CN107648179A - A kind of novel skin drug-delivery preparation for treating rheumatoid arthritis and preparation method thereof - Google Patents
A kind of novel skin drug-delivery preparation for treating rheumatoid arthritis and preparation method thereof Download PDFInfo
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- 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
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Abstract
A kind of novel skin drug-delivery preparation for treating rheumatoid arthritis and preparation method thereof, belong to the preparation for external application to skin technical field of field of pharmaceutical preparations.Said preparation is composed of the following components in parts by weight：80 120 parts of SIN HCl, 50 350 parts of phosphatidase 2,30 60 parts of cholesterol, 15 50 parts of volatile oil, 47 parts of vitamin E, 2,000 12 30 parts of DSPE PEG, 15,000 30000 parts of buffer solution.SIN HCl PEG modification carriers best in quality can be made in the present invention, wherein transmitting, body elasticity is larger, and entrapment efficiency is suitable, stability is high, particle diameter is suitable and is evenly distributed, and has good drug penetration through skin and synovial membrane targeting, can reach the purpose of Synergy and attenuation.
Description
Technical field
The invention belongs to the preparation for external application to skin technical field of field of pharmaceutical preparations, and in particular to one kind treats rheumatoid
Arthritic novel skin drug-delivery preparation and preparation method thereof.
Background technology
Cucoline（Sinomenine, SIN）It is from menispermaceous plants caulis sinomeniisinomenium acutum(thunb.)
Rehd. et Wils. or hair sinomenium acutumsinomenium scutum(Thunb.) Rehd.et Wils. var. cinereum
Rehd. the alkaloid monomer extracted in et Wils., chemistry are entitled
(9α,13α,14α)-7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinan-6-
One, its molecular formula are C19H23NO4, molecular weight 329.38.There is SIN anti-inflammatory, immunosupress, analgesia, decompression, the anti-rhythm of the heart to lose
Pharmacological action is often waited, medicinal is mostly its hydrochloride (sinomenine hydrochloride, SIN-HCl), is clinically used for treating
Rheumatism, rheumatoid arthritis and arrhythmia cordis etc..But the medicine bioavilability is low, half-life short, frequent drug administration is needed, through body circulation
It is few that diseased region dose is reached afterwards, and clinical long-term use easily causes fash, Neuroleptic Leukocytopenia, decrease of platelet and stomachache etc.
Gastrointestinal side effect, therefore, percutaneous dosing turn into its preferable administering mode.However, SIN-HCl（SIN）The transmission of itself
Skin is limited in one's ability, and playing it therapeutic action has larger limitation.
Carrier（Transfersomes, TFS）Also known as flexible nano-liposomes, it is a kind of new cutaneous penetration carrier, its
Main component is phosphatide and surfactant（Such as sodium taurocholate, deoxysodium cholate）, there is the morphotropism of height, can efficiently penetrate
The skin duct of several times smaller than itself and reach cutaneous penetration purpose.There are some researches show volatile oil can make the lipid of liposome double
Molecule tunic softening, while expansible skin aqueous channels, so as to promote carrier carried medicine to penetrate skin, therefore the present invention will
Edge activator of the limonene-citral Mixed chaotic sequences as carrier.
In addition, the present invention has carried out PEG modifications to carrier.Covered by submissive and hydrophilic PEG chain parts on carrier surface
Gai Hou, steric hindrance and hydrophily all greatly increase, and so can not only prevent carrier from assembling, and strengthen its stability, also have
Beneficial to carrier skin is penetrated by skin aqueous channels.Meanwhile the carrier after PEG modifications enter can reduce after blood it is netted
The identification and intake of endothelial system, extend its blood circulation time, may be used also for the blood vessel of RA inflammation synovial membranes position special construction
" infiltration and delay of enhancing " (enhanced permeability and retention, EPR) effect is produced, so as to realize
Targeting drug delivery.
The present invention constructs the PEG modification carrier transdermal delivery systems that a kind of stability is good and percutaneous permeability is strong, can
Medicine SIN-HCl is encapsulated in lipid bilayer, improves the ability of its Transdermal absorption, realizes that medicine is directed to RA inflammation synovial membranes
The targeted delivery at position, so as to play the effect of local treatment.
The content of the invention
The problem of existing for prior art, it is an object of the invention to design offer one kind to treat rheumatoid arthritis
Novel skin drug-delivery preparation and preparation method thereof technical scheme.
To achieve the object of the present invention, SIN-HCl PEG provided by the present invention modification carrier by following parts by weight into
It is grouped into：
SIN-HCl 80-120 parts
Phosphatidase 2 50-350 parts
Cholesterol 30-60 parts
Volatile oil 15-50 parts
Vitamin E 4-7 parts
The 12-30 parts of DSPE-PEG 2000
Buffer solution 15000-30000 parts
Currently preferred SIN-HCl carriers are made up of following parts by weight ingredient：
100 parts of SIN-HCl
00 part of phosphatidase 3
50 parts of cholesterol
20 parts of volatile oil
5 parts of vitamin E
2,000 18 parts of DSPE-PEG
25000 parts of buffer solution
SIN-HCl meets medicinal standard, commercially available product, manufacturer's such as limited public affairs of Hunan Zheng Qing pharmacy group share in the present invention
Department, still, is not restricted to this company.
As the phosphatide for forming carrier, natural phospholipid and synthetic phospholipid can be used.Natural phospholipid includes phosphatide
Acyl monoethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols, egg yolk lecithin, hydrogenated yolk lecithin, yolk phosphorus
Phosphatidyl glycerol, yolk phospholipid acyl serine, PI, soybean lecithin, hydrogenated soy phosphatidyl choline, hydrogenation lecithin
Fat, EPG, lecithin acyl serine and lecithin acyl inositol etc..Synthetic phospholipid is DOPC, two
DSPC, DPPC, DMPC, DLPC, two
Stearyl phosphatidyl glycerine, DPPG, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, PE and
Polyglycol derivatization phospholipid such as PEG-DSPE 2000（DSPE-mPEG2000）；Two soft esters
Acyl courage phosphatide-polyethylene glycol 2000（DPPG-mPEG2000）；HSPC-polyethylene glycol 2000（HSPC-
mPEG2000）；DOPC-polyethylene glycol 2000（DOPC-mPEG2000）Deng.
