CN107468638A - A kind of huperzine PLGA PEG PLFGA copolymer gels and preparation method thereof - Google Patents
A kind of huperzine PLGA PEG PLFGA copolymer gels and preparation method thereof Download PDFInfo
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- CN107468638A CN107468638A CN201710542259.2A CN201710542259A CN107468638A CN 107468638 A CN107468638 A CN 107468638A CN 201710542259 A CN201710542259 A CN 201710542259A CN 107468638 A CN107468638 A CN 107468638A
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- huperzine
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- 239000000499 gel Substances 0.000 title claims abstract description 75
- ZQPQGKQTIZYFEF-WCVJEAGWSA-N Huperzine Natural products C1([C@H]2[C@H](O)C(=O)N[C@H]2[C@@H](O)C=2C=CC=CC=2)=CC=CC=C1 ZQPQGKQTIZYFEF-WCVJEAGWSA-N 0.000 title claims abstract description 68
- 229920001577 copolymer Polymers 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 229920006022 Poly(L-lactide-co-glycolide)-b-poly(ethylene glycol) Polymers 0.000 title abstract 3
- 239000003814 drug Substances 0.000 claims abstract description 65
- 229940079593 drug Drugs 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims abstract description 5
- 230000006837 decompression Effects 0.000 claims abstract description 4
- 239000012153 distilled water Substances 0.000 claims abstract description 4
- 238000005374 membrane filtration Methods 0.000 claims abstract description 4
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 4
- 230000000694 effects Effects 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 5
- 239000000825 pharmaceutical preparation Substances 0.000 abstract description 4
- 238000013270 controlled release Methods 0.000 abstract description 3
- 238000002513 implantation Methods 0.000 abstract description 3
- 238000001647 drug administration Methods 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 239000000243 solution Substances 0.000 description 18
- 238000001727 in vivo Methods 0.000 description 13
- 210000004369 blood Anatomy 0.000 description 10
- 239000008280 blood Substances 0.000 description 10
- 238000010521 absorption reaction Methods 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- UBAPRWGDQPSCEH-UHFFFAOYSA-N Des-N-methyl-beta-obscurine Natural products N1CCCC2C3CC(C)CC21C(C=CC(=O)N1)=C1C3 UBAPRWGDQPSCEH-UHFFFAOYSA-N 0.000 description 8
- YYWGABLTRMRUIT-XHBSWPGZSA-N Huperzine B Natural products N1CCC[C@@H]2[C@@H]3C=C(C)C[C@]21C(C=CC(=O)N1)=C1C3 YYWGABLTRMRUIT-XHBSWPGZSA-N 0.000 description 8
- 238000013268 sustained release Methods 0.000 description 8
- 239000012730 sustained-release form Substances 0.000 description 8
- 241000700159 Rattus Species 0.000 description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 239000012738 dissolution medium Substances 0.000 description 5
- 230000008030 elimination Effects 0.000 description 5
- 238000003379 elimination reaction Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 5
- 229920000436 Poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) Polymers 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000003405 delayed action preparation Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 102100033639 Acetylcholinesterase Human genes 0.000 description 2
- 108010022752 Acetylcholinesterase Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 241000700157 Rattus norvegicus Species 0.000 description 2
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 2
- 229960004373 acetylcholine Drugs 0.000 description 2
- 229940022698 acetylcholinesterase Drugs 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229920001400 block copolymer Polymers 0.000 description 2
- 230000008499 blood brain barrier function Effects 0.000 description 2
- 210000001218 blood-brain barrier Anatomy 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
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- 238000010790 dilution Methods 0.000 description 2
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- 239000012535 impurity Substances 0.000 description 2
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- 206010002091 Anaesthesia Diseases 0.000 description 1
- 102000003914 Cholinesterases Human genes 0.000 description 1
- 108090000322 Cholinesterases Proteins 0.000 description 1
- 244000050510 Cunninghamia lanceolata Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 208000026139 Memory disease Diseases 0.000 description 1
- 108010067035 Pancrelipase Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 229940048961 cholinesterase Drugs 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
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- 238000009792 diffusion process Methods 0.000 description 1
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- 238000012377 drug delivery Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
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- 230000005284 excitation Effects 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
- 210000001652 frontal lobe Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
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- 229920002521 macromolecule Polymers 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4748—Quinolines; Isoquinolines forming part of bridged ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
Abstract
A kind of huperzine PLGA PEG PLFGA copolymer gels of the present invention and preparation method thereof, belong to pharmaceutical preparations technology field.Methods described is：Huperzine bulk drug 0.2g and PLGA PREG PLGA copolymer 1 2.0g are taken, 50 DEG C of water-bath decompression rotary evaporations in 500ml round-bottomed flasks are placed in after fully being dissolved with acetone 200ml and remove solvent；The copolymer for making to obtain forms film on round-bottomed flask wall；100ml distilled water is added into round-bottomed flask and dissolves copolymer at room temperature, after copolymer is completely dissolved, through 0.45 μm of membrane filtration, is produced.The huperzine PLGA PEG PLFGA copolymer gels are as obtained by prepared by the above method.The present invention has advantages below：Biocompatibility is preferable；Slow controlled-release effect is preferable；Drugloading rate is high, simple to operate；Convenient drug administration, it is not necessary to operation implantation.
