CN107049954A - A kind of drug regimen and its production and use - Google Patents
A kind of drug regimen and its production and use Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
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Abstract
The invention belongs to pharmaceutical technology field.The present invention relates to a kind of pharmaceutical composition, it includes active material, cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting substance.The invention further relates to a kind of method for the pharmaceutical composition for preparing the application, it includes：(a) cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting substance are dissolved in organic solvent；(b) active material is dissolved in the aqueous solution；(c) organic solvent for obtaining step (a) is mixed with the aqueous solution that step (b) is obtained.The present invention further relates to purposes of the pharmaceutical composition of the purposes and the present invention of oleic acid or linoleic acid in the Lung targeting for improving medicine in medicine is prepared.
Description
Technical field
The invention belongs to pharmaceutical technology field.It is related to a kind of pharmaceutical composition of lung's targeting, more particularly to a kind of lung
The cationic-liposome for being used to convey siRNA of targeting.
Background technology
Higher and in quick incremental development trend, the pulmonary infection in the PUD D incidence of disease such as China's lung cancer, pulmonary tuberculosis
It is very common and serious complication.In order to treat these diseases, substantial amounts of human and material resources and wealth has had been put into country
A series of power, and achieve in drug therapy field impressive progresses, but existing medicine be distributed in vivo it is big most without
Target selectivity, causes dosage big, cycle length and can produce serious toxicity and drug resistance.Therefore, lung is developed
The delivery system of targeting is the key for solving these problems.
Liposome (Liposome) is also known as vesica (Vesicle), is self-assembly of the lipoid molecule in aqueous phase.It contains
There are one or more lipid bilayers (Bilayer), in each double membrane structure, the hydrophobic part in lipoid molecule is by dredging
Aqueous phase interaction is combined together, and hydrophilic segment points to the aqueous phase in environment.Liposome is initially will by British scholar Bangham
Phosphatide is dispersed in water discovery when carrying out electron microscopic observation.Because liposome has the structure similar to cell membrane, therefore early stage
Liposome be used for cell membrane structure and function research.Gregoriadsi is equal to and is first used for liposome for 1971
The transhipment of bioactive substance.
Include natural quasi-ester and synthesis lipoid for preparing the lipoid molecule of liposome.It is using more in natural quasi-ester
Natural phospholipid, such as lecithin, cephalin etc.；Synthesis lipoid is mainly used for the lipoid molecule of gene transfer, such as DOTMA, DOPE,
DMRIE etc..In lipoid molecule it can be seen from the molecular structure of natural phosphatidyl choline and synthesis lipoid in addition to DC-Chol, point
The characteristics of minor structure has one jointly, i.e., be all made up of, most of class two hydrophobic tail chains and a hydrophilic head
Fat is to be chained up hydrophilic segment and hydrophobic part by ester bond, amido link, ehter bond etc. by the link part of glycerol backbone.
RNA interference (RNA interference, RNAi) is one kind by double-stranded RNA small molecule segment (small
Interfering RNAs, siRNA) cause silencing specific genes phenomenon with its homologous mRNA.Because RNAi has special, height
The features such as imitating and be lasting, it is studied and application turns into the focus in molecular biology research, while the technology is also to be related
The Gene intervention treatment of clinical disease opens a new way.
SiRNA is a relatively large molecule, and with strong negative electrical charge, hydrophily is high, it is impossible to directly through cell
Film enters cell.So, in order to for treating in PUD D, industry in the urgent need to a kind of efficient, low toxicity, high lung target
SiRNA transmission systems.
The content of the invention
It is an aspect of the invention to provide a kind of pharmaceutical composition, it includes active material, cationic phospholipid, poly- second two
The phosphatide and lung's targeting substance of alcohol modification.In some embodiments, the pharmaceutical composition of the application further comprises courage
Sterol.
Active material
Those skilled in the art can select suitable active material according to actual needs.
In some embodiments, the active material is used to alleviate, treat or prevent PUD D or illness.Alleviate,
The active material for treating or preventing PUD D or illness can be had been approved by various countries or regional Drug Administration
Be used for treat or prevent PUD D or the medicine of illness, such as Chinese food Drug Administration, U.S.'s food and medicine
Those of Surveillance Authority, Japanese Drug and medical instrument management office or European drugs administration approved.
Terminology used in the present invention " PUD D or illness " include it is known or known in future related to lung or
Disease or illness in lung occurs for person.In some embodiments, the PUD D or illness are pulmonary infection, tuberculosis
Or lung cancer.
In some embodiments, the active material is biomolecule, chemical molecular or its composition.In some implementations
In mode, the active material is single-stranded or double-stranded nucleic acid.In some embodiments, the length of the nucleic acid 5-200bp,
5-150bp、5-120bp、5-100bp、5-80bp、5-60bp、5-50bp、5-40bp、10-40bp、15-40bp、15-30bp、
Or 20-30bp.In some embodiments, the active material is siRNA.In some embodiments, the active material
It is the siRNA for needing lung to target, for example, treating or preventing the siRNA of PUD D.
In some embodiments, the siRNA of the application is the siRNA for alleviating, treating or preventing PUD D, its
The selectively targeted mRNA related to PUD D, by selective degradation mRNA so as to alleviate, treat or prevent PUD D.
By the technological means of this area, those skilled in the art can be found that related to alleviating, treating or preventing PUD D
MRNA, and design corresponding siRNA.
In some embodiments, the active material is to suppress Thorium Lung Burden secretion pulmonary surfactant
GAP-associated protein GAP A (pulmonary surfactantassociated protein, SPA) siRNA.In some embodiments
In, the active material is that have such as SEQ ID NO：SiRNA shown in 1 and 2, with such as SEQ ID NO：Shown in 3 and 4
SiRNA or with such as SEQ ID NO：SiRNA shown in 5 and 6.
Cation lipid
Such as invent the term " cation lipid " used and refer to that under the pH of selection (for example, physiological pH) carries arbitrary number
Net positive charge lipid.A variety of cation lipids were described in the literature, many of which is commercially available.