The present inventor has found that the egg yolk lecithin that commercially available PC contents are 60% ~ 98% is particularly suitable as base by research
Plinth phosphatide membrane material encapsulates active pharmaceutical ingredient, and so as to form high-quality carrier, the preferably PC contents of egg yolk lecithin are 80%
~90%。
In the carrier of the present invention, cholesterol plays a part of adjusting membrane fluidity, can improve lipid bilayer
The stability of film and the envelop rate of medicine.Cholesterol used meets the standard of pharmacopeia 2015.
It is edge activator from volatile oil in the carrier of the present invention, volatile oil is eucalyptus oil, menthol, lemon
Mixture more than one or both of terpene volatile oil such as aldehyde, limonene, preferably by limonene and citral according to weight
Measure ratio 1:4~4:1 mixes, more preferably by limonene and citral by weight 1:1 Mixed chaotic sequences mixed.
Relative to other surfaces activating agent class edge activator, formed carrier security is made using volatile oil as edge activator
Greatly improve, so that patient is possibly realized using this product for long periods.
The present inventor has found that both ratios can be 1 in limonene-citral Mixed chaotic sequences by research:4~4:1
（W/W）In the range of select, when Mixed chaotic sequences ratio be 1:When 1, the SIN-HCl PEG modifications formed transmit body elasticity most
Good, percutaneous permeability is optimal.
In the carrier of the present invention, vitamin E is added as antioxidant, prevents phosphatide to be oxidized.
Research is found, when SIN-HCl, the egg yolk lecithin using above-mentioned specified quantitative（PC contents 60% ~ 95%）, cholesterol,
Limonene-citral Mixed chaotic sequences（Limonene:Citral=1:4~4:1, W/W）, DSPE-PEG 2000 and during vitamin E,
It can be made SIN-HCl PEG modifications carrier best in quality, its envelop rate and have good stability, skin permeation rates are higher.
The preparation of SIN-HCl PEG modifications carrier provided by the present invention adopts the following technical scheme that：
The present invention is prepared using alcohol injection, and this method comprises the following steps：
（1）Phosphatide, cholesterol, volatile oil, vitamin E and the DSPE-PEG 2000 of formula ratio are weighed, is dissolved in absolute ethyl alcohol,
It is well mixed, obtain organic phase；
（2）SIN-HCl is dissolved in phosphate buffer, obtains aqueous phase；
（3）Under normal temperature condition, syringe holder is used（1）In organic phase be slowly expelled to（2）In aqueous phase in, this process is being stirred
Carried out under the conditions of mixing；
（4）After organic phase injection completely, continue to stir 5 min, obtain SIN-HCl PEG modification carrier suspensions, this is mixed
After suspension places 2-3 h, it is placed in liposome mini-extruder extrusion instrument, 100 nm polycarbonate membranes is squeezed through under 0.10MPa, then at
50 nm polycarbonate membranes are squeezed through under 0.38 MPa, produce carrier sample.
In view of the easily safety issue of loss and finished product of volatile oil in preparation process（The residual for referring mainly to organic solvent is asked
Topic）, the present invention is from alcohol injection preparation carrier.
The one kind of described cushioning liquid in phosphate buffer, citrate buffer and carbonate buffer solution, it is excellent
Select the phosphate buffer solution that pH value is 7.0.
Described mixing speed is 150-750 r/min, such as 150,350,550,750r/min, preferably 550r/min.
SIN-HCl of the present invention transmit preparation it is preferred in, carrier extrude when pressure used to carrier
Grain diameter influence is very big, and it is as follows to select more excellent pressure through test of many times：The pressure for squeezing through 100 nm polycarbonate membranes is 0.10MPa,
The pressure for squeezing through 50 nm polycarbonate membranes is 0.38MPa.
By the above method, SIN-HCl PEG modification carriers best in quality can be made, wherein transmit body elasticity compared with
Greatly, entrapment efficiency is suitable, and stability is high, and particle diameter is suitable and is evenly distributed, and drug penetration through skin is good.
Research finds, the particle diameter of carrier to its it is skin-penetrating have considerable influence, particle diameter is smaller, and carrier penetrability is got over
It is good；In addition, excellent elasticity is also to improve the essential condition of carrier percutaneous abilities.The SIN- being prepared by the method for the present invention
HCl PEG modification transmission body elasticities are good, and particle diameter is small and is evenly distributed, and this is the excellent principal element of its percutaneous permeability.
Brief description of the drawings
The transmission electron microscope picture of the optimal prescription SIN-HCl PEG modifications carriers of Fig. 1；
Each embodiment carriers of Fig. 2 and the isolated skin penetration curve of reference preparation liposome；
Fig. 3 each groups rat right hind leg toes volumetric ratio compared with（n=9）；
The measurement result of Fig. 4 each group rat TNF-α serum levels（n=9）, in figure:Compared with Control groups, ## P<
0.01；
Compared with Model groups, * * P< 0.01；Compared with PEG-TFS-1 groups, ▲ ▲ P< 0.01；
The measurement result of Fig. 5 each group rat IL-1 β serum levels（n=9）, in figure：Note:Compared with Control groups, ## P<
0.01；
Compared with Model groups, * * P< 0.01；Compared with PEG-TFS-1 groups, ▲ ▲ P< 0.01；
Fig. 6 SIN-HCl PEG modify influence of the carrier to rat ankle joint tectology；
Pharmaceutical concentration-time curve after Fig. 7 mouse percutaneous dosings in skin（n=5）；
Pharmaceutical concentration-time curve in Fig. 8 rabbit percutaneous dosing posterior joint chambers（n=5）；
The pharmaceutical concentration-time curve of blood after Fig. 9 rabbit percutaneous dosings（n=4）.
Embodiment
With reference to specific embodiment, the invention will be further elaborated.Methods described is routine unless otherwise instructed
Method, the raw material can obtain from open commercial sources unless otherwise instructed.
Described in the embodiment of the present invention and SIN-HCl PEG modification carrier particle diameter and particle diameter distribution, form, encapsulating
The evaluation method of the indexs such as rate, elasticity, percutaneous permeability is as follows：
1. particles size and distribution evaluation method
Above-mentioned SIN-HCl PEG modifications carrier is taken, is suitably diluted with distilled water, the grain of carrier is determined using laser particle analyzer
Footpath and its distribution situation.
