modulus steeply rises near 30 DEG C, the phase transition point of solution-gel is 34 DEG C, works as addition
After Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the maximum storage modulus after system phase in version does not occurs significantly to change, and phase transition temperature also becomes
Change；Fig. 4 (b) shows, the Copolymer-4 hydrogel solution of 25wt% at room temperature has less viscosity, in phase
Near transition point, system viscosity steeply rises, and after adding Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the viscosity after system phase in version there occurs significantly
Increase；Rheological property result of study shows, the addition of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] causes Copolymer-4 heat-induced gel broad perspectives
Change in matter, also demonstrates on molecular level, and Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and polymer there occurs common assembly behavior.
Embodiment 29
Take the thermic hydrogel aqueous solution of preparation in 0.5mL embodiment 18 and be placed in 10mL test tube (internal diameter 14mm) end
Portion, after in 37 DEG C of waters bath with thermostatic control, placement 15min forms it into gel, top adds the PBS buffering of 5mL equality of temperature
Solution (pH=7.4) is 1.5cm as release medium, release area2, put into water bath with thermostatic control shaking table, shaking bath turns
Speed is 50rpm, and every day, PBS above gel was all replaced by timing, and uses HPLC to detect release medium
The burst size of middle Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], operation repetitive 3 parts, finally it is plotted against time with accumulative release amount of medicine, prepares release song
Line (as shown in Figure 5)；
Following method is used to measure HPLC: the high performance liquid chromatograph of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]: Waters (e2695-2489) includes four
Unit's pump, UV-detector, column oven, automatic sampler and online degasser；Chromatographic column:C18(5
μm 100A 4.6*150mm) mobile phase A: 0.1% trifluoroacetic acid aqueous solution, Mobile phase B: acetonitrile；Use gradient
It is eluted in 0～20min: mobile phase A is dropped to 48% by 52%；20min～30min: mobile phase A rises to from 48%
52%；Detection wavelength: 220nm, column temperature: 30 DEG C, flow velocity: 1.0mL/min, sample size: 50 μ L；
Measurement result shows that ((as shown in Figure 5), medicine is had well by the thermic pharmaceutical hydrogel preparation of embodiment 18
Slow release effect, hypoglycemic medicine Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is in heat-induced gel pharmaceutical preparation, and the first day, the prominent amount of releasing was only 22.13%, medicine
The most steadily release was up to 9 days, and accumulative effectively release in the 9th day reaches 87.64%.
Embodiment 30
Take the thermic hydrogel aqueous solution of preparation in 0.5mL embodiment 19 and be placed in 10mL test tube (internal diameter 14mm) end
Portion, after in 37 DEG C of waters bath with thermostatic control, placement 15min forms it into gel, top adds the PBS buffering of 5mL equality of temperature
Solution (pH=7.4) is 1.5cm as release medium, release area2, put into water bath with thermostatic control shaking table, shaking bath turns
Speed is 50rpm, and every day, PBS above gel was all replaced by timing, and uses above-mentioned HPLC detection release
The burst size of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] in medium, operation repetitive 3 parts, finally it is plotted against time with accumulative release amount of medicine, is released
Putting curve (as shown in Figure 6), result shows, medicine is had by the thermic pharmaceutical hydrogel preparation of embodiments of the invention 19
Having good slow release effect, hypoglycemic medicine Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is in heat-induced gel pharmaceutical preparation, and the first day, the prominent amount of releasing was only
16.46%, medicine the most steadily discharges up to 9 days, and accumulative effectively release in the 9th day reaches 55.97%.