The cation lipid of the present invention includes dioleoyl propyl group chlorination trimethylammonium (" DOTMA "), the sweet ammonia of 5- carboxyspermine bases
Sour double octadecyl amides (" DOGS "), 1,2- dioleoyl epoxide -3- dimethylamino-propanes (" DODMA "), the sub- oil of 1,2- bis-
Base epoxide-N, N- dimethyl -3- aminopropanes (" DLinDMA "), sub-oleoyl epoxide-N, the N- dimethyl -3- amino of 1,2- bis-
Propane (" DLenDMA "), N- bis- oleyl-N, N- alkyl dimethyl ammonium chlorides (" DODAC "), didecyl Dimethy ammonium bromide
Oil base epoxide-the N- of (" DDAB "), 2,3- bis- [2 (spermine-carbamyl) ethyl]-N, the third ammoniums of N- dimethyl -1- (" DOSPA "),
1,2- dioleoyl -3- dimethylammonium-propanes (" DODAP "), dioleoyl trimethyl ammonium propane (" DOTAP "), 1,2- distearyls
Acyloxy-N, N- dimethyl -3- aminopropanes (" DSDMA "), N- (1,2- double myristyl epoxide propyl- 3- yls)-N, N- bis-
Methyl-N-hydroxy ethyl phosphonium bromide ammonium (" DMRIE "), 3- dimethylaminos -2- (cholesteric -5- alkene -3- β-epoxide butane -4- oxygen
Base) -1- (cis, cis-alkenyloxy groups of 9,12- 18) propane (" CLinDMA "), 2- [5 '-(cholesteric -5- alkene -3- β-oxygen
Base) -3 '-oxa- amoxy) -3- dimethyl -1- (it is cis, cis -9 ', the alkenyloxy groups of 1-2 '-ten eight or two) propane
(" CpLinDMA "), 1,2-N, the Asia oil base carbamyl -3- dimethylaminopropanecompounds of N '-two (" DLincarbDAP "), 1,2- bis-
Linolenyl carbamyl -3- dimethylaminopropanecompounds (" DLinCDAP "), N, the oil base epoxide benzylamine of N- dimethyl -3,4- bis-
(" DMOBA "), 1,2-N, the oil base carbamyl -3- dimethylaminopropanecompounds of N '-two (" DOcarbDAP "), flax acyls of 2,3- bis-
Base epoxide-N, N- dimethyl propylamine (" DLinDAP "), the Asias of 2,2- bis- oil base -4- dimethylaminomethyls-[1,3]-dioxolane
The Asia of (" DLin-K-DMA "), 2,2- bis- oil base -4- dimethyl aminoethyls-[1,3]-dioxolane (" DLin-K-XTC2-
DMA "), 2- (2,2- bis- ((9Z, 12Z)-ten eight -9,12- diene -1- bases)-DOX -4- bases)-N, N- dimethyl second
Amine (" DLin-KC2-DMA "), dimethylaminoethyl carbamyl-cholesterol (" DC-Chol "), Isosorbide-5-Nitrae-bis- (3-N- oleyl ammonia
Base-propyl group) piperazine or its mixture.
In some embodiments, the cation lipid is selected from didecyl Dimethy ammonium bromide (" DDAB "), two oil
Acyl trimethyl ammonium propane (" DOTAP "), 1,2- dioleoyl epoxide -3- dimethylamino-propanes (" DODMA "), dioleoyl propyl group
One or more in chlorination trimethylammonium (" DOTMA "), dimethylaminoethyl carbamyl-cholesterol (" DC-Chol ").One
In a little embodiments, the cation lipid is dioleoyl cation lipid.In some embodiments, the cationic lipid
Matter is DOTAP or DOTMA.
Polyethyleneglycol modified phosphatide
The term " polyethyleneglycol modified phosphatide " used is such as invented to refer to by the phosphorus of polyethylene glycol (" PEG ") covalent modification
Fat.A variety of polyethyleneglycol modified phosphatide were described in the literature, many of which is commercially available.
In some embodiments, the polyethyleneglycol modified phosphatide is to be by the length of polyethylene glycol covalent modification
The lipid of C6-C20 alkyl chain.In some embodiments, the polyethyleneglycol modified phosphatide is selected from polyethyleneglycol modified
DPPC (DPPC), 2- oleoyl -1- palm tin glycerol-3-phosphocholines (POPC), distearyl phosphatide
One kind or many in phatidylcholine (DSPC), DSPE (DSPE) and DOPC (DOPC)
Kind.In some embodiments, the polyethyleneglycol modified phosphatide is the ethanolamines phosphorus of polyethyleneglycol modified alkyl chain
Fat.In some embodiments, the polyethyleneglycol modified phosphatide is PEG-DSPE.
In some embodiments, the molecular weight of the polyethylene glycol higher than 100,200,300,400,500,600,700,
800th, 900,1000,1200,1500,1700 or 1900.In some embodiments, the molecular weight of the polyethylene glycol is less than
20000th, 15000,13000,10000,8000,7000,5000,4000,3000,2500 or 2200.In some embodiments,
The molecular weight of the polyethylene glycol is 100-20000,200-15000,300-13000,400-10000,500-8000,600-
7000th, 700-5000,800-4000,900-3000,1000-2500,1200-2200,1500-2200,1700-2200 or
1900-2200。
Lung's targeting substance
The present invention lung's targeting substance can be it is any can increase the material that pharmaceutical composition is distributed in lung, for example
Antibody, part, acceptor, small-molecule substance etc..
In some embodiments, lung's targeting substance is selected from oleic acid, linoleic acid, cholesterol acid ester, cholesterol
One or more in linoleate.In some embodiments, lung's targeting substance is oleic acid or linoleic acid.
In some embodiments, lung's targeting substance is mixed into medicine group as a component of pharmaceutical composition
In compound, but not with any other composition in pharmaceutical composition (for example, active material, cationic phospholipid or polyethylene glycol
The phosphatide of modification) it is covalently attached.