2. morphologic observation
Take above-mentioned SIN-HCl PEG modifications carrier appropriate, be diluted with water, drip on copper mesh, with 1% Salkowski's solution negative staining, filter
Paper draws excess stain liquid, and transmission electron microscope observation is used after drying.
3. the assay method of envelop rate
It is measured using centrifugal ultrafiltration method.Carrier sample to be measured is taken, is shaken up, takes a small amount of progress observation by light microscope, really
Recognize precision after being wherein free of drug crystallization and measure 200 μ L, be placed in centrifugal ultrafiltration pipe（The molecular cut off of milipore filter is 100000
Da）, carry out refrigerated centrifuge（RCF：14000 g, temperature：4 ℃）, all outer aqueous phases are collected, are transferred to suitable volumes
Measuring bottle in, with mobile phase constant volume, shake up, using SIN-HCl content as index, with HPLC methods determine carrier sample in and pass
Index components content in external aqueous phase is passed, envelop rate is calculated as follows：
EE=（W
S
-W
EA
）/W
S
×100%
In formulaEEFor envelop rate；W S For the content (mg) of SIN-HCl in sampling amount；W EA For the content of SIN-HCl in outer aqueous phase
(mg)。
4. the assay method of elasticity
Precision measures 10 mL carriers, is placed in liposome mini-extruder extrusion instrument, squeezes through 50 nm polycarbonate membranes, pressure 0.38
MPa, by sample, all the time used in extrusion is designated as its elasticity.（This law bibliography：Zaafarany G. M. E., Avad
G. A. S., Holayel S. M. et al. Role of egde activators and surface charge in
developing ultradeformable vesicles with enhanced skin delivery [J]. Int J
Pharm, 2010,397(1-2):164-172.）
5. isolated skin Penetration Signature investigates method
Take SD rats, cervical dislocation to be placed in after putting to death in fixed plate, cut off rat clavicle to the hair of belly with operating scissors, cut big
Mouse skin of abdomen, carefully removes hypodermis, ensures the integrality of keratoderma, and with physiological saline rinsed clean, flattening is simultaneously
The moisture of attachment is sucked with filter paper, is sandwiched in aluminium foil, -70 DEG C of freezings are standby.First skin is thawed at room temperature during experiment, cut
Take suitable size and be fixed between supply pool and acceptance pool（Effective skin area is 0.785 cm2, acceptance pool volume is 5
mL）, epidermis is put into acceptable solution towards supply pool（0.9% physiology salt water-ethanol（4:1）Mixed liquor）, make liquid level and skin inner layer
Contact, the bubble in acceptance pool is drained, acceptance pool bath temperature is controlled at 32 DEG C, and magnetic agitation rotating speed is 400 r/ in pond
Min, balance is after 15 minutes, and the acceptable solution that more renews gives 250 μ L carrier samples to be measured, loading records the time as 0 simultaneously
Time, in the several μ L of point in time sampling 200 of 0.5,1,2,4,6,8,10,12,24 h, and isometric fresh connect is added in time
By liquid, take 10 μ L samples to carry out HPLC analyses, calculate drug accumulation transit dose.
0.785 is transdermal penetration effective area in formula（cm2）, 5 be acceptance pool volume（mL）, 0.2 is that transdermal penetration is tested
Effective volume（mL）, Cn is the drug concentration that n-th of sample point measures（mg/mL）, Ci be the sample point before each point measure it is dense
Degree（mg/mL）.
The calculating of stable state transdermal penetration rates：Using the drug accumulation transit dose of each sample point as ordinate, the time is horizontal seat
Mark, transdermal penetration curve is drawn, to steady-state permeation section in curve（Straight line portion）Linear regression is carried out, the slope of gained straight line is
For stable state transdermal penetration ratesJ(mg/cm2·h)。
Embodiment 1
SIN-HCl PEG modify carrier（PEG-TFS-1）It is made up of following parts by weight ingredient：
100 parts of SIN-HCl
300 parts of egg yolk lecithin
50 parts of cholesterol
Limonene-citral Mixed chaotic sequences (1:1, W/W) 20 parts
5 parts of vitamin E
2,000 18 parts of DSPE-PEG
25000 parts of phosphate buffer
The specification of main constituents and source：SIN-HCl（Meet medicinal standard）, the limited public affairs of Hunan Zheng Qing pharmacy group share
Department；Egg yolk lecithin（PC contents are 80%, injection stage）, Shanghai Advanced viecle Technology Co., Ltd.；Cholesterol（Meet China
The standard of pharmacopeia 2015）, Shanghai Advanced viecle Technology Co., Ltd.；Limonene, citral, the poly- roc natural perfume material oil of Ji'an City have
Limit company；Vitamin E（Meet the standard of Chinese Pharmacopoeia 2015）, Zhejiang NHU Company Ltd；DSPE-PEG 2000, on
Hai Aiweite Pharmaceutical Technology Co., Ltd；Phosphate buffer solution is 1/15 mol/L potassium dihydrogen phosphates and 1/15 mol/L
Disodium phosphate soln by volume 53.4:The mixed liquor of 46.6 mixing.
SIN-HCl PEG modification carrier preparation method be：
Prepared using alcohol injection, this method comprises the following steps：
（1）Egg yolk lecithin, cholesterol, citral, limonene, vitamin E and the DSPE-PEG 2000 of formula ratio are weighed, it is molten
In absolute ethyl alcohol, it is well mixed, obtains organic phase；
（2）SIN-HCl is dissolved in phosphate buffer, is well mixed, obtains aqueous phase；
（3）Under normal temperature condition, with micro syringe handle（1）In organic phase be slowly expelled to（2）In aqueous phase in, this process
Carry out under agitation；
（4）After organic phase is injected, continue to stir 5 min, obtain a suspension, after this suspension is placed into 2-3 h, put
In liposome mini-extruder extrusion instrument, 100 nm polycarbonate membranes are squeezed through under 0.10 MPa, the poly- carbon of 50 nm is squeezed through under 0.38 MPa
Acid esters film, is produced.