Description
Technical field
The invention belongs to pharmaceutical preparations technology field, and in particular to a kind of huperzine PLGA-PEG-PLFGA copolymers coagulate
Glue and preparation method thereof.
Background technology
Huperzine is a kind of invertibity choline zymase inhibitor, has selective inhibitory to true cholinesterase.
Bioactivity is high, has higher fat-soluble, and molecule is small, easily through blood-brain barrier, into maincenter after be more distributed in brain
Frontal lobe, temporal lobe, hippocampus etc. have the brain area of close ties with learning and memory, have under low dosage to acetylcholinesterase (AChE)
Powerful inhibitory action, make acetylcholine (ACh) content in nerve synapse gap in distributed area significantly raised, so as to strengthen nerve
First excitatory transmission, intensified learning and the excitation of memory brain area, play and improve cognitive function, enhancing memory is kept and promotes note
Recall the effect of reproduction.Available in treatment, old benign memory deficits and various dull-witted, Function of memory cognition and emotion-directed behavior barrier
Hinder.
The general preparation of huperzine is tablet or capsule, and oral absorption is rapid and complete, bioavilability 96%, 10-
Up to blood peak concentration of drug, distribution is also fast, easily passes through blood-brain barrier within 30 minutes.It is 4 hours to eliminate half-life period.Though short of soak time
Effect is rapid, but drug treating time is also shorter, it is necessary to be repeated continuously administration.
The content of the invention
Situ-gel (in situ-forming gel) delivery system be one kind can be injected in vivo with liquid condition and to
Medicine portion forms the carrier delivery systems of the gel of semisolid without powerless or chemical change, and the system has good tissue phase
Capacitive, grown in the medicine-feeding part holdup time；Storage medicine can be played simultaneously, prevents medicine is affected by environment from waiting effect, Neng Gouqi
To the effect of long-term sustained release administration.This delivery system has the advantages of liquid preparation and gel preparation concurrently, as preparation technology is simple,
Medicine can be carried in a mixed way；Administration is inconvenient, it is not necessary to operation implantation；Bio-compatible type and slow controlled-release effect are good etc..PLGA-
PEG-PLGA copolymer gels are a kind of temperature-sensitive situ-gels, are a kind of new medicinal macromolecules, are less than phase in temperature
During transition temperature, polymeric material is water-soluble to form the liquid flowed freely, when temperature is higher than phase transition temperature, forms non-chemical friendship
The gel of connection.
The preparation method of PLGA-PEG-PLFGA copolymer gels is a lot, including：Physically trapping method, dialysis, emulsification are molten
Agent volatility process and film dispersion method etc..The drugloading rate of wherein film dispersion method is high, simple to operate.This preparation is disperseed from film
Method, huperzine PLGA-PEG-PLFGA copolymer gels are prepared, investigated each technique, the shadow that formulation factors discharge to gel
Ring, have selected the factor being had a great influence to gel release, and investigated by a series of prescription, technological factor, finally given most
Good prescription and preparation technology.