Oleic acid in the present invention is a kind of single unsaturation Omega-9 aliphatic acid, and molecular formula is CH3(CH2)7CH=CH (CH2)7COOH。
Linoleic acid in the present invention is a kind of aliphatic acid, chemical entitled linoleic acid, molecular formula
For CH3(CH2)4CH=CHCH2CH=CH (CH2)7COOH。
It is not wishing to be bound by theory, inventor is unexpected to be found to target by adding lung into pharmaceutical composition
Material (for example, oleic acid or linoleic acid) can greatly improve the distribution in lung after pharmaceutical composition is applied, and be controlled so as to improve
The effect of PUD D is treated or prevented, required dosage is effectively reduced.
Pharmaceutical composition
In some embodiments, the pharmaceutical composition of the application exists in the form of lipidic nanoparticles.In some realities
Apply in mode, the pharmaceutical composition of the application exists in the form of liposome.In some embodiments, the medicine group of the application
Compound exists in the form of cationic-liposome.In some embodiments, the pharmaceutical composition of the application is used for Formulations for systemic administration.
In some embodiments, the pharmaceutical composition of the application is used for intravenous administration.
In some embodiments, the pharmaceutical composition of the application include as active material siRNA, be used as cation
The DOTAP or DOTMA of lipid, the PEG-DSPE of polyethyleneglycol modified phosphatide, oleic acid or linoleic acid as Lung targeting material
And cholesterol.
In some embodiments, the mass ratio of the active material and cation lipid is 0.001: 1~1: 1.
In some embodiments, the mass ratio of the active material and cation lipid is 0.002: 1~1: 1,0.003: 1~1: 1,
0.004: 1~1: 1,0.005: 1~1: 1,0.01: 1~1: 1,0.02: 1~1: 1,0.03: 1~1: 1,0.04: 1~1: 1,
0.05: 1~1: 1,0.06: 1~1: 1,0.07: 1~1: 1,0.08: 1~1: 1,0.09: 1~1: 1,0.1: 1~1: 1,0.002:
1~0.8: 1,0.002: 1~0.5: 1,0.002: 1~0.4: 1,0.002: 1~0.3: 1,0.002: 1~0.2: 1,0.002: 1
~0.15: 1,0.002: 1~0.12: 1 or 0.002: 1~0.1: 1.In some embodiments, active material and cation are stated
The mass ratio of lipid is 0.05: 1~0.2: 1.
In some embodiments, the mol ratio of polyethyleneglycol modified phosphatide and cation lipid is 0.01: 1~1: 1.
In some embodiments, the mol ratio of polyethyleneglycol modified phosphatide and cation lipid is 0.01: 1~0.9: 1,0.01: 1
~0.8: 1,0.01: 1~0.7: 1,0.01: 1~0.6: 1,0.01: 1~0.5: 1,0.01: 1~0.4: 1,0.01: 1~0.3:
1st, 0.01: 1~0.2: 1,0.01: 1~0.1: 1,0.01: 1~0.08: 1,0.01: 1~0.06: 1,0.01: 1~0.05: 1,
0.02: 1~0.05: 1,0.03: 1~0.05: 1,0.04: 1~0.05: 1,0.02: 1~0.08: 1,0.02: 1~0.1: 1,
0.02: 1~0.2: 1,0.02: 1~0.3: 1,0.02: 1~0.4: 1 or 0.02: 1~0.5: 1.In some embodiments, gather
The phosphatide and the mol ratio of cation lipid that ethylene glycol is modified are 0.02: 1~0.08: 1.
In some embodiments, the mol ratio of lung's targeting substance and cation lipid is 0.2: 1~5: 1.At some
In embodiment, the mol ratio of lung's targeting substance and cation lipid is 0.2: 1~5: 1,0.2: 1~4: 1,0.2: 1~3:
1st, 0.2: 1~2: 1,0.2: 1~1: 1,0.2: 1~0.9: 1,0.3: 1~1: 1,0.4: 1~1: 1,0.5: 1~1: 1,0.6: 1~
1: 1,0.7: 1~1: 1 or 0.7: 1~0.9: 1.In some embodiments, mole of lung's targeting substance and cation lipid
Than for 0.4: 1~1: 1.
In some embodiments, the mol ratio of lung's targeting substance and polyethyleneglycol modified phosphatide is 0.01: 1~1:
1.In some embodiments, the mol ratio of lung's targeting substance and polyethyleneglycol modified phosphatide be 0.01: 1~0.9: 1,
0.01: 1~0.8: 1,0.01: 1~0.7: 1,0.01: 1~0.6: 1,0.01: 1~0.5: 1,0.01: 1~0.4: 1,0.01: 1
~0.3: 1,0.01: 1~0.2: 1,0.01: 1~0.1: 1,0.01: 1~0.08: 1,0.02: 1~0.08: 1,0.03: 1~
0.08: 1,0.04: 1~0.08: 1,0.05: 1~0.08: 1,0.06: 1~0.08: 1,0.02: 1~0.1: 1,0.02: 1~0.2
: 1,0.02: 1~0.3: 1,0.02: 1~0.4: 1 or 0.02: 1~0.5: 1.In some embodiments, lung's targeting substance with
The mol ratio of cation lipid is 0.03: 1~0.08: 1.
In some embodiments, the mol ratio of cholesterol and cation lipid is 0.1: 1~5: 1.In some embodiment party
In formula, the mol ratio of cholesterol and cation lipid is 0.1: 1~5: 1,0.1: 1~4: 1,0.1: 1~3: 1,0.1: 1~2: 1,
0.1: 1~1: 1,0.1: 1~0.9: 1,0.1: 1~0.8: 1,0.2: 1~0.8: 1,0.3: 1~0.8: 1,0.4: 1~0.8: 1,
0.5: 1~0.8: 1 or 0.5: 1~0.7: 1.In some embodiments, the mol ratio of cholesterol and cation lipid is 0.3: 1
~1: 1.
In some embodiments, the pharmaceutical composition of the application includes：Cationic phospholipid：1；Active material：0.001-
1；Polyethyleneglycol modified phosphatide：0.01-1；Lung's targeting substance：0.1-5；And cholesterol：(above-mentioned data are represented 0.2-5
The mol ratio of each component).