Embodiment 2
SIN-HCl PEG modify carrier（PEG-TFS-2）It is made up of following parts by weight ingredient：
80 parts of SIN-HCl
250 parts of egg yolk lecithin
30 parts of cholesterol
Limonene-citral Mixed chaotic sequences (3:1, W/W) 15 parts
4 parts of vitamin E
2,000 12 parts of DSPE-PEG
5000 parts of phosphate buffer 1
The specification of main constituents and source：SIN-HCl（Meet medicinal standard）, the limited public affairs of Hunan Zheng Qing pharmacy group share
Department；Egg yolk lecithin（PC contents are 80%, injection stage）, Shanghai Advanced viecle Technology Co., Ltd.；Cholesterol（Meet country
The standard of pharmacopeia 2015）, Nanjing Xinbai Pharmaceutical Co；Limonene, citral, the limited public affairs of the poly- roc natural perfume material oil of Ji'an City
Department；Vitamin E（Meet the standard of NF 2015）Zhejiang NHU Company Ltd；DSPE-PEG 2000, Shanghai Ai Wei
Special Pharmaceutical Technology Co., Ltd；Phosphate buffer solution is 1/15 mol/L potassium dihydrogen phosphates and 1/15 mol/L phosphoric acid hydrogen
Two sodium solutions by volume 53.4:The mixed liquor of 46.6 mixing.
SIN-HCl PEG modification carrier preparation method be：
Prepared using alcohol injection, this method comprises the following steps：
（1）Egg yolk lecithin, cholesterol, citral, limonene, vitamin E and the DSPE-PEG 2000 of formula ratio are weighed, it is molten
In absolute ethyl alcohol, it is well mixed, obtains organic phase；
（2）SIN-HCl is dissolved in phosphate buffer, is well mixed, obtains aqueous phase；
（3）Under normal temperature condition, with micro syringe handle（1）In organic phase be slowly expelled to（2）In aqueous phase in, this process
Carry out under agitation；
（4）After organic phase is injected, continue to stir 5 min, obtain a suspension, after this suspension is placed into 2-3 h, put
In liposome mini-extruder extrusion instrument, 100 nm polycarbonate membranes are squeezed through under 0.10 MPa, the poly- carbon of 50 nm is squeezed through under 0.38 MPa
Acid esters film, is produced.
Embodiment 3
SIN-HCl PEG modify carrier（PEG-TFS-3）It is made up of following parts by weight ingredient：
120 parts of SIN-HCl
350 parts of egg yolk lecithin
60 parts of cholesterol
Limonene-citral Mixed chaotic sequences (1:4, W/W) 50 parts
7 parts of vitamin E
2,000 30 parts of DSPE-PEG
30000 parts of phosphate buffer
The specification of main constituents and source：SIN-HCl（Meet medicinal standard）, the limited public affairs of Hunan Zheng Qing pharmacy group share
Department；Egg yolk lecithin（PC contents are 80%, injection stage）, Shanghai Advanced viecle Technology Co., Ltd.；Cholesterol（Meet country
The standard of pharmacopeia 2015）, Nanjing Xinbai Pharmaceutical Co；Limonene, citral, the limited public affairs of the poly- roc natural perfume material oil of Ji'an City
Department；Vitamin E（Meet the standard of NF 2015）Zhejiang NHU Company Ltd；DSPE-PEG 2000, Shanghai Ai Wei
Special Pharmaceutical Technology Co., Ltd；Phosphate buffer solution is 1/15 mol/L potassium dihydrogen phosphates and 1/15 mol/L phosphoric acid hydrogen
Two sodium solutions by volume 53.4:The mixed liquor of 46.6 mixing.
SIN-HCl PEG modification carrier preparation method be：
Prepared using alcohol injection, this method comprises the following steps：
（1）Egg yolk lecithin, cholesterol, citral, limonene, vitamin E and the DSPE-PEG 2000 of formula ratio are weighed, it is molten
In absolute ethyl alcohol, it is well mixed, obtains organic phase；
（2）SIN-HCl is dissolved in phosphate buffer, is well mixed, obtains aqueous phase；
（3）Under normal temperature condition, with micro syringe handle（1）In organic phase be slowly expelled to（2）In aqueous phase in, this process
Carry out under agitation；
（4）After organic phase is injected, continue to stir 5 min, obtain a suspension, after this suspension is placed into 2-3 h, put
In liposome mini-extruder extrusion instrument, 100 nm polycarbonate membranes are squeezed through under 0.10 MPa, the poly- carbon of 50 nm is squeezed through under 0.38 MPa
Acid esters film, is produced.
Embodiment 4
SIN-HCl PEG modify carrier（PEG-TFS-4）It is made up of following parts by weight ingredient：
100 parts of SIN-HCl
270 parts of egg yolk lecithin
40 parts of cholesterol
Limonene-citral Mixed chaotic sequences (4:1, W/W) 30 parts
7 parts of vitamin E
2,000 20 parts of DSPE-PEG
20000 parts of phosphate buffer
The specification of main constituents and source：SIN-HCl（Meet medicinal standard）, the limited public affairs of Hunan Zheng Qing pharmacy group share
Department；Egg yolk lecithin（PC contents are 80%, injection stage）, Shanghai Advanced viecle Technology Co., Ltd.；Cholesterol（Meet country
The standard of pharmacopeia 2015）, Nanjing Xinbai Pharmaceutical Co；Limonene, citral, the limited public affairs of the poly- roc natural perfume material oil of Ji'an City
Department；Vitamin E（Meet the standard of NF 2015）Zhejiang NHU Company Ltd；DSPE-PEG 2000, Shanghai Ai Wei
Special Pharmaceutical Technology Co., Ltd；Phosphate buffer solution is 1/15 mol/L potassium dihydrogen phosphates and 1/15 mol/L phosphoric acid hydrogen
Two sodium solutions by volume 53.4:The mixed liquor of 46.6 mixing.
SIN-HCl PEG modification carrier preparation method be：
Prepared using film dispersion method, this method comprises the following steps：
（1）Weigh SIN-HCl, egg yolk lecithin, cholesterol, citral, limonene, vitamin E and the DSPE-PEG of formula ratio
2000, it is dissolved in organic solvent [methylene chloride-methanol（5:2,V/V）Mixed organic solvents] in, it is transferred in eggplant-shape bottle, depressurizes
Rotation boils off organic solvent, obtains dry film；
（2）With phosphate buffer hydration step（1）Obtained dry film, shaking, makes the complete aquation of lipid film, obtains a suspension；
（3）After above-mentioned suspension is placed into 2-3 h, it is placed in liposome mini-extruder extrusion instrument, 100 nm is squeezed through under 0.10 MPa
Polycarbonate membrane, 50 nm polycarbonate membranes are squeezed through under 0.38 MPa, are produced.