It is an object of the invention to disclose a kind of preparation method of huperzine PLGA-PEG-PLFGA copolymer gels.
Second object of the present invention is to disclose a kind of huperzine PLGA- for preparing gained by above-mentioned preparation method
PEG-PLFGA copolymer gels.
The purpose of the present invention is achieved through the following technical solutions：
A kind of preparation method of huperzine PLGA-PEG-PLFGA copolymer gels, wherein, methods described includes following
Step：
(1) huperzine bulk drug 0.2g and PLGA-PREG-PLGA copolymer 1 2.0g, is taken, it is fully molten with acetone 200ml
50 DEG C of water-bath decompression rotary evaporations in 500ml round-bottomed flasks are placed in after solution and remove solvent；
(2) copolymer for, obtaining step (1) forms film on round-bottomed flask wall；
(3), copolymer is dissolved at room temperature toward addition 100ml distilled water in round-bottomed flask, after copolymer is completely dissolved,
Through 0.45 μm of membrane filtration, huperzine PLGA-PEG-PLFGA copolymer gels are produced.
The huperzine PLGA-PEG-PLFGA copolymers that gained is prepared as the preparation method described in above-mentioned technical proposal coagulate
Glue.
The invention has the advantages that：
1st, copolymer gel of the invention is compared with the existing temperature sensitive type block gel having been commercialized, biocompatibility compared with
It is good.
2nd, copolymer gel of the invention is compared with the existing temperature sensitive type block gel having been commercialized, slow controlled-release effect compared with
It is good.
3rd, copolymer gel preparation method film dispersion method of the present invention, with other preparation method (such as physics bags
Bury method, dialysis, emulsion-solvent evaporation method etc.) to compare, drugloading rate is high, simple to operate.
4th, situ-gel drug delivery sys tems of the present invention, compared with other vivo medicine-feeding systems, convenient drug administration,
Need not be performed the operation implantation.
Brief description of the drawings：
1st, Fig. 1 is influence of the different dissolution mediums to huperzine copolymer gel insoluble drug release.
2nd, Fig. 2 is influence (n=3) of the different rotating speeds to huperzine copolymer gel insoluble drug release.
3rd, Fig. 3 is the influence of the concentration against drug of PLGA-PEG-PLGA copolymers.
4th, Fig. 4 is influence of the drug concentration to release.
5th, Fig. 5 is the release profiles of huperzine copolymer gel.
6th, Fig. 6 is the typical HPLC spectrograms that blank plasma (a), plasma sample (b) and plasma sample add internal standard (c).
7th, Fig. 7 be five mouse after hypodermic injection, mean blood plasma concentration-time graph (n=5).
8th, Fig. 8 is the linear relationship chart of body absorption percentage and the external cumulative release percentage that directly releases the drug.
Embodiment：
To readily appreciate technical scheme, below in conjunction with specific embodiment to a kind of huperzine of the present invention
PLGA-PEG-PLFGA copolymer gels and preparation method thereof are further described.
Embodiment 1：A kind of preparation method of huperzine PLGA-PEG-PLFGA copolymer gels：
(1) huperzine bulk drug 0.2g and PLGA-PREG-PLGA copolymer 1 2.0g, is taken, it is fully molten with acetone 200ml
50 DEG C of water-bath decompression rotary evaporations in 500ml round-bottomed flasks are placed in after solution and remove solvent；
(2) copolymer for, obtaining step (1) forms film on round-bottomed flask wall；
(3), copolymer is dissolved at room temperature toward addition 100ml distilled water in round-bottomed flask, after copolymer is completely dissolved,
Through 0.45 μm of membrane filtration, huperzine PLGA-PEG-PLFGA copolymer gels are produced.
Illustrate the determination of preparation process of the present invention and possessed beneficial effect below by way of specific experiment example.
Experimental example 1：The determination of huperzine PLGA-PEG-PLFGA copolymer gels technique and prescription：
First, instrument and reagent：
(1), instrument：
UV-1700 type ultraviolet specrophotometers；85-2A type constant temperature magnetic force heating stirrers；HS3210D supersonic cleaning machines；
80-2 centrifuges；PHS-3B type precision pH meters；FA1004N electronic balances；RCD-3 medicament dissolution instruments.
