CN105412015B - Entecavir Liposomal formulation of enoxolone modification and preparation method thereof - Google Patents
Entecavir Liposomal formulation of enoxolone modification and preparation method thereof Download PDFInfo
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- CN105412015B CN105412015B CN201511009226.9A CN201511009226A CN105412015B CN 105412015 B CN105412015 B CN 105412015B CN 201511009226 A CN201511009226 A CN 201511009226A CN 105412015 B CN105412015 B CN 105412015B
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- entecavir
- enoxolone
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- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- 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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
- A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
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- 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
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- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
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Abstract
The invention belongs to field of pharmaceutical preparations, and in particular to a kind of Entecavir Liposomal formulation of enoxolone modification and preparation method thereof.The Entecavir Liposomal formulation of the present invention is modified phosphatide and is made by enoxolone and polyethylene glycol.This Entecavir liposome hepatic targeting is good, small toxicity, and envelop rate is high, and difference is small between uniform particle sizes, and batch.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, it is related to a kind of entecavir medicinal composition, relates in particular to a kind of sweet
Entecavir Liver targeting Liposomal formulation of careless hypo acid modification and preparation method thereof.
Background technology
Entecavir (Entecavir) is a kind of oral antiviral medicament, there is good hepatitis B inhibitory action.Initially
Developed by Bristol-Myers Squibb Co. (Bristol-Myers Squibb), entecavir tablets are in China within 2 months 2006
It is approved listing, [4- hydroxyls -3- (methylol) -2- is sub- by entitled [1S- (1 α, 3 α, 4 β)] -2- amino -1, the 9- dihydros -9- of its chemistry
Methylcyclopentyl] -6H- purine-6-ones, the slightly soluble in water, solubility is 2.4mg/mL, with following structural formula：
The pharmaceutical preparation of Entecavir listed both at home and abroad at present is peroral dosage form, into digestive system after easily by
The destruction of digestive juice so that the blood concentration of Entecavir is relatively low, reduces therapeutic effect.On the other hand, the grace listed at present is replaced
Card Wei oral formulations, lack the targeting for liver, and 60%-70%, through kidney excretion, is also easy to produce renal toxicity in human body.
Further, since content of the Entecavir in single dose is very low so that the content of active ingredient is difficult to protect between each single dose
Hold constant.
Therefore it provides a kind of good absorbing effect, toxicity are low, hepatic targeting is good, content is uniform and batch between the small grace of difference
There is greater significance for card Wei pharmaceutical composition.
The content of the invention
It is an object of the invention to provide a kind of good absorbing effect, toxicity is low, hepatic targeting is good, content is uniform and batch between
The small entecavir medicinal composition of difference.
The present invention provides a kind of Entecavir liposome of enoxolone modification, including Entecavir, arginine, GA-
PEG-DPPC, phosphatide, cholesterol.
In the present invention, GA-PEG-DPPC is obtained by sweet by enoxolone, PEG, DPPC reaction
The DPPC of careless hypo acid-PEG modifications.As one embodiment of the present invention, the PEG is PEG2000
Or/and PEG800, preferably PEG2000 and PEG800 mixture, most preferably the ratio between amount of material is 1:1 PEG2000
With PEG800 mixture.PEG2000 and PEG800 of the present invention mixture, refer to that enoxolone-PEG2000 is modified
DPPC and enoxolone-PEG800 modification DPPC mixture.
It is used as one embodiment of the present invention, Entecavir, arginine, phosphatide, cholesterol, GA-PEG-DPPC materials
The ratio between amount be 1:4-8:20-80:4-10:1-5, preferably 1:6:30:6:4.
Phosphatide of the present invention may be selected from lecithin, soybean lecithin, cephalin, sphingomyelins, hydrogenated soya phosphatide, two Pork and beans
One or more in dimyristoyl phosphatidylcholine, DOPC, phosphatidyl-ethanolamine, DOPE
Mixture, it is a kind of preferably in lecithin, hydrogenated soya phosphatide, DOPC and DPPC
Or several mixtures, most preferably DPPC.