The preparation of reference preparation
Reference preparation SIN-HCl conventional liposomes（LPS）It is made up of following parts by weight ingredient：
100 parts of SIN-HCl
300 parts of egg yolk lecithin
50 parts of cholesterol
5 parts of vitamin E
25000 parts of phosphate buffer
The specification of main constituents and source：SIN-HCl（Meet medicinal standard）, the limited public affairs of Hunan Zheng Qing pharmacy group share
Department；Egg yolk lecithin（PC contents are 80%, injection stage）, Shanghai Advanced viecle Technology Co., Ltd.；Cholesterol（Meet country
The standard of pharmacopeia 2015）, Shanghai Advanced viecle Technology Co., Ltd.；Vitamin E（Meet the standard of NF 2015）Zhejiang is new
With into limited company；Phosphate buffer solution is 1/15 mol/L potassium dihydrogen phosphates and 1/15 mol/L phosphoric acid hydrogen two
Sodium solution by volume 53.4:The mixed liquor of 46.6 mixing.
The preparation method of SIN-HCl conventional liposomes is：
Prepared using film dispersion method, this method comprises the following steps：
（1）Weigh SIN-HCl, egg yolk lecithin, cholesterol, the vitamin E of formula ratio, be dissolved in organic solvent [dichloromethane-
Methanol（5:2, V/V）Mixed organic solvents] in, it is transferred in eggplant-shape bottle, decompression rotation boils off organic solvent, obtains dry film.
（2）With phosphate buffer hydration step（1）Obtained dry film, shaking, makes the complete aquation of lipid film, obtains a suspension
Liquid.
（3）After above-mentioned suspension is placed into 2-3 h, it is placed in liposome mini-extruder extrusion instrument, 100 is squeezed through under 0.10 MPa
Nm polycarbonate membranes, 50 nm polycarbonate membranes are squeezed through under 0.38 MPa, are produced.
Elasticity is carried out to the SIN-HCl PEG modification carriers obtained by above-described embodiment and SIN-HCl conventional liposomes
Evaluation, particle diameter and entrapment efficiency determination, morphologic observation and isolated skin Penetration Signature are investigated, acquired results be shown in Table 1, table 2, Fig. 1,
Fig. 2.As a result show, the rounded or similar round of SIN-HCl PEG modification carriers, size and distribution uniform, between particle
Adhesion and clustering phenomena are had no, there is good elasticity and transdermal penetration rates.
The SIN-HCl PEG of table 1 modify particle diameter, elasticity and the envelop rate of carrier
The SIN-HCl PEG of table 2 modify the stable state transdermal penetration rates of carrier
The pharmacodynamic experiment research of invention formulation treatment rheumatoid arthritis
1. materials and methods
1.1 medicine：SIN-HCl PEG modify carrier, by SIN-HCl, egg yolk lecithin, cholesterol, limonene-citral
Mixed chaotic sequences, vitamin E, DSPE-PEG 2000, phosphate buffer composition, are carried out from the sample obtained by embodiment 1
Pharmacodynamics is investigated, and is provided by Zhejiang University of Traditional Chinese Medicine's Chinese medicine preparation laboratory；Brufen liniment, the holy first limited public affairs of medicine company in Shenyang
Department, Chinese medicines quasi-word H10940235.
The foundation of 1.2 model of rheumatoid arthritis
Wistar rat arthritis models are established by antigen induction mode, comprised the following steps that：Precision weighs oxCollagen Type VI
10mg, it is dissolved in 5ml 0.05mol/L acetic acid, is gently mixed（Avoid producing excessive heat influence albumen）4 DEG C of refrigerators of juxtaposition
In dissolve overnight, produce concentration be 2mg/mL collagen solution.
0th day, collagen solution and complete Freund's adjuvant are mixed into emulsion in equal volume, using 3 injections in big rat-tail
Inject 0.4 ml in root（Containing the mg of collagen 0.4）Emulsion, this is initial immunity.After 14 days, by collagen solution and incomplete Freund
Agent is mixed into emulsion in equal volume, and 0.2 ml is injected in rat-tail root using 2 injections（Containing the mg of collagen 0.2）Emulsion, this is
Booster immunization.Rat articular scoring is recorded after initial immunity weekly, to judge whether modeling succeeds.
Reference literature（Song H P, Xin L, Yu R, et al. Phenotypic characterization of
type II collagen-induced arthritis in Wistar rats[J]. Experimental and
Therapeutic Medicine, 2015. 10(4): 1483-1488.）, using 0-4 levels The joint evaluation method to rat kind wind
Wet arthritis are scored：0 point, no erythema and arthroncus sign；1 point, there is erythema or slight at ankle-joint or sole middle part
Swelling；2 points, ankle-joint to sole middle part has erythema and swelling；3 points, there are erythema and moderate swelling in ankle-joint and vola pedis joint；4
Point, ankle-joint to pin has erythema and serious swelling.Joint scores are four limbs scoring summation, use arthritis index（arthritis
Index, AI）Represent, be considered as modeling success as AI ＞ 4（AI≤16）.
1.3 packets and administration
The successful rat of modeling is randomly divided into five groups, respectively model control group, Sin-HCl carrier groups（Test group）、
Sin-HCl conventional liposome groups（Reference preparation group）, blank carrier group（Matrix control group）With brufen liniment group（It is positive right
According to group）.The another rat for setting one group of non-modeling is as Normal group, totally 6 groups, every group 10.
Start to be administered after modeling success（Administering mode is percutaneous dosing, and medicine-feeding part is unified for rat right hind leg, administration
Area is the cm of 1 cm × 1）, test group gives the optimal μ L/d of prescription Sin-HCl carriers 720（Dosage is 10 mg/kg/d, with
Sin is counted）, matrix control group gives the μ L/d of blank carrier 720, and reference preparation group gives the μ L/d of conventional liposome 720（Sin
Dosage is 10 mg/kg/d）, positive controls give the μ L/d of brufen liniment 720（Dosage is 200 mg/kg/d）,
Model control group gives the μ L/d of physiological saline 720 respectively with Normal group.Each experimental group dosage of one day, which divides 2 times, to be given, every time
360 μ L, it is administered 2 weeks altogether.