(2), reagent：
Huperzine bulk drug；
Huperzine reference substance；
Polylactide-co-glycolide-polypropylene glycol-polylactide-co-glycolide block copolymer (PLGA-PEG-PLGA)；
Pure water；
Other reagents are all to analyze pure (A.R.).
2nd, the quality evaluating method of huperzine PLGA-PEG-PLFGA copolymer gels：
(1), release：
The release of medicine is a key index of huperzine PLGA-PEG-PLFGA copolymer gel quality controls.
(1), the assay method of release：
The huperzine PLGA-PEG-PLFGA copolymer gels 1ml that Example 1 prepares gained is placed in tool plug glass examination
Guan Zhong, then test tube is put into digestion instrument, bath temperature is 37 DEG C.After solution forms gel completely, add 4ml releases and be situated between
Matter (i.e. the PBS solution of water and pH7.4), rotating speed 4ml, release area are 0.40cm2.Whole dissolutions are taken out in the predetermined time
Medium, while the fresh dissolution medium of same volume is added to ensure sink conditions.Release amount of medicine is accumulated to be plotted against time, has obtained
To release profiles, Fig. 1 is as a result seen.
As seen from Figure 1, dissolution medium difference has a significant impact to insoluble drug release.In the dissolution medium of PBS solution, medicine
Release is more slowly lasting.
(2), influence of the digestion instrument rotating speed to insoluble drug release：
Using pH7.4 PBS solution as dissolution medium, rotating speed is controlled to investigate rotating speed to insoluble drug release row for 55,75r/min
For influence, as a result see Fig. 2.
From Figure 2 it can be seen that rotating speed has a significant impact to insoluble drug release.With the increase of rotating speed, faster drug release.Copolymer
In vivo without agitation after gel injection, it is contemplated that the sink conditions of In vitro-in vivo correlation and release in vitro, select less
Rotating speed, as 55r/min.
3rd, huperzine PLGA-PEG-PLFGA copolymer gels prescription single factor exploration：
(1), influence of the copolymer concentration to insoluble drug release：
Huperzine is shown in Fig. 3 from the release profiles in the copolymer gel that concentration is 8%, 10% and 12% (W/W).
As a result show, the rate of release of medicine reduces with the increase of the concentration of copolymer.Increase the concentration of polymer, it is molten
The quantity of micella increases therewith in liquid.Correspondingly, the quantity increase of micella in gel structure is formed, glue distance between clusters shortens, medicine
Thing molecule is decreased by intermicellar diffusion rate, shows as the reduction of rate of releasing drug.
(2), influence of the drug concentration to release：
The release profiles that drugloading rate is 0.14%, 0.20% and 0.30% in same copolymer gel are shown in Fig. 4.
As a result show, the rate of release of medicine increases with the increase of drug concentration.Increase the concentration of medicine, solution micella
The increase therewith of middle medicine.Correspondingly, the drug concentration increase in gel structure in micella is formed, shows as the increasing of rate of releasing drug
Add.
4th, the determination of copolymer gel prescription：
(1), the determination of more excellent prescription：
While the volume of copolymer gel medication is reduced as far as possible, slow down the release of medicine as far as possible, so the increasing that should try one's best
The concentration of big copolymer gel, the optimal prescription for determining copolymer gel are：The copolymer gel that concentration is 12% contains 0.20%
Medicine.
(2), copolymer gel prescription and technique reappearance are investigated：
Three batches of copolymer gels are prepared by more excellent prescription, and quality evaluation is carried out to it, the results are shown in Table 1 and Fig. 5.
From table 1 and Fig. 5, there was no significant difference for the medicament contg and release of the three batches of copolymer gels, shows prescription
It is good with technique reappearance.