Embodiment 31
Take the thermic hydrogel aqueous solution of preparation in 0.5mL embodiment 20 and be placed in 10mL test tube (internal diameter 14mm) end
Portion, after in 37 DEG C of waters bath with thermostatic control, placement 15min forms it into gel, top adds the PBS buffering of 5mL equality of temperature
Solution (pH=7.4) is 1.5cm as release medium, release area2, put into water bath with thermostatic control shaking table, shaking bath turns
Speed is 50rpm, and every day, PBS above gel was all replaced by timing, and uses above-mentioned HPLC detection release
The burst size of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] in medium, operation repetitive 3 parts, finally it is plotted against time with accumulative release amount of medicine, is released
Put curve (as shown in Figure 7),
Result shows, the thermic pharmaceutical hydrogel preparation of embodiments of the invention 20 has good slow release effect to medicine,
Hypoglycemic medicine Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is in heat-induced gel pharmaceutical preparation, and the first day, the prominent amount of releasing was only 19.02%, and medicine is the most steadily released
Lengthening and reach 9 days, accumulative effectively release in the 9th day reaches 69.66%.
Embodiment 32
There is the feature of significant concentration of glucose dependency blood sugar lowering based on GLP-1 analog Polypeptide-k, in the present embodiment
Using ICR mice to carry out carbohydrate tolerance test as animal model, give glucose by gavage after being administered, dynamically detection is dynamic
Thing change of blood sugar, investigates heat-induced gel pharmaceutical preparation and drops hypoglycemic effect and persistent period；
Using normal male ICR mice 24, body weight 30 ± 2g, laboratory animal is randomly divided into 4 groups (n=6): point
It is not: Blank group (Normal group does not inject any solution)；NaCl group (blank normal saline group)；Solution
Group (positive control Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] solution group)；Gel group (Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] gel group)；
Solution group: be dissolved in by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] in PBS buffer solution, is configured to 1mg/mL mother solution, uses front dilution
10 times；
Gel group: the product that embodiment 18 prepares；
By each treated animal fasting 4h (freely drinking water), tail point blood sampling next day (20 μ L) measures blood glucose, afterwards subcutaneous injection
Being administered (7.5mL/kg), after 15min, gavage gives glucose (3g/10mL/kg), and in glucose load 30min and 60
After min, tail point blood sampling (20 μ L) measures blood glucose (blood sugar detection uses glucose oxidase method, lower same), experimental result
As shown in Figure 8；
Afterwards, each treated animal the most upon administration the 1st, 2,3,4,5,6,7,9:00 in 8 and 9 day morning starts to prohibit
Food (freely drinking water) 4h, then tail point is taken a blood sample, and gavage gives glucose (ibid), respectively after glucose load 30min,
60min tail point is taken a blood sample, and measures blood glucose, and experimental result is as shown in Fig. 9 to Figure 18；
Measuring Mouse Weight record the 0th～9 day morning, result is as shown in figure 19；
The oral glucose tolerance test result of experiment mice shows, each group mouse subcutaneous injection is administered gavage glucose after 15min
Blood glucose over time, compared with NaCl group, Solution group and Gel group after oral glucose 30min and
Each time point of 60min all can significantly reduce mouse blood sugar, owing to the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] half-life in animal body is about 12h,
Quickly it is degraded in Mice Body, therefore substantially rises, with NaCl at drug administration by injection Solution next day group mouse blood sugar
The blood glucose unknown significance difference of group mice, and Gel group mouse blood sugar is can be effectively reduced after drug administration by injection, with NaCl
Group mouse blood sugar is significant difference (p ＜ 0.05)；Within continuous 9 days, observe change of blood sugar knot after each group of mice glucose load every day
Fruit display, compared to NaCl group, the ICR mouse blood sugar after Gel group single-dose is effectively controlled after glucose load,
Blood sugar content is less than NaCl group (p ＜ 0.05, have significant difference), sustainable one week of internal blood sugar reducing function, it was demonstrated that real
The product executing example 18 has good slow release effect in vivo, has reached the slow release blood sugar lowering target of a week, and body
Interior pharmacodynamic results is consistent with external release kinetics results；Each group ICR mice body weight change during testing such as figure
Shown in 18, compared with Blank group and NaCl group, in 9 days, the ICR Mouse Weight of Gel group is decreased obviously, it was demonstrated that real
The product executing example 18 has slimming effect, and during zoopery, mice, without any untoward reaction, thus proves
The safety of the heat-induced gel pharmaceutical preparation of the present invention is good.