In some embodiments, the pharmaceutical composition of the application includes：Cationic phospholipid：1；Active material：0.1-
0.5；Polyethyleneglycol modified phosphatide：0.02-0.5；Lung's targeting substance：0.4-2；And cholesterol：0.3-1 (above-mentioned data
Represent the mol ratio of each component).
In some embodiments, the particle diameter of the pharmaceutical composition of the application is in 10-500nm.In some embodiments,
The particle diameter of the nano particle is in 50-200nm.In some embodiments, the particle diameter of the nano particle 10-400nm,
10-300nm、10-250nm、20-200nm、30-200nm、40-200nm、50-200nm、60-200nm、70-200nm、80-
200nm、90-200nm、100-200nm、110-200nm、120-200nm、130-200nm、140-200nm、150-200nm、
160-200nm, 50-300nm, 80-250nm or 100-200nm.In some embodiments, the particle diameter of the nano particle exists
110-200nm。
Method commonly used in the art can be used to measure particle diameter, such as ESEM method, light scattering method.In some embodiment party
In formula, particle diameter is detected using light scattering method.In some embodiments, particle diameter is detected using dynamic Laser scatterometer.
The application nanoparticle has the acceptable coefficient of dispersion.In some embodiments, the application nanoparticle is scattered
Coefficient is not more than 0.3,0.2,0.19 or 0.18.
Those skilled in the art know, the application composition further can be modified.In some embodiments
In, group (for example, antibody, part, specific substrates etc.) can be targetted or other in the surface modification of the application composition
Macromolecule improves the targeting or other kinetic parameters of application composition with further, or for entering to the application composition
Row spike.
Those skilled in the art know, in addition to mentioned component, and described pharmaceutical composition also includes pharmaceutically acceptable
Other compositions.In some embodiments, the other compositions include freeze drying protectant, including but not limited to lactose, sweet dew
Sugar, dextran, sucrose and glycine.In some embodiments, the other compositions include solution, including but not limited to chlorine
Change sodium solution, glucose solution, PBS or ethanol solution etc..
Term " pharmaceutically acceptable " used in this application refers to such compound, raw material, composition and/or agent
Type, they are in the range of rational medicine judgement, it is adaptable to anti-without excessive toxicity, excitant, metamorphosis with patient tissue contacts
Should or with the rational disproportionate other problemses of interests/Hazard ratio and complication, and effective for given application.
Beneficial effect
It is not wishing to be bound by theory, the pharmaceutical composition of the application has following one or more advantages：1st, whole body is given
After medicine there is excellent lung to target；2nd, particle diameter distribution evenly；3rd, stability is more excellent；4th, deeper into entrance tumour；
5th, higher drug effect.
Preparation method
It is another aspect of the invention to provide a kind of method for the pharmaceutical composition for preparing the application, it includes：(a) will
Cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting substance are dissolved in organic solvent；(b) active material is dissolved in
In the aqueous solution；(c) organic solvent for obtaining step (a) is mixed with the aqueous solution that step (b) is obtained.
In some embodiments, the present processes further comprise what step (d) dialysis or ultrafiltration step (c) were obtained
Mixture.
In some embodiments, the step of the application (a) further comprises the aqueous solution injection without active material
Step into organic solvent.
In some embodiments, the organic solvent in step (a) is ethanol.
In some embodiments, the aqueous solvent in step (b) is phosphate buffer, citrate buffer solution or contained
The DEPC aqueous solution.
In some embodiments, the ratio for the aqueous solution that the organic solvent and step (b) that step (a) is obtained are obtained is 1:
10~1: 1.In some embodiments, the ratio for the aqueous solution that the organic solvent and step (b) that step (a) is obtained are obtained is 1:
9~1: 1,1: 8~1: 1,1: 7~1: 1,1: 6~1: 1,1: 6~1: 2 or 1: 6~1: 3.In some embodiments, step (a)
The ratio for the aqueous solution that the organic solvent and step (b) of acquisition are obtained is 1: 5~1: 3.
Preparing the illustrative methods of the pharmaceutical composition of the application includes：
Dialysis：Cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting substance are dissolved separately in ethanol
In, lipid mixture ethanol solution is mixed to get in proportion.Then the buffer solution containing active material is injected into lipid mixed solution
In, vortex mixed 2 seconds；The liquid room temperature that is mixed to get is static to be put 10 minutes, and then mixed solution is transferred in dialysis tubing,
Dialysed in HEPES cushioning liquid 1~2 hour and form cationic-liposome.
Physical mixed method：Cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting substance are dissolved separately in
In ethanol, lipid mixture ethanol solution is mixed to get in proportion.Then plain buffer is injected in lipid mixed solution, mixed
Close uniform, obtain blank liposomes liquid solution.Blank liposomes liquid solution is added in the buffer solution containing active material with liquid-transfering gun,
Gently blow and beat, mix；The liquid room temperature that is mixed to get is static to put 20 minutes, obtains cationic-liposome.
Ultrafiltration：Cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting substance are dissolved separately in ethanol
In, lipid mixture ethanol solution is mixed to get in proportion.Then the buffer solution containing active material is injected into lipid mixed solution
In, vortex mixed 2 seconds；The liquid room temperature that is mixed to get is static to put 10 minutes, is placed in after taking-up in super filter tube, centrifugal ultrafiltration
(4000 revs/min, 15 minutes), cationic-liposome is obtained after concentration.
Purposes in Lung targeting
Another aspect of the invention be to provide purposes in the Lung targeting for improving medicine of oleic acid or linoleic acid or
Purposes of the person in Lung targeting composition is prepared.
In some embodiments, compared with being added without the composition of oleic acid or linoleic identical prescription, Lung targeting
Effect improve at least 1%, 5%, 10%, 15%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%th, 300%, 400% or 500%.
Pharmaceutical applications, methods for the treatment of diseases and therapeutical uses
Another aspect of the present invention is purposes of the pharmaceutical composition for providing the application in medicine is prepared.