1.4 toes volumetric measurements
After modeling success, the toes volume of each group rat right hind leg is measured with toes capacity measurer, before comparing modeling, after modeling
And administration after rat right hind leg toes volume.
1.5 inflammatory factors determine
Successive administration two weeks, in the 15th day abdominal aortic blood 3ml, 3000 left heart 10min, serum are separated, using ELISA method
TNF-α and IL-1 β in serum are measured respectively.
1.6 Histomorphological
Put to death rat within the 15th day after administration, take rat ankle joint, dyed through fixation, dehydration, embedding, section and HE after optics
Micro- Microscopic observation ankle-joint synovial membrane, cartilage, the pathological change of bone, and from inflammatory cell infiltration, pannus formed, cartilage destruction
Pathological analysis is carried out with four aspects of bone erosion.
1.7 statistical method
All measure numerical value are with means standard deviation（±s）Represent, Dan Yin is carried out to all data using SPSS22.0 softwares
Plain variance analysis.
2. experimental result
2.1 Sin-HCl PEG modify influence of the carrier to rat articular swelling degree
Germicidal efficacy finds that after being administered 2 weeks, each group rat articular swelling degree has different degrees of decline, wherein with experiment
Group and positive controls are the most notable, and reference preparation control group takes second place, and model control group and bare substrate control group are slightly weak.Show
Sin-HCl PEG modifications carrier can mitigate arthroncus caused by rheumatoid arthritis.As a result Fig. 3 is seen.
2.2 Sin-HCl PEG modify the carrier influence horizontal to TNF-α, IL-1 β in rat blood serum
Experimental result shows that after being administered 2 weeks, TNF-α, the horizontal sequences of IL-1 β are as follows in each group rat blood serum：Model comparison
Group ＞ bare substrate control group ＞ reference preparation control group ＞ positive controls ＞ test group ＞ Normal groups.Wherein, model
The TNF-α of control group and bare substrate control group, the horizontal highests of IL-1 β, reference preparation control group slightly drop compared with model control group
It is low, but still maintain higher level；Positive controls decline more obvious；The reduction of test group group is the most obvious, close normal right
According to group.Show that Sin-HClPEG modifications carrier can mitigate inflammatory reaction caused by rheumatoid arthritis.As a result Fig. 4, figure are seen
5。
2.3 Sin-HCl PEG modify carrier to the morphologic influence of rat tissue
Rats in normal control group ankle-joint synovial membrane backing layer eucaryotic cell structure is complete, queueing discipline（1-2 layers, more in individual layer）, synovial membrane
Visible multilayer adipocyte under cell；Have no that inflammatory cell infiltration and pannus are formed；Cartilage is transparent and surface is smooth, has no soft
Osteoclasia and bone erosion, joint cavity gap is, it is apparent that articulation structure is complete（See Fig. 6-A）.Model control group rat ankle joint
Synovial cell proliferation, plumpness；Intrasynovial has a large amount of cell infiltrations, is formed with pannus；Articular surface hyaline cartilage by
Destroy, bone erosion is serious, and joint cavity gap is invisible（See Fig. 6-B）.Test group is compared with model control group, synovial hyperplasia and inflammation
Property cellular infiltration significantly reduces, and has no that pannus is formed；Cartilage and osteoclasia are lighter；Adipocyte and joint cavity gap clearly may be used
See, recover good compared with Normal group（See Fig. 6-C）.Positive controls are compared with model control group, synovial hyperplasia and inflammation
Property cellular infiltration and pannus formed and improved, but still visible cartilage surface destroys, bone erosion is serious, articular cavity narrow gaps
（See Fig. 6-D）.Reference preparation control group and bare substrate control group are still shown in synovial cell proliferation, plumpness, with a large amount of inflammatories
Cellular infiltration exists and pannus generation；Articular surface hyaline cartilage destroys and bone erosion is obvious, and joint cavity gap is invisible, extensive
It is multiple ineffective（See Fig. 6-E, 6-F）.Show that Sin-HCl PEG modify carrier to rheumatoid arthritis in rats ankle-joint group
Knitting form has good therapeutic action.
3. experiment conclusion
Physical efficiency is transmitted in Sin-HClPEG modifications significantly reduces the arthroncus degree of model of rheumatoid arthritis rat, hence it is evident that subtracts
Subinflammation is reacted, and also has good repair to its Pathomorphologic.Invention formulation is to rat rheumatoid joint
Inflammation has definite therapeutic effect.
Invention formulation in healthy animal body pharmacokinetic studies
1. materials and methods
1.1 material
Medicine：SIN-HCl PEG modify carrier, are mixed by SIN-HCl, egg yolk lecithin, cholesterol, limonene-citral
Volatile oil, vitamin E, DSPE-PEG 2000, phosphate buffer composition are closed, medicine is carried out from the sample obtained by embodiment 1
Effect, which is learned, to be investigated, and is provided by Zhejiang University of Traditional Chinese Medicine's Chinese medicine preparation laboratory；Conventional liposome, by SIN-HCl, yolk lecithin
Fat, cholesterol, vitamin E, phosphate buffer composition, carry out pharmacokinetics investigation, by Zhejiang from the sample obtained by embodiment 5
River university of TCM Chinese medicine preparation laboratory provides；Ondansetron hydrochloride, Huangyan, Zhejiang macrobiosis pharmaceutical factory.
Experimental animal：Cleaning grade ICR mouse（35±5）G, male, tested by Zhejiang University of Traditional Chinese Medicine and provided diligently, permitted
Can the number of card：（Shanghai）2013-0016；Rabbit（2.0-2.5）Kg, male, Hangzhou Yuhang Ke Liantu industry Specialty Co-operative Organization.