As one embodiment of the present invention, the present invention provides a kind of Entecavir liposome of enoxolone modification
Preparation method, comprises the following steps：
(1) enoxolone, PEG and DCC heating response in organic solvent, products therefrom and succinic anhydride and two palmityls
Phosphatidyl choline reacts, and GA-PEG-DPPC is made；
(2) it is Entecavir and arginine is soluble in water, add DPPC, cholesterol, GA-PEG-
DPPC, ethanol are mixed, and after water bath sonicator concussion, are moved into reactor, supercritical carbon dioxide are passed through, in 15-30MPa pressure
With 40-60 DEG C at a temperature of react, be made enoxolone modification Entecavir liposome.
As one embodiment of the present invention, in above-mentioned preparation method, organic solvent is DMF in step (1).
As one embodiment of the present invention, in above-mentioned preparation method, enoxolone and PEG materials in step (1)
The ratio between amount is 0.7-1:1.
As one embodiment of the present invention, in above-mentioned preparation method, Entecavir in step (2)：Arginine：Two palm fibres
Palmitic acid phosphatidyl choline：Cholesterol：GA-PEG-DPPC：Ethanol=1:6:30:6:4:10.
As one embodiment of the present invention, in above-mentioned preparation method, step is passed through supercritical carbon dioxide in (2)
Afterwards, at reaction condition is 25MPa pressure and 40 DEG C, react 40 minutes.
Compared with prior art, advantage is the entecavir medicinal composition of the present invention：(1) Entecavir is improved
Hepatic targeting and drug effect, reduce, envelop rate uniform to toxic and side effect (2) Entecavir liposomal particle size of other histoorgans
Height, improves the scattered uniformity of medicine, it is ensured that stability is high between low dose of Entecavir formulation batch.
The present invention uses following initialisms：DMF represents dimethylformamide；PEG represents polyethylene glycol；GA represents Radix Glycyrrhizae time
Acid；DPPC represents DPPC；DCC represents 1,3- dicyclohexyl carbodiimides.
Embodiment
The present invention is described in further detail with specific embodiment below, but present invention is not limited to these implementations
Example.
The Entecavir method for preparing lipidosome of the enoxolone of embodiment 1 modification
1) GA-PEG2000-DPPC preparation
Under nitrogen protection, enoxolone (10g, 21.2mmol) is added in 1000mL flasks, DMF (400mL) is added, stirs
Mix dissolving.DCC (5.2g, 25.4mmol), PEG2000 (50.8g, 25.4mmol) are added afterwards, and 40 DEG C are reacted 12 hours.Reaction
After end, organic layer uses aqueous hydrochloric acid solution, sodium bicarbonate aqueous solution and brine It successively, and gained organic layer is dried, decompression
Concentration.Gained concentrate purifies (eluant, eluent with silica gel column chromatography：Ethyl acetate/petroleum ether), obtain yellow solid 38.0g.
Upper step products therefrom (30g, 12.2mmol) is added in 500mL flasks under nitrogen protection, adds DMF
(200mL), stirring and dissolving.Succinic anhydride (3.66g, 36.6mmol) is added afterwards, and 70 DEG C are reacted 2 hours.Reaction solution is used respectively
Sodium bicarbonate aqueous solution and brine It, gained organic layer are dried.
Up walk and DPPC (11.6g, 15.8mmol) is added in organic layer, DCC (3.2g,
15.8mmol), 40 DEG C are reacted 24 hours.Organic layer uses aqueous hydrochloric acid solution, sodium bicarbonate aqueous solution and brine It, institute successively
Obtain organic layer to dry, be concentrated under reduced pressure.Gained concentrate is purified with silica gel column chromatography, eluant, eluent：Ethyl acetate/petroleum ether), obtain
Yellow solid 32.0g, is GA-PEG2000-DPPC.
