CN102579437B - Tacrolimus composition containing alcohol and preparation method of tacrolimus composition - Google Patents
Tacrolimus composition containing alcohol and preparation method of tacrolimus composition Download PDFInfo
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- CN102579437B CN102579437B CN201110331382.2A CN201110331382A CN102579437B CN 102579437 B CN102579437 B CN 102579437B CN 201110331382 A CN201110331382 A CN 201110331382A CN 102579437 B CN102579437 B CN 102579437B
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Abstract
The invention discloses a tacrolimus composition containing alcohol and a preparation method of the tacrolimus composition. The tacrolimus composition disclosed by the invention comprises 0.001 to 0.05 wt% of tacrolimus, 0.2 to 10 wt% of phospholipid, 10 to 60 wt% of alcohol, 0 to 0.5 wt% of cholesterol, and the balance of water. The tacrolimus composition which is provided by the invention and can be used for local skin is an ethosome-type preparation, and not only can overcome the defects of unpleasant viscosity and greasiness which are generated by greasing bases of commercially available ointment and difficulty in cleaning of the commercially available ointment, but also can improve the release rate of medicaments and increase retention of the medicaments in the skin, so that the curative effect is strengthened.
Description
Technical field
The present invention relates to a kind of compositions of the tacrolimus that contains alcohol, and their preparation method.The alcohol composition that contains of novelty of the present invention can be used as local skin drug-supplying system, for example, can be used as ethosome preparation for percutaneous drug delivery.
Background technology
Atopic dermatitis (atopic dermatitis, AD, also referred to as atopic eczema) be that a kind of chronic, recurrent of take that violent pruritus is feature, inflammatory skin are sick, its cause of disease and pathogenesis are still not clear, but generally believe relevant with immunologic dysfunction.Owing to lacking specific treatment means, larger on patients ' life quality impact, and its sickness rate rising, therefore become study hotspot in recent years.Over nearly 40 years, external corticosteroid has become the primary treatment method of AD, but there is obvious limitation in this class Drug therapy AD, prolonged application easily causes the untoward reaction such as atrophoderma attenuation, telangiectasis, thereby only limit to short term therapy, and hormone preparation can not be for face and cervical region.Therefore in the urgent need to a kind of non-cortical steroid, and medicine safely and effectively.
Tacrolimus (Tacrolimus, FK506), commodity are called Prograf (PullockReusable), it is the potent immunosuppressant of a kind of Macrolide that Japanese Teng Ze group (Fujisawa) was isolated from the fermentation medium of tsukubaensis streptomycete (the fungi antibacterial of a kind of class, is found in soil sample by Tsukuba) in 1984.The immunosuppressive action of tacrolimus is stronger 10～100 times than ciclosporin A (CsA), thereby greatly reduces clinical using dosage, and untoward reaction simultaneously also obviously reduces.
Tacrolimus ointment (tacrolimus ointment, trade name: Pood that) be the whole world first granted non-cortical steroid external immunomodulator.In December, 2000 U.S. FDA approval 0.03% and 0.1% tacrolimus ointment goes on the market in the U.S., and in April, 2005 is in Discussion on Chinese Listed.The indication of tacrolimus ointment because of potential danger should not use hormone therapy or not good enough to hormone therapy curative effect, maybe cannot tolerate in to severe atopic dermatitis patients, as short-term or intermittent long-term treatment.Wherein, 0.03% tacrolimus ointment is approved for 2 years old and above child and AD in adults patient, and 0.1% tacrolimus ointment is approved for AD in adults patient.
At present, gone on the market and only had above-mentioned ointment a kind of for clinical tacrolimus external preparation.According to relevant bibliographical information, because this ointment is greasing base, therefore there is viscosity beastly and greasy, and be not easy to clean, the more important thing is ointment base characteristic limitations medicine from release wherein, affected the timely performance (Kudla RM.Topical ointment.US 4279901) of drug effect.
With regard to tacrolimus, existing product is still oleaginous base ointment, and release of the proper property of product such as medicine, absorption etc. having no are to some extent taken on a new look.Therefore, this area still needs to have and is suitable for tacrolimus administration particularly by the compositions of percutaneous drug delivery, especially can avoid using oleaginous base and be desirably in the compositions that there is change the aspects such as release such as medicine, absorption.