1.2 method
1.2.1 skin microdialysis
Healthy ICR mouse 10 are taken, male, are randomly divided into two groups, every group 5, the h of fasting 12, free water before experiment.With 2%
（W/V）Yellow Jackets（20 mL·kg-1）After intraperitoneal injection of anesthesia, its four limbs is fixed, belly upward, is placed in 37-38
On DEG C heating cushion, mouse breastbone is removed to the hair of belly with shaver and elbow scissors.Using pull needle as guiding, microdialysis is visited
Pin inserts mouse part skin（Skin corium）Under, then pull needle is extracted, and probe effective film is determined after subcutaneous location, use is medical
Adhesive tape is fixed, and is 3.14 cm by effective area2, depth be 0.8 cm PA tube be fixed on mouse web portion with 502 glue
Adding medicine on skin.
After operation technique, 1 h is balanced with not drug containing ringer's solution perfusion, perfusion rate is 2 μ Lmin-1, every 30
Min collects 1 part of dialyzate, first collects 1 blank sample, then loading, applied sample amount are 720 μ L, and sample is uniformly applied into polypropylene
In pipe effective area, the skin microdialysis sampling time, totally 10 h, the sample collected were placed in automatic constant-temperature freezer unit.Thoroughly
It is as follows to analyse sample handling processes：60 μ L internal standard OND solution is added into 60 μ L microdialysis samples of collection（10 ng·mL-1）, it is abundant that 1 min that is vortexed mixes it, obtains solution A；20 μ L solution A is taken again, and the internal standard OND for adding 980 μ L thereto is molten
Liquid（5ng·mL-1）, solution A is obtained, for SIN concentration mensurations.
1.2.2 articular cavity microdialysis
Healthy rabbits 10 are taken, male, are randomly divided into two groups, every group 5, the h of fasting 12, free water before experiment.With 20%（W/
V）Urethane（4-5 mL·kg-1）After auricular vein injecting anesthetic, allow it to lie on one's side and four limbs are fixed, be placed in 37-38 DEG C of heating
On pad, rabbit knee and surrounding hair are removed with shaver and elbow scissors.With pull needle from shin bone node peak and kneecap
The ligamentum patellae both sides recess at lower edge line midpoint pierces through articular cavity for 45 ° obliquely（Feel obvious and break through sense）, will be micro-
Analysis probe penetrates at pull needle pin hole along same angle, extracts pull needle, then determines probe effective film at articular cavity position,
And fixed with medical adhesive tape.
After operation technique, 1 h is balanced with blank ringer's solution perfusion, perfusion rate is 2 μ Lmin-1, every 30 min
1 part of dialyzate is collected, first collects 1 blank sample, then loading, total applied sample amount are 720 μ L, are divided 6 times, every μ of 30 min loadings 120
L, sample is uniformly applied to knee joint position, the articular cavity microdialysis sampling time, totally 10 h, the sample collected were placed in
In automatic constant-temperature freezer unit.Sample handling processes of dialysing are as follows：Added into 60 μ L microdialysis samples of collection in 60 μ L
Mark OND solution（10 ng·mL-1）, it is abundant that 1 min that is vortexed mixes it, B solution is obtained, for SIN concentration mensurations.
1.2.3 Blood Microdialysis
Healthy rabbits 8 are taken, male, are randomly divided into two groups, every group 4, the h of fasting 12, free water before experiment.With 20%（W/
V）Urethane（4-5 mL·kg-1）After auricular vein injecting anesthetic, allow it to lie on one's side and four limbs are fixed, be placed in 37-38 DEG C of heating
On pad, wiping auricular vein with alcohol swab makes its expansion, extracts syringe needle after being pierced into auricular vein with syringe needle, tear is managed
Blood vessel is inserted along pin hole, the fixed-wing of hand-held Blood Microdialysis probe is inserted a probe into rapidly in tear pipe and pushed ahead, so
Carefully tear pipe is removed afterwards, probe activity film is infiltrated in blood, medical proof fabric is fixed, and finally uses shaver and elbow again
Cut off and remove rabbit knee and surrounding hair.In whole process, prevent probe from coming off, sliding, as rabbit has pain, struggle or fiber crops
When liquor-saturated effect will disappear, anaesthetic is supplemented as one sees fit.
After operation technique, 1 h is balanced with blank ringer's solution perfusion, perfusion rate is 2 μ Lmin-1, every 30 min
1 part of dialyzate to be collected, first collects 1 blank sample, then is administered, total dose is 720 μ L, is divided 6 times, every μ L of 30 min loadings 120,
Sample is uniformly applied to knee joint position, the Blood Microdialysis sampling time, totally 10 h, the sample collected were placed in automatically
In constant temperature chiller.Sample handling processes of dialysing are as follows：60 μ L internal standard OND is added into 60 μ L microdialysis samples of collection
Solution（10 ng·mL-1）, it is abundant that 1 min that is vortexed mixes it, C solution is obtained, for SIN concentration mensurations.
2. experimental result
Experimental result is shown, in skin pharmacokinetics, drug concentration fluctuation of the carrier with liposome in skin is smaller, and
Higher concentration is presented；Skin pharmacokinetics parameter shows, the AUC of carrier0-tAnd CssIt is 8.2,8.7 times of liposome respectively,
And MRT0-infIt is slightly shorter compared with liposome.Show that carrier can rely on its own advantage, promote medicine more efficiently to penetrate skin.
In articular cavity pharmacokinetics, carrier also fluctuates with drug concentration of the liposome in articular cavity without obvious peak valley, and small
Drug concentration in skin；Articular cavity pharmacokinetic parameters show, the AUC of carrier0-tAnd CssRespectively liposome 2.5,
2.1 times, and MRT0-infIt is slightly shorter compared with liposome.Illustrate compared with conventional liposome, carrier has certain synovial membrane targeting.
From blood concentration-time curve and pharmacokinetic parameters, on the one hand, carrier, the blood concentration of liposome（Css）Respectively less than
Its drug concentration in skin and articular cavity（Css）, illustrate that there is both of which articular cavity to be distributed targeting；On the other hand, two carry
The body magnitude relationship of drug concentration and their drug concentration magnitude relationships in skin and articular cavity in blood（Carrier is big
In liposome）On the contrary, the blood concentration i.e. after carrier administration（Css）And AUC0-tRespectively less than liposome --- the former AUC0-t
And CssThe 1/3 of the latter is each about, is further proved, compared with conventional liposome, synovial membrane targeting that carrier has.In addition,
Blood pharmacokinetic parameters are shown, compared with skin, articular cavity, medicine has longer MRT in blood0-inf, illustrate medicine to complete
The transhipment of body blood circulation system is less, it is possible to reduce the whole body toxic side effect of medicine, and then prove research system from another angle
Agent has Targeting distribution characteristic.