Claims (10)
1. a thermic hydrogel hypoglycemic medicine preparation, it is characterised in that containing medicine and injectable polymer thermic
Hydrogel；
Described medicine is Polypeptide-k；
Described thermic hydrogel is made up of Amphipathilic block polymer and solvent, and wherein amphipathic nature block polymer is hydrophilic
Block is Polyethylene Glycol (PEG), and hydrophobic block is degradable polyester or polyamino acid；Described thermic hydrogel its coagulate
Colloid ties up to be in solution state under room temperature or low temperature, and under human body temperature, Spontaneous conversion is gel.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that described polymer heat
Cause the sol-gel transition temperature of its polymer water system of hydrogel between 4-40 DEG C.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that described is amphipathic
In block copolymer:
1) mean molecule quantity of hydrophilic Polyethylene Glycol block A is 400-8000, and content is 10-90wt%；
2) mean molecule quantity of hydrophobic polyester block or polyamino acid B block is 500-40000, and content is 90-10
Wt%；
Wherein, hydrophobic polyester block selected from poly DL-lactide, poly-D-lactide, PLLA, PGA,
Poe, poly-epsilon-caprolactone, poly-ε-alkyl replaces caprolactone, poly-δ-valerolactone, poly-Isosorbide-5-Nitrae, 8-trioxa spiral shell [4.6]-9-
Hendecanone, poly-para-dioxanone, polyesteramide, Merlon, polyacrylate, polyether ester or above-mentioned each polyester
In the copolymer that formed of two or more polyester；
Wherein, hydrophobic polyamino acid block selected from polyalanine, polyphenylalanine, poly-leucine, polylysine,
The copolymer that two or more polyamino acid in polyglutamic acid, poly-aspartate or above-mentioned each polyamino acid is formed；
3) described block copolymer be the triblock copolymer of ABA or BAB type, the di-block copolymer of AB type,
The graft copolymer of A-g-B or B-g-A type, (A-B)nOr (B-A)nStar block copolymer and A (BA)nOr
B(AB)nThe segmented copolymer of configuration, wherein n is the integer of 2 to 10；
4) the part or all of end of described block polymer is connected to function end group, and end group is hydrophilic hydroxyl, amino, carboxylic
Any one of base, imidazole radicals, aldehyde radical, cyano group, nitro；Or hydrophobic alkyl, sterin, alkoxyl, virtue
Any one of perfume base, aromatic heterocyclic, amide ester group, halogen atom, trichloromethyl, ester group, sulfydryl.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that described polymer is single
The amphipathic nature block polymer of one, or be the combination of the amphipathic nature block polymer of two or more different molecular structures
Thing.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that described blood sugar lowering is many
Peptide is selected from Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], albiglutide, Li Sina peptide, Du Lalu peptide, one or more in Suo Malu peptide.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that described medicine
Content accounts for the 0.01～5% of thermic pharmaceutical hydrogel preparation.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that described polymer exists
Content in preparation is 3-50wt%.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that described polymer is molten
Solvent in liquid is pure water, normal saline, buffer solution, tissue culture medium, cell culture fluid, animals and plants or human body
Body fluid or be other aqueous solution and not medium based on organic solvent.
9., according to a preparation method for the thermic hydrogel hypoglycemic medicine preparation described in claim 1-8, its feature exists
In, comprising: first at dissolution in low temperature polymer in aqueous solution, then at-20 DEG C or following store for future use, use
Front redissolution is also directly added into described Polypeptide-k, is mixed evenly to prepare as solution injection；
Prepared pharmaceutical preparation forms thermal reversion hydrogel when temperature is higher than sol-gel transition temperature；
Described low temperature refers to the sol-gel transition temperature less than polymer.
Thermic hydrogel hypoglycemic medicine preparation the most according to claim 1, it is characterised in that use subcutaneous or flesh
Meat tissue injection system uses.
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