Summary of the invention
The object of the present invention is to provide a kind of tacrolimus compositions for local skin, to overcome at least one item of the following shortcoming of existing tacrolimus ointment: 1) greasing base, there is viscosity beastly and greasy, and be not easy to clean; 2) characteristic limitations of ointment base medicine from release wherein, affected the timely performance of drug effect.The inventor finds, take water as substrate, makes tacrolimus and phospholipid and alcohol combination, and the aqueous liquid composition of acquisition can overcome at least one above-mentioned shortcoming effectively.The present invention is based on this discovery and be accomplished.
Summary of the invention:
First aspect present invention provides a kind of compositions of tacrolimus, wherein comprises the water of 0.001～0.5wt% tacrolimus, 0.2～10wt% phospholipid, 10～60wt% alcohol, 0～0.5wt% cholesterol and surplus.
Second aspect present invention provides the method for preparing compositions described in first aspect present invention, and it is injection method or film dispersion method.
Detailed Description Of The Invention:
First aspect present invention provides a kind of compositions of tacrolimus, wherein comprises the water of 0.001～0.5wt% tacrolimus, 0.2～10wt% phospholipid, 10～60wt% alcohol, 0～0.5wt% cholesterol and surplus.
In an embodiment of compositions described in first aspect present invention, wherein contain 0.005～0.25wt% tacrolimus, or contain 0.01～0.2wt% tacrolimus, or contain 0.01～0.1wt% tacrolimus, or contain 0.02～0.1wt% tacrolimus, or contain 0.03～0.1wt% tacrolimus.
In an embodiment of compositions described in first aspect present invention, wherein said phospholipid is to be selected from one or more in natural phospholipid or synthetic phospholipid.In one embodiment, wherein said natural phospholipid is to be selected from one or more in following: soybean lecithin, Ovum Gallus domesticus Flavus lecithin, cephalin, creatinine, lipositol, lysophosphatide, phosphatidic acid, phosphatidyl glycerol.In one embodiment, wherein said synthetic phospholipid is to be selected from one or more in following: stearoyl/palmityl/oleoyl phosphatidylcholine, or stearoyl/palmityl/oleoyl PHOSPHATIDYL ETHANOLAMINE and derivant thereof.In one embodiment, wherein said synthetic phospholipid is to be selected from one or more in following: stearoyl and/or palmityl and/or oleoyl phosphatidylcholine, or stearoyl and/or palmityl and/or oleoyl PHOSPHATIDYL ETHANOLAMINE and derivant thereof.In one embodiment, wherein said phospholipid is to be selected from one or more in following: phosphatidylcholine (phosphatidylcholine, PC), hydrogenation PC, phosphatidic acid (phosphatidic acid, PA), Phosphatidylserine (phosphatidylserine, PS), PHOSPHATIDYL ETHANOLAMINE (phosphatidylethanolamine, PE), phosphatidyl glycerol (phosphatidyglycerol, PPG) and phosphatidylinositols (phosphatidylinositol, PI).In one embodiment, wherein said phospholipid is to be selected from one or more in following: soybean lecithin, cephalin, DSPE (DSPE), oleoyl phosphatidylcholine (POPC), Ovum Gallus domesticus Flavus lecithin, oleoyl phosphatidylcholine (POPC).In one embodiment, wherein said phospholipid is soybean lecithin.
In an embodiment of compositions described in first aspect present invention, wherein contain 0.4～8wt% phospholipid, or contain 0.5～6wt% phospholipid, or contain 0.5～5wt% phospholipid, or contain 1～4wt% phospholipid, or contain 2～3wt% phospholipid.
In an embodiment of compositions described in first aspect present invention, wherein said alcohol is to be selected from one or more in following: the Polyethylene Glycol that ethanol, isopropyl alcohol, propylene glycol, ethylene glycol, molecular weight are 200-600 (for example Macrogol 200, Liquid Macrogol, PEG400, Macrogol 600).In one embodiment, wherein said alcohol is selected from ethanol, propylene glycol and combination thereof.
In an embodiment of compositions described in first aspect present invention, wherein contain 10～50wt% alcohol, or contain 10～45wt% alcohol, or contain 15～45wt% alcohol, or contain 20～45wt% alcohol, or contain 30～40wt% alcohol.