To sum up, by blood and skin, articular cavity Pharmacokinetic Results comparative analysis, we can be drawn to draw a conclusion：Give
After medicine, the drug concentration of carrier in skin is maximum, and articular cavity takes second place, minimum in blood（Only the 1/15 of articular cavity）, and
Though drug concentration of the liposome in skin, articular cavity and blood also presents and carrier identical trend, it is in blood
Drug concentration differ relatively small with articular cavity（About the 1/2 of articular cavity）, illustrate that carrier has certain synovial membrane targeting
Property；Compared with liposome, the MRT of carrier0-infShorter at skin, blood and articular cavity position, this is to a certain extent
Illustrate that carrier can rely on the advantage of itself, promote medicine to realize faster Transdermal absorption.Pharmacokinetic Results be shown in Table 3, table 4,
Table 5, Fig. 7, Fig. 8, Fig. 9.
Skin pharmacokinetic parameters after the mouse percutaneous dosing of table 3（n=5）
Articular cavity pharmacokinetic parameters after the rabbit percutaneous dosing of table 4（n=5）
Blood pharmacokinetic parameters after the rabbit percutaneous dosing of table 5（n=4）
3. experiment conclusion
After the skin pharmacokinetics of carrier and liposome, articular cavity pharmacokinetics and blood pharmacokinetics fully demonstrate PEG modifications
Sin-HCl carriers have good transdermal penetration characteristic and synovial membrane targeting, can reach the purpose of Synergy and attenuation.
Claims (6)
1. a kind of novel skin drug-delivery preparation for treating rheumatoid arthritis, it is characterised in that by following parts by weight into packet
Into：
SIN-HCl 80-120 parts, phosphatidase 2 50-350 parts, cholesterol 30-60 parts, volatile oil 15-50 parts, vitamin E 4-7 parts,
The 12-30 parts of DSPE-PEG 2000, buffer solution 15000-30000 parts, described volatile oil be eucalyptus oil, menthol, citral,
Mixture more than one or both of terpene volatile oil such as limonene, preferably by limonene and citral according to weight ratio
1:4~4:1 mixes, and more preferably limonene-citral Mixed chaotic sequences are by limonene and citral by weight 1:1 is mixed
Conjunction forms.
A kind of 2. novel skin drug-delivery preparation for treating rheumatoid arthritis as claimed in claim 1, it is characterised in that by
The composition composition of following parts by weight：
100 parts of SIN-HCl, 00 part of phosphatidase 3,50 parts of cholesterol, 20 parts of volatile oil, 5 parts of vitamin E, DSPE-PEG 2000
18 parts, 25000 parts of buffer solution.
A kind of 3. novel skin drug-delivery preparation for treating rheumatoid arthritis as claimed in claim 1 or 2, it is characterised in that
The one kind of described cushioning liquid in phosphate buffer, citrate buffer and carbonate buffer solution, preferable ph are
7.0 phosphate buffer solution.
4. a kind of preparation method for the novel skin drug-delivery preparation for treating rheumatoid arthritis as claimed in claim 1 or 2,
It is characterized in that comprise the following steps that：
（1）Phosphatide, cholesterol, volatile oil, vitamin E and the DSPE-PEG 2000 of formula ratio are weighed, is dissolved in absolute ethyl alcohol,
It is well mixed, obtain organic phase；
（2）SIN-HCl is dissolved in buffer solution, is well mixed, obtains aqueous phase；
（3）Under normal temperature condition, with micro syringe handle（1）In organic phase be added dropwise to（2）In aqueous phase in, this process
Carry out under agitation；
（4）After organic phase is added dropwise completely, continue to stir 5 min, obtain SIN-HCl PEG modification carrier suspensions, this is mixed
After suspension places 2-3 h, it is placed in liposome mini-extruder extrusion instrument, squeezes through 100 nm polycarbonate membranes, then squeeze through the poly- carbonic acid of 50 nm
Ester film, produce carrier sample.
5. a kind of preparation method for the novel skin drug-delivery preparation for treating rheumatoid arthritis as claimed in claim 4, its
It is characterised by described step（3）Middle mixing speed is 150-750 r/min, preferably 150,350,550,750 r/min, more
It is preferred that 550 r/min.
6. a kind of preparation method for the novel skin drug-delivery preparation for treating rheumatoid arthritis as claimed in claim 4, its
It is characterised by described step（4）Pressure used during middle carrier extrusion：The pressure for squeezing through 100 nm polycarbonate membranes is 0.10
MPa, the pressure for squeezing through 50 nm polycarbonate membranes are 0.38 MPa.
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|---|---|---|---|---|
| CA2507067A1 (en) * | 2002-11-28 | 2004-06-10 | Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences | Sinomenine and sinomenine compounds, synthesis and use |
| CN1660121A (en) * | 2004-12-27 | 2005-08-31 | 广州中医药大学 | Paste agent of penetrating through skin for treating arthritid and preparation method |
| CN102085182A (en) * | 2007-09-30 | 2011-06-08 | 湖南正清制药集团股份有限公司 | Sinomenine hydrochloride slow-release injection and preparation method thereof |
| CN104906042A (en) * | 2015-05-28 | 2015-09-16 | 浙江中医药大学 | Novel percutaneous drug delivery preparation for treating acute close traumatism of soft tissue and preparation method of preparation |
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2017
- 2017-09-21 CN CN201710859751.2A patent/CN107648179A/en active Pending
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| CA2507067A1 (en) * | 2002-11-28 | 2004-06-10 | Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences | Sinomenine and sinomenine compounds, synthesis and use |
| CN1660121A (en) * | 2004-12-27 | 2005-08-31 | 广州中医药大学 | Paste agent of penetrating through skin for treating arthritid and preparation method |
| CN102085182A (en) * | 2007-09-30 | 2011-06-08 | 湖南正清制药集团股份有限公司 | Sinomenine hydrochloride slow-release injection and preparation method thereof |
| CN104906042A (en) * | 2015-05-28 | 2015-09-16 | 浙江中医药大学 | Novel percutaneous drug delivery preparation for treating acute close traumatism of soft tissue and preparation method of preparation |
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