The quality evaluation (n=3) of huperzine copolymer gel prepared by 1 more excellent prescription of table and technique
5th, conclusion：
(1), release in vitro condition is selected：
It is molten that PBS of the huperzine PLGA-PEG-PLFGA copolymer gels in water, pH7.4 has been investigated according to preformulation study
Solubility in liquid, show that pH7.4 PBS solution dissolution medium meets sink conditions, available for the external of huperzine preparation
Release research.Rotating speed also has a significant impact to insoluble drug release.With the increase of rotating speed, faster drug release.Copolymer gel
Less rotating speed, as 55r/min are selected after injection without agitation in vivo.
(2), the single factor exploration of copolymer gel prescription：
Investigate the influence of copolymer concentration, drug concentration to release, it is contemplated that the factor such as dosage, slow release effect,
More excellent prescription is determined.
By copolymer gel single factor exploration, the more excellent prescription for determining copolymer gel is：Concentration is 12% copolymerization
Thing gel contains 0.20% medicine.
Experimental example 2：Huperzine copolymer gel rat Internal pharmacokinetics are studied：
Interior medicine dynamics research is to evaluate the important indicator of sustained-release preparation, and medicine is needed by acting in vivo after all
To play curative effect.Although difference in genera be present, animal experiment can conduct as the trial test before human trial, its result
Human trial provides certain reference.Applicant is using rat as animal pattern, using high effective liquid chromatography for measuring blood concentration,
Preliminary pharmacokinetic studies have been carried out to homemade huperzine copolymer gel.
First, laboratory apparatus and material：
(1), instrument：
Supersonic cleaning machine；
Centrifuge；
Automatic vortex mixer；
High performance liquid chromatograph；
(2), reagent：
Huperzine bulk drug；
Huperzine reference substance；
Huperzine B reference substance；
Polylactide-co-glycolide-polypropylene glycol-polylactide-co-glycolide block copolymer (PLGA-PEG-PLGA)；
Triethylamine；
Methanol；
Pure water.
Huperzine PLGA-PEG-PLFGA copolymer gels (prepared by embodiment 1)
(3), animal：
Wistar rats, body weight are 190~200g.
2nd, experimental method and result：
(1), the foundation of huperzine analysis in vivo：
(1), chromatographic condition：
The content of huperzine in plasma sample is detected using HPLC UV, condition is as follows：
Chromatographic column：ODSC18Chromatographic column (TEDAChorm, 250 × 4.6mm)；
Mobile phase：Methanol：Water：Triethylamine (50:50:0.02)；
Internal standard：Huperzine B；
Detection wavelength：311nm；
Column temperature：25℃；
Flow velocity：0.8ml/min；
Sample size：20μl.
(2), the processing of plasma sample：
The configuration of inner mark solution：Precision weighs Huperzine B 25mg into 50ml capacity product, with methanol dilution and is settled to
Scale, it is 500 μ g/ml solution 1 to obtain concentration, and precision pipettes solution 6.4ml and is settled to quarter with methanol dilution into 50ml volumetric flasks
Spend and produce internal standard contrast solution.
Plasma treatment process：The μ l of rat plasma 200 are taken to be separately added into 0.1mol/L NaOH solutions in 2ml centrifuge tubes
After the μ l of internal standard reference substance solution 10, vortex 3min, dichloromethane 1ml is added, centrifuged after vortex 1min (3000rpm ×
5min), the μ l of lower floor's organic phase 600 are drawn, in N2Lower drying, residue are dissolved with 50 μ l methanol, vortex mixed 3min, centrifugation,
The μ l sample introductions of supernatant 20 are taken to determine.Record chromatogram and peak area.
(3), method specificity is investigated：
The μ l of rat blank plasma 100 are taken, by " the processing of (2), plasma sample：" operation is handled under item, but is not added with internal standard；Separately
Two parts of administration plasma samples are taken, by " the processing of (2), plasma sample：" under item operation handle, a copy of it is not added with internal standard, gained
Typical spectrum is shown in Fig. 6.As can be seen here, the impurity peaks separating degree in huperzine, Huperzine B and blood plasma is good, eliminates impurity pair
The interference of measure.Illustrate using Huperzine B as internal standard, be feasible using the huperzine in Plasma By Hplc
's.The appearance time of huperzine and Huperzine B is respectively 10.7min and 12.9min or so, and endogenous substance in plasma
The measure of huperzine is not disturbed.