In an embodiment of compositions described in first aspect present invention, wherein contain 0～0.25wt% cholesterol, or contain 0～0.20wt% cholesterol, or contain 0～0.1wt% cholesterol, or not containing cholesterol.
In an embodiment of compositions described in first aspect present invention, wherein contain the water of 0.001～0.5wt% tacrolimus, 0.5～5wt% phospholipid, 10～45wt% alcohol, 0～0.5wt% cholesterol and surplus.
In an embodiment of compositions described in first aspect present invention, wherein contain 0.03～0.1wt% tacrolimus, 1～4wt% phospholipid, 30～40wt% alcohol, the water of 0～0.1wt% cholesterol and surplus.
According to the compositions described in first aspect present invention, examine under a microscope, wherein contain the vesicle particle that mean diameter is less than 500nm.In one embodiment, in said composition, containing mean diameter is less than 400nm, is less than 300nm, is less than 250nm, is less than 200nm or is less than the vesicle particle of 100nm.In one embodiment, in said composition, contain the vesicle particle that mean diameter is 50-500nm, 50-400nm, 50-250nm, 50-200nm or 50-100nm.
According to the compositions described in first aspect present invention, examine under a microscope, wherein contain and be substantially irregular spherical vesicle particle.According to the compositions described in first aspect present invention, examine under a microscope, wherein contain substantially vesicle particle spherical in shape.According to the compositions described in first aspect present invention, examine under a microscope, wherein contain the vesicle particle that is substantially elliposoidal.
According to the compositions described in first aspect present invention, wherein can also contain one or more and be selected from following adjuvant: thickening agent is (such as but not limited to tragcanth, arabic gum, pectin, alginic acid, sodium alginate, potassium alginate, xanthan gum, agar, polyvinylpyrrolidone, polyacrylic acid, sodium polyacrylate, polyvinyl alcohol, carbomer, methylcellulose, sodium carboxymethyl cellulose, hydroxyethyl-cellulose, hydroxypropyl emthylcellulose, glycerol), coloring agent is (such as but not limited to caramel, alizarin dish is red, Yihong, lemon yellow, carmine and indigo, methylene blue), pH adjusting agent is (such as but not limited to hydrochloric acid, sulphuric acid, phosphoric acid, acetic acid, boric acid, citric acid, malic acid, sodium hydroxide, sodium bicarbonate, sodium hydrogen phosphate, diethylamine, triethanolamine, sodium citrate, Borax).It will be appreciated by those skilled in the art that tacrolimus, phospholipid, alcohol, the cholesterol percetage by weight in final composition is constant when there are these adjuvants, and as long as suitably regulate simply the consumption of water.In addition, the selection of these adjuvants and add method in those skilled in the art's technical scope, for example when compositions formulated, reduce in right amount the consumption of water, final formation, have after intended shape and big or small vesicle particle, then water-soluble thickener (such as hydroxypropyl emthylcellulose, carbomer, sodium alginate, xanthan gum, the arabic gum etc.) solution that supplements water preparation is to compositions full dose.
Second aspect present invention provides the method for preparing compositions described in first aspect present invention any one, and it is injection method or film dispersion method.
In an embodiment of method described in second aspect present invention, the method is injection method, and it comprises the steps:
I) tacrolimus, phospholipid and optional cholesterol (for example, under constant temperature and/or airtight condition) are dissolved in alcohol;
Ii) make step I) gained mixture (for example, under constant temperature and/or airtight condition, for example with step I) under identical constant temperature and/or airtight condition) inject (for example injecting with uniform flow velocity) water; With
Iii) making step I i) gained mixture carries out homogeneous dispersion (for example by ultrasonic method and/or Filtration and/or squeezing and pressing method), obtains compositions.
In an embodiment of described injection method, described step I i) constant temperature is 30～50 ℃, is preferably 40～50 ℃.
In an embodiment of described injection method, described step I ii) by mixture by homogeneous dispersion after, resulting composition is examined under a microscope, and wherein contains the vesicle particle that mean diameter is less than 500nm.In one embodiment, in said composition, containing mean diameter is less than 400nm, is less than 300nm, is less than 250nm, is less than 200nm or is less than the vesicle particle of 100nm.In one embodiment, in said composition, contain the vesicle particle that mean diameter is 50-500nm, 50-400nm, 50-250nm, 50-200nm or 50-100nm.