The application another further aspect is related to use of the pharmaceutical composition of the application for alleviating, treating or preventing disease or illness
On the way.
On the other hand the application is related to a kind of method alleviated, treat or prevent disease or illness, including to needing its
Object uses the pharmaceutical composition of the application of effective dose.
" alleviation ", " treatment " or " prevention " to certain disease or symptom includes prevention or mitigates certain situation, reduces certain
The situation of kind is risen or the speed of development, reduces the risk for developing certain situation, prevention or the delay disease related to certain situation
Shape develops, and reduces or terminates the symptom related to certain situation, produce the complete or partial reverse of certain situation, cure certain
Situation, or more combination.
Term " effective dose " used herein refers to, it is possible to achieve the disease or symptom of object, or can prevent
Property suppress prevent disease or symptom occur medicine amount.Effective dose can be by one or more diseases of object or disease
Shape alleviates the amount of medicine to a certain extent；Can be by those with disease or the related one or more physiology of the symptom origin cause of formation or life
Thing chemistry argument section or the amount for being completely recovered to normal medicine；And/or the possibility that disease or symptom occur can be reduced
Medicine amount.
The effective dose of composition provided herein depends on many factors well known in the art, such as body weight, year
Age, passing medical history, the treatment currently received, the intensity of the health status of object and drug interaction, allergy, it is super quick and
Side effect, and method of administration and the degree of disease development.One skilled in the art (such as doctor or animal doctor) can be according to these
Or other conditions or the corresponding reduction of requirement or rise dosage.
In some embodiments, the composition that the application is provided can be in treatment effective dose about 0.01mg/kg to about
Administration is (for example, about 0.01mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 5mg/kg, about between 100g/kg
10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/
Kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about
85mg/kg, about 90mg/kg, about 95mg/kg, about 100mg/kg, about 200mg/kg, about 500mg/kg, about 1g/kg, about 5g/kg,
About 10g/kg, about 20g/kg, about 50g/kg, about 70g/kg, about 90g/kg or about 100g/kg).A certain given dose can be divided into many
Minor tick is administered, for example once a day, twice daily or more, monthly twice or more, once in a week, once every two weeks, often
Once in three weeks, monthly or every two months or more the moons once.In some embodiments, dosage can become with treatment process
Change.For example, in some embodiments, initial dosages are high than subsequent dose dosage.In some embodiments, it is administered
Dosage is adjusted in treatment process according to the reaction of administration object.
Dosage regimen can be optimal reaction (for example, treatment is responded) by adjustment.For example, can carry out single dose administration or
In the dosage administration of a period of time point multiple separations.
In some embodiments, the object of the application is mammal, such as rat, mouse, rabbit, chicken, pig.One
In a little embodiments, the object is non-human mammal.In some embodiments, the object is people.
Brief description of the drawings
The grain size distribution of siRNA cationic-liposomes prepared by Fig. 1 embodiments 1 and embodiment 2.
The grain size distribution of siRNA cationic-liposomes prepared by Fig. 2 embodiments 3-6.
Fig. 3 determines obtained naked siRNA, QH-10 and QH-9 living imaging figure and mouse tissue fluorescence according to embodiment 7
Intensity histograms.
Fig. 4 determined according to embodiment 7 obtained QH-9, QH-9-2, QH-16, QH-17 and QH-18 living imaging figure and
Mouse tissue fluorescence intensity block diagram.
Fig. 5 determines obtained cell transfecting effect fluorescence results figure according to embodiment 8.
Fig. 6 determines obtained fluorescent quantitative PCR curve map according to embodiment 9.
Embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.Following examples will be helpful to this area
Technical staff further understands the present invention, but the invention is not limited in any way.
Embodiment 1：Dialysis prepares siRNA cationic-liposomes (QH-9)
Weigh dioleoyl trimethyl ammonium propane (DOTAP) 11.03mg, linoleic acid 3.52mg, cholesterol 3.66mg, distearyl
Acyl phosphatidyl-ethanolamine-polyethylene glycol 2000 (PEG2000- DSPE) 1.79mg, it is dissolved in 1.0ml absolute ethyl alcohols and is made into phosphatide second
Alcoholic solution (20mg/ml).By siRNA with being made into the siRNA solution that concentration is 10mg/ml in DEPC water.Used in embodiment 1-6
SiRNA be adjust VEGF siRNA.
Prepare two 2ml centrifuge tubes, managed labeled as A pipes and B.45 μ l phosphatide ethanol solutions are added into A pipes with liquid-transfering gun,
Add 105 μ l absolute ethyl alcohols and be diluted to suitable concn.350 μ l DEPC water are added in B pipes, 5 μ l siRNA solution are added
(10mg/ml) obtains 355 μ l liquid.The siRNA solution in B pipes is added in A pipes with liquid-transfering gun, gently piping and druming 10 times, slightly
Micro-vortex is mixed 2 seconds.The liquid room temperature that is mixed to get is static to be put 10 minutes, and mixed solution then is transferred into 15ml dialysis tubings
In, dialysed 1~2 hour in HEPES cushioning liquid, the filter membrane of subsequent 0.22 μm of mistake is degerming.Resulting liposome code name is
QH-9。
Detected, cation using granularity potentiometer (Malvern nano particle size potentiometer, ZetasizerNano ZS)
Liposome QH-9 average grain diameter is in 156.00 ± 6.3nm scopes, and the coefficient of dispersion is 0.19, and current potential is in 35.97 ± 2.2mV models
Enclose, particle diameter distribution result is as shown in Figure 1.
Embodiment 2：Physical mixed method prepares siRNA cationic-liposomes (QH-9-2)
Weigh dioleoyl trimethyl ammonium propane (DOTAP) 11.03mg, linoleic acid 3.52mg, cholesterol 3.66mg, distearyl
Acyl phosphatidyl-ethanolamine-polyethylene glycol 2000 (PEG2000-DSPE) 1.79mg, is dissolved in 1.0ml absolute ethyl alcohols and is made into phosphatide second
Alcoholic solution (20mg/ml).By siRNA with being made into the siRNA solution that concentration is 3mg/ml in DEPC water.