(4), the drafting of standard curve：
Precision measures the μ l of blank plasma 100, sequentially adds the system that concentration is 0.2,0.5,1,2,4,8,10,20 μ g/ml
The μ l of row huperzine methanol solution 20, then according to being operated under " processing of (2), plasma sample " item.
With the ratio between the peak area of huperzine and internal standard Huperzine B (AHupA/AHupB) corresponding drug concentration is mapped, obtain
Calibration curve equation：AHupA/AHupB=0.0307C+0.0013 (R2=0.9956).As can be seen here, in 0.2~20 μ g/ml concentration
In the range of it is linear good.
(5), precision and the rate of recovery：
(a), precision：
Replication 5 times in the plasma sample one day containing high, medium and low concentration huperzine are taken, calculate in a few days standard deviation
Difference, METHOD FOR CONTINUOUS DETERMINATION 5 days, calculate standard deviation in the daytime.As a result 2 and table 3 are shown in Table respectively.As a result show：In a few days determine basic, normal, high
Three kinds of drug concentration plasma sample RSD are respectively 6.67%, 3.51% and 11.07%, meet methodology withinday precision requirement
(RSD<15%)；In the daytime it is respectively 4.27%, 13.20% and to determine basic, normal, high three kinds of drug concentration plasma samples RSD
8.20%, meet methodology withinday precision requirement (RSD<15%).
The in a few days precision (n=5) of the HPLC measure of huperzine in the plasma sample of table 2
The precision in the daytime (n=5) of the HPLC measure of huperzine in the plasma sample of table 3
(b), extraction recovery：
A certain amount of huperzine reference substance solution is separately added into blank plasma samples, high, medium and low three kinds of medicines are made
The plasma sample of thing concentration, i.e. concentration are respectively 20,4,2 μ g/ml, every kind of concentration mensuration 3 times, calculate medicine under each concentration and reclaim
Rate, it the results are shown in Table 4.As a result show：The rate of recovery of basic, normal, high three kinds of drug concentration plasma samples is all higher than 60%, meets method
Learn and require.
The rate of recovery detection (n=3) of huperzine in the plasma sample of table 4
(6), minimum detection limit and quantitative limit are investigated：
Under above-mentioned chromatographic condition, it is equal to 3 and 10 with signal to noise ratio respectively and calculates, lowest detection is limited to 0.2 μ g/ml；It is minimum
Quantitatively it is limited to 0.6 μ g/ml.
(2), huperzine PLGA-PEG-PLFGA copolymer gels pharmacokinetic study：
(1), administering mode：
Wistar rats 5, raised one week under laboratory condition.Ether light anesthesia is used before administration, is subcutaneously injected respectively
Huperzine sustained-release gel 0.5ml.
(2), plasma sample collection and processing：
In taking blood 1ml or so, anticoagulant heparin with timing eye socket after administration, 6000rpm centrifuges 10min, isolated blood
Slurry.- 20 DEG C of freezen protectives, until measure.
Blood specimen collection point is as follows：Later before injection and 1,3,6,12,24h after injection, blood sampling every 24h once.
(3), determination of plasma concentration result：
By " the foundation of (one), huperzine analysis in vivo：(4), the drafting of standard curve：" standard curve meter under item
Calculate the content of sample huperzine.Plasma concentration v. time experimental data is shown in Table 5, and blood concentration, which is plotted against time, sees Fig. 7.
Plasma drug level-time experimental data (n=5, ng/ of huperzine sustained-release gel formulations is subcutaneously injected in table 5
ml)
(3), the processing and analysis of intra-body data：
(1), DAS methods compartment model is fitted：
According to residual sum of squares (RSS), r2With AIC methods come judge the model of linear pharmacokinetics and to make by oneself injectable sustained-release
The blood concentration-time curve of gel carries out single compartment and double compartment fitting.
Using DAS programs, the model for judging huperzine injectable gel according to degree of fitting and AIC belongs to, the results showed that
Meet single compartment model, weight 1/C.