In an embodiment of described injection method, described step I ii) by mixture by homogeneous dispersion after, resulting composition is examined under a microscope, and wherein contains and is substantially irregular spherical vesicle particle.According to the compositions described in first aspect present invention, examine under a microscope, wherein contain the vesicle particle that is substantially elliposoidal.
In an embodiment of method described in second aspect present invention, the method is film dispersion method, and it comprises the steps:
A) for example, at suitable container (flask, for example, round-bottomed flask), tacrolimus, phospholipid and optional cholesterol are for example dissolved in, in organic solvent (chloroform), and then gained solution evaporation is removed organic solvent, to form uniform lipid film on chamber wall;
B) by adding the mixture of alcohol and water in the step container that comprises lipid film a), make described lipid film hydration;
C) making step b) gained mixture carries out homogeneous dispersion (for example by ultrasonic method and/or Filtration and/or squeezing and pressing method), obtains compositions.
In an embodiment of described film dispersion method, described step c) by mixture by homogeneous dispersion after, resulting composition is examined under a microscope, and wherein contains the vesicle particle that mean diameter is less than 500nm.In one embodiment, in said composition, containing mean diameter is less than 400nm, is less than 300nm, is less than 250nm, is less than 200nm or is less than the vesicle particle of 100nm.In one embodiment, in said composition, contain the vesicle particle that mean diameter is 50-500nm, 50-400nm, 50-250nm, 50-200nm or 50-100nm.
In an embodiment of described film dispersion method, described step c) by mixture by homogeneous dispersion after, resulting composition is examined under a microscope, and wherein contains and is substantially irregular spherical vesicle particle.According to the compositions described in first aspect present invention, examine under a microscope, wherein contain the vesicle particle that is substantially elliposoidal.
According to the method described in second aspect present invention, wherein at step I ii) and step c) in, described ultrasonic method and/or Filtration and/or squeezing and pressing method are the combination that adopts following any one method or several different methods independently of one another: 1) adopt probe type ultrasonic instrument ultrasonic 1-30min in ice bath to carry out meticulous homogeneity; Use again 0.45 μ m or 0.22 μ m filtering with microporous membrane, obtain the compositions (can be described as in this article ethosome) that finally contains vesicle particle; 2) adopt bath formula Ultrasound Instrument ultrasonic 1-30 minute in ice-water bath, then use 0.45 μ m or 0.22 μ m filtering with microporous membrane, obtain the compositions that finally contains vesicle particle; 3) adopt extruder at 55 ℃, push continuously (or individually) by aperture the polycarbonate leaching film of 200nm, 100nm and 80nm respectively 5 times, obtain the compositions that finally contains vesicle particle.
In either side of the present invention, the feature wherein having between two or more embodiments arbitrarily can combine mutually, as long as they can be not conflicting, certainly when combining each other, necessary words can be done suitably to modify to individual features.
Be further described with feature to various aspects of the present invention below.
All documents that the present invention quotes from, their full content is incorporated to herein by reference, and if when the expressed implication of these documents and the present invention are inconsistent, be as the criterion with statement of the present invention.In addition, various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art, nonetheless, the present invention still wishes at this, these terms and phrase to be described in more detail and to be explained, the term of mentioning and phrase, if any inconsistent with known implication, are as the criterion with the implication that the present invention was explained.
As described herein, term " compositions ", it can also refer to pharmaceutical composition or can refer to cosmetic composition, is used in experimenter and realizes and treat, prevent, alleviate and/or alleviate disease of the present invention or disease or adverse health situation.
As described herein, term " experimenter " can refer to that patient or other accept the present composition to treat, to prevent, to alleviate and/or to alleviate the animal of disease of the present invention or disease, particularly mammal, such as people, Canis familiaris L., monkey, cattle, horse etc.
The compositions that the object of this invention is to provide a kind of tacrolimus, said composition presents the feature of ethosome preparation, therefore in this article, the tacrolimus compositions that the present invention obtains also can be described as tacrolimus ethosome or tacrolimus ethosome preparation, its have particle diameter less and be evenly distributed, good stability and/or the strong feature of skin penetrating power.