Prepare two 2ml centrifuge tubes, managed labeled as A pipes and B.2000 μ l DEPC water are added into A pipes with liquid-transfering gun, then
22.5 μ l phosphatide ethanol solutions are added with injection needle tube, is well mixed, obtains blank liposomes liquid solution.With liquid-transfering gun into B pipes
10 μ l siRNA solution are added, then the blank liposomes liquid solution in A pipes is added in pipe B, is blown and beaten, obtained repeatedly with liquid-transfering gun
Product room temperature it is static put 20 minutes, the filter membrane of subsequent 0.22 μm of mistake is degerming.Resulting liposome code name is QH-9-2.
Detected, cation using granularity potentiometer (Malvern nano particle size potentiometer, Zetasizer Nano ZS)
Liposome QH-9-2 average grain diameter is in 192.1 ± 0.13nm scopes, and the coefficient of dispersion is 0.13, and current potential is in 34.1 ± 2.8mV models
Enclose, particle diameter distribution result is as shown in Figure 1.
Embodiment 3：The siRNA cationic-liposomes (QH-16) of other prescriptions
Weigh dioleoyl trimethyl ammonium propane (DOTAP) 11.00mg, linoleic acid 3.26mg, cholesterol 3.62mg, distearyl
Acyl phosphatidyl-ethanolamine-polyethylene glycol 2000 (PEG2000-DSPE) 2.13mg, is dissolved in 1.0ml absolute ethyl alcohols and is made into phosphatide second
Alcoholic solution (20mg/ml).By siRNA with being made into the siRNA solution that concentration is 10mg/ml in DEPC water.
Prepare two 2ml centrifuge tubes, managed labeled as A pipes and B.45 μ l phosphatide ethanol solutions are added into A pipes with liquid-transfering gun,
Add 105 μ l absolute ethyl alcohols and be diluted to suitable concn.5 μ l siRNA solution are added in B pipes, 300 μ l DEPC are then added
Water is diluted to suitable concn.The siRNA solution in B pipes is added in A pipes with liquid-transfering gun, gently blows and beats 10 times, is somewhat vortexed
Mixing 2 seconds.The liquid room temperature that is mixed to get is static to put 10 minutes, and the liquid room temperature lucifuge being mixed to get is static to put 10 minutes, so
Mixed solution is transferred in 15ml dialysis tubings afterwards, lucifuge is dialysed 1~2 hour in HEPES cushioning liquid, subsequent 0.22 μm of mistake
Filter membrane it is degerming.Resulting liposome code name is QH-16.Using granularity potentiometer (Malvern nano particle size potentiometer,
Zetasizer Nano ZS) detected, cationic-liposome QH-16 average grain diameter is divided in 144.63 ± 8.4nm scopes
It is 0.14 to dissipate coefficient, and current potential is in 36.57 ± 3.1mV scopes, and particle diameter distribution result is as shown in Figure 2.
Embodiment 4：The siRNA cationic-liposomes (QH-17) of other prescriptions
Weigh dioleoyl trimethyl ammonium propane (DOTAP) 11.16mg, oleic acid 3.15mg, cholesterol 3.59mg, distearyl
Phosphatidyl-ethanolamine-polyethylene glycol 2000 (PEG2000- DSPE) 2.11mg, it is dissolved in 1.0ml absolute ethyl alcohols and is made into phosphatide ethanol
Solution (20mg/ml).By siRNA with being made into the siRNA solution that concentration is 10mg/ml in DEPC water.
Prepare two 2ml centrifuge tubes, managed labeled as A pipes and B.45 μ l phosphatide ethanol solutions are added into A pipes with liquid-transfering gun,
Add 105 μ l absolute ethyl alcohols and be diluted to suitable concn.5 μ l siRNA solution are added in B pipes, 350 μ l DEPC are then added
Water is diluted to suitable concn.The siRNA solution in B pipes is added in A pipes with liquid-transfering gun, gently blows and beats 10 times, is somewhat vortexed
Mixing 2 seconds.The liquid room temperature that is mixed to get is static to be put 10 minutes, and mixed solution is transferred in 15ml dialysis tubings, slow in HEPES
Rush lucifuge in solution to dialyse 1~2 hour, the filter membrane of subsequent 0.22 μm of mistake is degerming.Resulting liposome code name is QH-17.
Detected, cation using granularity potentiometer (Malvern nano particle size potentiometer, ZetasizerNano ZS)
Liposome QH-17 average grain diameter is in 181.83 ± 4.1nm scopes, and the coefficient of dispersion is 0.15, and current potential is 38.6 ± 3.9mV.Grain
Footpath distribution results are as shown in Figure 2.
Embodiment 5：The siRNA cationic-liposomes (QH-18) of other prescriptions
1,2- dioleoyls epoxide -3- dimethylamino-propanes (DODMA) 11.97mg, cholesterol 3.68mg is weighed, two is hard
Acyl phosphatidyl-ethanolamine-polyethylene glycol (PEG2000-DSPE) 2000 2.16mg, linoleic acid 3.2mg, is dissolved in 1.0ml anhydrous
Phosphatide ethanol solution (20mg/ml) is made into ethanol.SiRNA is molten with the siRNA that concentration is 10mg/ml is made into DEPC water
Liquid.
Prepare two 2ml centrifuge tubes, managed labeled as A pipes and B.45 μ l phosphatide ethanol solutions are added into A pipes with liquid-transfering gun,
Add 105 μ l absolute ethyl alcohols and be diluted to suitable concn.5 μ l siRNA solution are added in B pipes, 350 μ l DEPC are then added
Water is diluted to suitable concn.The siRNA solution in B pipes is added in A pipes with liquid-transfering gun, gently blows and beats 10 times, is somewhat vortexed
Mixing 2 seconds.The liquid room temperature that is mixed to get is static to be put 10 minutes, and the liquid being mixed to get is transferred in 15ml dialysis tubings,
Lucifuge is dialysed 1~2 hour in HEPES cushioning liquid, and the filter membrane of subsequent 0.22 μm of mistake is degerming.Resulting liposome code name is
QH-18。
Detected, cation using granularity potentiometer (Malvern nano particle size potentiometer, ZetasizerNano ZS)
Liposome QH-16 average grain diameter is in 138.2 ± 5.4nm scopes, and the coefficient of dispersion is 0.16, current potential in 40.03 ± 21mV scopes,
Particle diameter distribution result is as shown in Figure 2.