(2), statistics is away from calculating pharmacokinetic parameters：
Calculation pharmacokinetic parameters are asked away from theory using statistics.If up to Cmax Cmax and peak time Tmax from curve
Observation obtains；Existing property recurrence is carried out to time t by the logarithm value of the plasma drug concentration data of 4 points of Drug-time curve afterbody, by straight line
Slope seeks the elimination rate constant λ z (d-1) for calculating single exponent ring-down equation, and area under the drug-time curve AUC0-t is calculated with trapezoidal method
(ng·h·ml-1), zeroth order origin is away from S0(area under the drug-time curve AUC0- ∞, nghml-1) and single order origin away from S1
(ng·d2·ml-1)；And calculate mean residence time MRT (h), half-life period t1/2And elimination rate constant k (h (h)-1).As a result see
Table 6.
AUC0-∞=AUC0-t+CT/λz
t1/2=0.693MRT
K=1/MRT
Blood concentration when CT is most end sample time T in formula, λ z are that curve end is fitted to single exponent ring-down equation
Elimination rate constant.
Pharmacokinetic parameter after table 6 is subcutaneously injected in blood plasma
(4), In vitro-in vivo correlation research (IVIVC)：
Relationship between in vivo and in vitro test refers between inside and outside drug-eluting, release characteristics and internal drug absorption characteristic
Relation research.If correlation is good, it is possible to says the row of inside and outside dissolution, release test result to prediction medicine in vivo
To be significant, it is possible to tested with vitro test instead of vivo biodistribution availability, the weight as evaluation pharmaceutical preparation quality
Want index.Therefore, outer correlation research is developed to pharmaceutical preparation inside preparation, and the particularly exploitation of sustained-release preparation is extremely heavy
Will.
At present, FDA Guide Books have established three kinds of In vitro-in vivo correlation models, wherein horizontal A models are will be external molten
Go out characteristic inside being calculated with plasma drug concentration data by coil stripping integration method compared with absorption characteristic.Due to which employs
The whole time course data of In Vitro Dissolution and body absorption, thus be considered as that can apply being given birth in the heart wants approval process
Outer correlation models inside thing immunity.In the research of horizontal A models, the research method of body absorption characteristic have model according to
Method and coil stripping integration method, coil stripping integration method is relied to carry out sexual intercourse models fitting, directly mathematically really to test
Data calculate body absorption fraction, and outer correlation is ground inside the chamber that is particularly suitable for use in simulation difficult medicine and sustained-release preparation
Study carefully.
Preparation plasma drug concentration data is fitted with DAS compartment models, huperzine meets one compartment model in rat.Using
Wagner-Nelson methods calculate the absorption fraction of sustained release preparation each time in rat body, and calculation formula is as follows：
In formula：CTFor the blood concentration of T time；KeFor elimination rate constant；WithRespectively 0~T and 0~
Area under the Drug-time curve at ∞ moment.
Absorption percentage inside gained will be calculated and carry out linear fit with the external cumulative release percentage directly to release the drug,
As a result Fig. 8 is seen, correlative relationship equation is Y=1.5463X-23.188, r=0.9965 inside foundation.Free degree df=n-2
=6, critical value r6,0.001=0.823, r>r6,0.001Show that there is the correlation (P of highly significant in preparation body<0.001).
The pharmacokinetic parameter of huperzine injectable sustained-release gel preparation：TmaxFor 3h；CmaxFor (113.06 ±
10.23)ng·ml-1；AUC0→TFor (2330.85 ± 365.02) nghml-1；AUC0→∞For (3015.89 ± 401.06)
ng·h·ml-1；t1/2For (39.435 ± 2.38) h.
3rd, discussion of results：
(1), vivo approaches are established：
The present invention establishes inside huperzine analysis method and using Huperzine B as internal standard, determines stone in rat plasma
Assay method inside China fir alkali.Method high sensitivity, accurately and reliably, respectively in 0.2~20 μ g/ml (HPLC methods, R2=
0.9956) it is linear good in concentration range.When blood plasma is handled, mobile phase is used for the influence at elimination solvent peak before sample introduction
Weight soluble drug residue, but dissolubility is bad, applicant is molten using methanol solution weight, and retention time is suitable under liquid-phase condition,
Reach satisfied separating effect.