2) GA-PEG800-DPPC preparation
Under nitrogen protection, enoxolone (10g, 21.2mmol) is added in 1000mL flasks, DMF (400mL) is added, stirs
Mix dissolving.DCC (5.2g, 25.4mmol), PEG800 (20.3g, 25.4mmol) are added afterwards, and 40 DEG C are reacted 12 hours.Reaction
After end, organic layer uses aqueous hydrochloric acid solution, sodium bicarbonate aqueous solution and brine It successively, and gained organic layer is dried, decompression
Concentration.Gained concentrate purifies (eluant, eluent with silica gel column chromatography：Ethyl acetate/petroleum ether), obtain yellow solid 18.7g.
Upper step products therefrom (15g, 12.0mmol) is added in 250mL flasks under nitrogen protection, adds DMF
(100mL), stirring and dissolving.Succinic anhydride (3.60g, 3.6mmol) is added afterwards, and 70 DEG C are reacted 2 hours.Reaction solution uses carbon respectively
Sour hydrogen sodium water solution and brine It, gained organic layer are dried.
Up walk and DPPC (11.5g, 15.6mmol) is added in organic layer, DCC (3.2g,
15.6mmol), 40 DEG C are reacted 24 hours.Organic layer uses aqueous hydrochloric acid solution, sodium bicarbonate aqueous solution and brine It, institute successively
Obtain organic layer to dry, be concentrated under reduced pressure.Gained concentrate is purified with silica gel column chromatography, eluant, eluent：Ethyl acetate/petroleum ether), obtain
Yellow solid 20.9g, is GA-PEG800-DPPC.
3) preparation of liposome
Accurate the ratio between the amount of material that weighs is Entecavir：Arginine：DPPC：Cholesterol：GA-
PEG-DPPC：Ethanol=1:6:30:6:4:10 raw material.Entecavir and arginine are dissolved in deionized water first, then with
Other compositions are mixed, and ultrasonic vibration moves into reactor, be passed through supercritical carbon dioxide after 10 minutes under water-bath 100w,
At a temperature of 25MPa pressure and 40 DEG C, react 40 minutes, the Entecavir liposome of enoxolone modification is made.
Influence of the embodiment 2 from different types of polyethylene glycol to obtained liposome encapsulation and particle diameter
Zetasozer3000HS laser particle analyzers determine the particle diameter of liposome.
A certain amount of obtained liposome turbid liquor is taken, after Sephadex-G50 post separations, by determining and calculating
The free drug concentration C into proliposome suspensionTrip, a certain amount of liposome, anhydrous alcohol solution and constant volume are separately taken, by surveying
Fixed and calculating obtains total drug concentration C in proliposome suspensionAlways.Computational envelope rate：Envelop rate (%)=[(CAlways-CTrip)/CAlways]
× 100%
| Polyethylene glycol species | Average grain diameter/nm | Envelop rate/% |
| Polyethylene glycol 2000 | 187.0±15.6 | 80.1±5.8 |
| Polyethylene glycol-800 | 158.5±13.3 | 79.2±6.3 |
| Polyethylene glycol 2000：Polyethylene glycol-800=1:1 | 161.1±9.2 | 93.2±3.1 |
From experimental result, when polyethylene glycol 2000 is mixed with polyethylene glycol-800, particularly with 1:The amount of 1 material
The ratio between mixing when, obtained enoxolone modification Entecavir liposome, with polyethylene glycol 2000, polyethylene glycol is used alone
Compared when 800, evenly, envelop rate is higher for particle diameter, and sample room difference is small.
Internal pharmacodynamics of the Entecavir liposome of the enoxolone of embodiment 3 modification in hepatitis B infection duckling model
Experiment
1 age in days Beijing duckling is chosen, shin intravenous injection duck hepatitis B virus (DHBV)-DNA positive serums 0.2ml makes
Mould, after one week, vena jugularis externa takes blood, separates serum, and PCR methods detection serum DHBV-DNA filters out hepatitis B virus infection positive
Animal, random packet every group 6, is used to test as model group and each administration group.Sieve and raised one week after mould, start experiment.