Embodiment 6：Do not include the siRNA cationic-liposomes (QH-10) of lung's targeting substance
Weigh dioleoyl trimethyl ammonium propane (DOTAP) 11.03mg, cholesterol 3.52mg, distearoylphosphatidyl ethanol
Amine-polyethylene glycol 2000 (PEG2000- DSPE) 3.67mg, it is dissolved in 1.0ml absolute ethyl alcohols and is made into phosphatide ethanol solution (20mg/
ml).By siRNA with being made into the siRNA solution that concentration is 10mg/ml in DEPC water.
Prepare two 2ml centrifuge tubes, managed labeled as A pipes and B.45 μ l phosphatide ethanol solutions are added into A pipes with liquid-transfering gun,
Add 105 μ l absolute ethyl alcohols and be diluted to suitable concn.5 μ l siRNA solution are added in B pipes, 350 μ l DEPC are then added
Water is diluted to suitable concn.The siRNA solution in B pipes is added in A pipes with liquid-transfering gun, gently blows and beats 10 times, is somewhat vortexed
Mixing 2 seconds.The liquid room temperature that is mixed to get is static to be put 10 minutes, and the liquid being mixed to get is transferred in 15ml dialysis tubings,
Lucifuge is dialysed 1~2 hour in HEPES cushioning liquid, and the filter membrane of subsequent 0.22 μm of mistake is degerming.Resulting liposome code name is
QH-10。
Detected, cation using granularity potentiometer (Malvern nano particle size potentiometer, ZetasizerNano ZS)
The average grain diameter of liposome is in 215.33 ± 7.2m scopes, and the coefficient of dispersion is 0.23, and current potential is 51.2 ± 2.8mV, particle diameter distribution
As a result it is as shown in Figure 2.
Embodiment 7：The living imaging experiment of Cy5-siRNA cationic-liposomes
5 μ l siRNA solution (10mg/ml) in embodiment 1 and 6 are replaced with 6 μ l Cy5-siRNA (10mg/ml), made
Standby Cy5-siRNA cationic-liposomes.The ICR mouse 12 of health are taken, are divided into 3 groups, respectively the μ l Cy5- of tail vein injection 200
SiRNA cationic-liposomes QH-9,200 μ l Cy5-siRNA cationic-liposomes QH-10 and the 200 naked Cy5-siRNA of μ l is (dense
Spend 60 μ g/ml), the heart, liver, spleen, lung, kidney, brain tissue are won immediately within 4 hours, carry out living imaging instrument fluorescence and take pictures upon administration
(Japanese Shimadzu Corporation, Clairvivo OPT).The excitation wavelength that fluorescence is taken pictures is 658nm, and the time for exposure is 1s.According to fluorescence
Intensity determines the fluorescence distribution situation of internal internal organs, and obtained mouse tissue living imaging photo and fluorescence intensity data does column
Distribution map, as shown in Figure 3.
5 μ l siRNA solution (10mg/ml) in Examples 1 and 2-5 are replaced with 6 μ l Cy5-siRNA (10mg/ml),
Prepare Cy5-siRNA cationic-liposomes.The ICR mouse 15 of health are taken, are divided into 5 groups, respectively the μ l of tail vein injection 200
Cy5-siRNA cation lipid liquid solutions, win the heart, liver, spleen, lung, kidney, brain tissue in 4 hours upon administration, carry out live body immediately
Imager fluorescence is taken pictures (Japanese Shimadzu Corporation, Clairvivo OPT).The excitation wavelength that fluorescence is taken pictures is 658nm, time for exposure
For 1s.The fluorescence distribution situation of internal internal organs is determined according to fluorescence intensity, obtained mouse tissue living imaging photo and fluorescence
Intensity data does column distribution map, as shown in Figure 4.
Embodiment 8：The checking of siRNA in-vitro transfections
In order to verify the siRNA gene transfections of cationic-liposome, using mouse Thorium Lung Burden and
Fluorescein-labeled siRNA is used as appraisement system.Cell is inoculated with：Day before transfection inoculating cell, per the μ L culture mediums of hole 500, no
Antibody can be added, make cell transfection when density in 30-50%.
SiRNA-FAM is wrapped up using the method for embodiment 2, the culture of the cell cultivated containing 0.5mL is then added it to
Kong Zhong.Culture plate is gently rocked, is mixed.37 DEG C are cultivated 18 hours, with micro- sem observation fluorescence.Cell transfecting effect fluorescence results
See Fig. 5.
Embodiment 9：Carry the gene silencing checking of siRNA cationic-liposome
Three kinds of siRNA have been synthesized, have compared its disturbed condition to Thorium Lung Burden SP secretion A.
siRNA1：Sequence (5 ' to 3 ')
GCUUCAGACUGCACUCUACTT
GUAGAGUGCAGUCUGAAGCTT
siRNA2：Sequence (5 ' to 3 ')
CCAAUGGGCAGUCAGUCAATT
UUGACUGACUGCCCAUUGGTT
SiRNA3：Sequence (5 ' to 3 ')
CCAUCGCAAGCAUUACAAATT
UUUGUAAUGCUUGCGAUGGTT
Cell is inoculated with：Day before transfection Mice Inoculated Thorium Lung Burden cell, can not per the μ L culture mediums of hole 500
Plus antibody, make cell transfection when density in 30-50%.
SiRNA-FAM is wrapped up using the method for embodiment 2, the culture of the cell cultivated containing 0.5mL is then added it to
Kong Zhong.Culture plate is gently rocked, is mixed.37 DEG C are cultivated 48 hours, are collected cell detection and are suppressed efficiency.