(2), Internal pharmacokinetics are studied：
The present invention is studied pharmacokinetics inside huperzine injectable sustained-release gel.As a result medicine is shown
It can be sustained in vivo 3~4 days.The tablets in vitro of injectable gel is slightly longer than internal release time in this research, and inventor recognizes
For due in vivo contain substantial amounts of enzyme may promote gel corrosion but insoluble drug release.
4th, conclusion：
(1) the HPLC analysis methods of huperzine in plasma sample, are established, method specificity is strong, detection sensitivity
Height, accurate, reliable analysis method is provided for interior medicine dynamics.
(2) interior medicine dynamics research, is carried out to self-control huperzine injectable sustained-release gel, the results showed that gel
Blood concentration is more steady, and action time is obviously prolonged, and has reached the purpose of preparation.
(3), calculated using Wagner-Nelson methods, outer correlation shows inside huperzine injectable sustained-release gel
Write.
It is described above, only presently preferred embodiments of the present invention, not the present invention is made it is any in form and substantial limit
System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above skill
Art content, and the equivalent variations for a little variation, modification and evolution made, it is the equivalent embodiment of the present invention；Meanwhile it is all according to
The variation, modification and evolution for any equivalent variations made according to the substantial technological of the present invention to above example, still fall within this
In the range of the technical scheme of invention.
Claims (2)
1. a kind of preparation method of huperzine PLGA-PEG-PLFGA copolymer gels, it is characterised in that methods described includes
Following step：
(1) huperzine bulk drug 0.2g and PLGA-PREG-PLGA copolymer 1 2.0g, is taken, after fully being dissolved with acetone 200ml
It is placed in 50 DEG C of water-bath decompression rotary evaporations in 500ml round-bottomed flasks and removes solvent；
(2) copolymer for, obtaining step (1) forms film on round-bottomed flask wall；
(3), copolymer is dissolved at room temperature toward addition 100ml distilled water in round-bottomed flask, after copolymer is completely dissolved, warp
0.45 μm of membrane filtration, produce huperzine PLGA-PEG-PLFGA copolymer gels.
2. the huperzine PLGA-PEG-PLFGA copolymer gels of gained are prepared as the preparation method described in claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110613687A (en) * | 2019-10-30 | 2019-12-27 | 北京利普松生物科技有限公司 | Compound furacilin nano liposome and preparation method thereof |
| CN114088901A (en) * | 2021-11-19 | 2022-02-25 | 江苏科技大学 | Universal degradable drug-loaded membrane in-vitro release data optimization analysis method |
| US20230287352A1 (en) * | 2022-03-09 | 2023-09-14 | Healthina Stem Cell Industry Platform (Tianjin) Limited | Method for non-enzymatic 3d culture and amplification of mesenchymal stem cells |
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| US6352715B1 (en) * | 1998-02-19 | 2002-03-05 | Sagittarius Life Science Corp | Transdermal rate-controlled delivery of Huperzine A for treatment of alzheimer's disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6352715B1 (en) * | 1998-02-19 | 2002-03-05 | Sagittarius Life Science Corp | Transdermal rate-controlled delivery of Huperzine A for treatment of alzheimer's disease |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110613687A (en) * | 2019-10-30 | 2019-12-27 | 北京利普松生物科技有限公司 | Compound furacilin nano liposome and preparation method thereof |
| CN114088901A (en) * | 2021-11-19 | 2022-02-25 | 江苏科技大学 | Universal degradable drug-loaded membrane in-vitro release data optimization analysis method |
| CN114088901B (en) * | 2021-11-19 | 2023-12-22 | 江苏科技大学 | General degradable drug-carrying film in-vitro release data optimization analysis method |
| US20230287352A1 (en) * | 2022-03-09 | 2023-09-14 | Healthina Stem Cell Industry Platform (Tianjin) Limited | Method for non-enzymatic 3d culture and amplification of mesenchymal stem cells |
| US11946073B2 (en) * | 2022-03-09 | 2024-04-02 | Healthina Stem Cell Industry Platform (Tianjin) Limited | Method for non-enzymatic 3D culture and amplification of mesenchymal stem cells |
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