Each group gavage gives the medicine of respective concentration, and model group gavage gives physiological saline, successive administration 14 days, 1,3,7,14 days periods
Vena jugularis externa takes blood, separates serum, PCR methods detection serum DHBV-DNA levels.
As a result represented with the percentage with the detected value of administration first day：
DHBV-DNA% (Day x)=[DHBV-DNA titres (Day x)/DHBV-DNA titres (Day 1)] × 100%.
From experimental result, compared with the free Entecavir of Isodose, the Entecavir fat of enoxolone modification
Plastid is more preferable to the inhibition of hepatitis B in Hepatitis B Animal Model.
Claims (9)
1. a kind of Entecavir liposome of enoxolone modification, it is characterized in that, including Entecavir, arginine, GA-PEG-
DPPC, phosphatide, cholesterol, the PEG are PEG2000 and PEG800 mixture, and the phosphatide is two palmityl phosphatidyl courages
Alkali.
2. the Entecavir liposome of enoxolone modification as claimed in claim 1, it is characterized in that, PEG2000 and PEG800
The ratio between amount of material is 1:1.
3. the Entecavir liposome of enoxolone as claimed in claim 1 modification, it is characterized in that, Entecavir, arginine,
The ratio between phosphatide, cholesterol, amount of GA-PEG-DPPC materials are 1:4-8:20-80:4-10:1-5.
4. the Entecavir liposome of enoxolone as claimed in claim 3 modification, it is characterized in that, Entecavir, arginine,
The ratio between phosphatide, cholesterol, amount of GA-PEG-DPPC materials are 1:6:30:6:4.
5. the preparation method of the Entecavir liposome of any one of the claim 1-4 enoxolone modifications, including following step
Suddenly：
(1) enoxolone, PEG and DCC heating response in organic solvent, products therefrom and succinic anhydride and two palmityl phosphatide
Phatidylcholine reacts, and GA-PEG-DPPC is made；
(2) it is Entecavir and arginine is soluble in water, add DPPC, cholesterol, GA-PEG-DPPC,
Ethanol is mixed, and after water bath sonicator concussion, is moved into reactor, supercritical carbon dioxide is passed through, in 15-30MPa pressure and 40-
Reacted at a temperature of 60 DEG C, the Entecavir liposome of enoxolone modification is made.
6. preparation method as claimed in claim 5, it is characterized in that, organic solvent is DMF in step (1).
7. preparation method as claimed in claim 6, it is characterized in that, the ratio between enoxolone and amount of PEG materials are in step (1)
0.7-1:1。
8. preparation method as claimed in claim 7, it is characterized in that, Entecavir in step (2)：Arginine：Two palmityl phosphorus
Phosphatidylcholine：Cholesterol：GA-PEG-DPPC：The ratio between amount of ethanol material is 1:6:30:6:4:10.
9. preparation method as claimed in claim 8, it is characterized in that, step is passed through after supercritical carbon dioxide in (2), reacts bar
At a temperature of part is 25MPa pressure and 40 DEG C, react 40 minutes.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201511009226.9A CN105412015B (en) | 2015-12-28 | 2015-12-28 | Entecavir Liposomal formulation of enoxolone modification and preparation method thereof |
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| CN201511009226.9A CN105412015B (en) | 2015-12-28 | 2015-12-28 | Entecavir Liposomal formulation of enoxolone modification and preparation method thereof |
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| CN105412015B true CN105412015B (en) | 2017-07-18 |
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| CN102336802B (en) * | 2010-07-16 | 2013-11-06 | 四川大学 | Glycyrrhetinic acid-modified lipid, liver targeting liposome, micelle and compound, and their preparation method |
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