The cell extraction RNA of collection is taken, the expression quantity of SPA genes, fluorescent quantitation are detected by the method for real-time quantitative PCR
PCR amplification curves such as Fig. 6.As a result show, three siRNA are contained by cation lipid can suppress the expression of SPA genes,
Wherein siRNA2 suppresses efficiency more a height of 55% (table 1).
Table 1
| GAPDH | SPA | ΔCT | ΔΔCT | 2-ΔΔCT | Suppress efficiency | |
| Blank group | 18.55 | 24.62 | 6.07 | 0 | 1 | 0 |
| siRNA1 | 19.52 | 26.59 | 7.07 | 1.00 | 0.50 | 50% |
| siRNA2 | 19.02 | 26.23 | 7.21 | 1.14 | 0.45 | 55% |
| siRNA3 | 17.95 | 24.92 | 6.97 | 0.90 | 0.54 | 46% |
Claims (21)
1. a kind of pharmaceutical composition, it includes active material, cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting
Material.
2. pharmaceutical composition as claimed in claim 1, it exists in the form of liposome；Optionally, it is with cation lipid
The form of body is present.
3. pharmaceutical composition as claimed in claim 1, wherein lung's targeting substance is selected from oleic acid, linoleic acid, cholesterol
One or more in oleate, cholesterol linoleate；Optionally, lung's targeting substance is oleic acid or linoleic acid.
4. pharmaceutical composition as claimed in claim 1, wherein the active material is used to alleviate, treat or prevent PUD D
Or illness；Optionally, the PUD D is selected from pulmonary infection, tuberculosis and lung cancer；Optionally, the active material is biological
Molecule, chemical molecular or its composition；Optionally, the active material is single-stranded or double-stranded nucleic acid；Optionally, the nucleic acid
Length is in 5-200bp, 5-150bp, 5-120bp, 5-100bp, 5-80bp, 5-60bp, 5-50bp, 5-40bp, 10-40bp, 15-
40bp, 15-30bp or 20-30bp；Optionally, the active material is siRNA.
5. pharmaceutical composition as claimed in claim 1, wherein the cation lipid is selected from didecyl Dimethy ammonium bromide
(DDAB), dioleoyl trimethyl ammonium propane (DOTAP), 1,2- dioleoyl epoxide -3- dimethylamino-propanes (DODMA), two oil
One or more in acyl propyl group chlorination trimethylammonium (DOTMA), dimethylaminoethyl carbamyl-cholesterol (DC-Chol)；Can
Choosing, the cation lipid is dioleoyl cation lipid；Optionally, the cation lipid is DOTAP or DOTMA.
6. pharmaceutical composition as claimed in claim 1, wherein the polyethyleneglycol modified phosphatide is selected from polyethyleneglycol modified
DPPC (DPPC), 2- oleoyl -1- palm tin glycerol-3-phosphocholines (POPC), distearyl phosphatide
One kind or many in phatidylcholine (DSPC), DSPE (DSPE) and DOPC (DOPC)
Kind；Optionally, the polyethyleneglycol modified phosphatide is PEG-DSPE.
7. pharmaceutical composition as claimed in claim 1, it further comprises cholesterol.
8. pharmaceutical composition as claimed in claim 1, it includes siRNA as active material, as cation lipid
DOTAP or DOTMA, the PEG-DSPE of polyethyleneglycol modified phosphatide, oleic acid or linoleic acid and courage as Lung targeting material
Sterol.
9. pharmaceutical composition as claimed in claim 1, wherein the mass ratio of the active material and cation lipid is
0.001: 1~1: 1, optionally, ratio is 0.05: 1~0.2: 1.
10. pharmaceutical composition as claimed in claim 1, wherein polyethyleneglycol modified phosphatide and the mol ratio of cation lipid
For 0.01: 1~1: 1；Optionally, ratio is 0.02: 1~0.08: 1.
11. pharmaceutical composition as claimed in claim 1, the wherein mol ratio of lung's targeting substance and cation lipid are 0.2:
1~5: 1；Optionally, ratio is 0.4: 1~2: 1.
12. the mol ratio of pharmaceutical composition as claimed in claim 7, wherein cholesterol and cation lipid is 0.1: 1~5:
1；Optionally, ratio is 0.3: 1~1: 1.
13. pharmaceutical composition as claimed in claim 1, it includes：
Cationic phospholipid：1；
Active material：0.001-1；
Polyethyleneglycol modified phosphatide：0.01-1；
Lung's targeting substance：0.1-5；And
Cholesterol：0.2-5.
Above-mentioned data represent the mol ratio of each component.
14. pharmaceutical composition as claimed in claim 1, it includes：
Cationic phospholipid：1；
Active material：0.1-0.5；
Polyethyleneglycol modified phosphatide：0.02-0.5；
Lung's targeting substance：0.4-2；And
Cholesterol：0.3-1.
Above-mentioned data represent the mol ratio of each component.
15. a kind of method for preparing the pharmaceutical composition any one of claim 1-14, it includes：
(a) cationic phospholipid, polyethyleneglycol modified phosphatide and lung's targeting substance are dissolved in organic solvent；
(b) active material is dissolved in the aqueous solution；
(c) organic solvent for obtaining step (a) is mixed with the aqueous solution that step (b) is obtained.
16. the method according to preparing claim 15, it further comprises that step (d) dialysis or ultrafiltration step (c) are obtained
Mixture.
17. the method according to preparing claim 15, the wherein organic solvent in step (a) is ethanol.
18. the method according to preparing claim 15, the wherein aqueous solvent in step (b) is phosphate buffer, lemon
Acid buffer or the aqueous solution containing DEPC.
19. the organic solvent that the method according to preparing claim 15, wherein step (a) are obtained is obtained with step (b)
The ratio of the aqueous solution is 1: 10~1: 1；Optionally, ratio is 1: 5~1: 3.
20. the purposes of oleic acid or linoleic acid in the Lung targeting for improving medicine.
21. purposes of the pharmaceutical composition any one of claim 1-14 in medicine is prepared.
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