In one embodiment of the invention, the composition of described tacrolimus compositions and weight percentage are: 0.001～0.5wt% tacrolimus, 0.5～5wt% phospholipid, 10～45wt% low-molecular-weight alcohol, and 0～0.5wt% cholesterol, all the other are distilled water.
In one embodiment of the invention, composition and the weight percentage of described tacrolimus compositions are preferably: 0.03～0.1wt% tacrolimus, 2～3wt% phospholipid, 30～40wt% low-molecular-weight alcohol, 0～0.1wt% cholesterol, all the other are distilled water.
In the present invention, in mentioning the present composition during amount of water, use in " water of surplus ", phrases such as " all the other are distilled water ", it does not represent that wish of the present invention meaning limits the present invention's formula for enclosed, and the amount that is interpreted as water when this type of phrase is modified is to add to 100% formula full dose (can be the percent of w/w percent or weight/volume percent or any other form).Such as containing pigment, pH adjusting agent, skin keratin softening agent etc. in the present composition, in these cases, add after these additives, can add " water of surplus " so that the present composition reaches the full dose of formula 100%.
" wt% " in the present invention, as do not indicated in addition, represents w/w percent.
According to the present invention, phospholipid is the main filmogen that forms ethosome.Described phospholipid comprises one or more in natural phospholipid or synthetic phospholipid.Natural phospholipid comprises soybean lecithin, Ovum Gallus domesticus Flavus lecithin, cephalin, creatinine, lipositol, lysophosphatide, phosphatidic acid, phosphatidyl glycerol etc.; Synthetic phospholipid comprises stearoyl/palmityl/oleoyl phosphatidylcholine, or stearoyl/palmityl/oleoyl PHOSPHATIDYL ETHANOLAMINE and derivant thereof.Soybean lecithin in preferred natural phospholipid.
Alcohol (for example low-molecular-weight alcohol and liquid polyethylene glycol) is also the component that forms a kind of necessity of ethosome, its objective is and may increase medicine at cuticular dissolubility, change the phase transition temperature of Stratum corneum lipids, strengthen its mobility, pliability, can also strengthen flexibility and the mobility of ethosome film, ethosome is deformed in percutaneous transmittance process, more easily by the gap less than its particle diameter, arrive deep skin.Described low-molecular-weight alcohol is monohydric alcohol or the polyhydric alcohol (such as but not limited to ethanol, isopropyl alcohol, propylene glycol, ethylene glycol) that contains 2-4 carbon atom; Described liquid polyethylene glycol is for example that molecular weight is the Polyethylene Glycol (for example PEG200, PEG300, PEG400, Macrogol 600) of 200-600.Preferred alcohol is the two combination of ethanol or propylene glycol or this.
Cholesterol is a kind of component often adding in conventional liposome, its objective is the phase transition temperature that changes lipid film, strengthens the stability of liposome membrane; But meanwhile, cholesterol add the rigidity that also can increase film, make it not yielding; And flexibility and morphotropism that an important feature of ethosome is film, therefore, the addition of cholesterol can not be too much, also can not add in some cases.
One of preparation method of tacrolimus ethosome of the present invention is injection method, comprise the steps: to take tacrolimus and phospholipid, optional cholesterol in prescription ratio, be placed in vial, (vial is connected with syringe is airtight to add wherein the low-molecular-weight alcohol of prescription ratio, by syringe, add alcohols, can avoid its volatilization), under constant temperature, airtight condition, by magnetic stirrer, extremely dissolve completely.Then, under identical constant temperature, airtight condition, the above-mentioned alcoholic solution containing medicine and phospholipid is injected to the water of recipe quantity with uniform flow velocity, after having noted, continue to stir, adopt again suitable ultrasonic and filter method to process, obtain the ethosome finally homogenizing.
The another kind of preparation method of tacrolimus ethosome of the present invention is film dispersion method, comprise the steps: to take tacrolimus and phospholipid, optional cholesterol in prescription ratio, put in round-bottomed flask, after adding a small amount of chloroform to dissolve, be placed in Rotary Evaporators, under constant temperature, rotary evaporation, except desolventizing, forms uniform lipid film in flask walls.In prescription ratio preparation alcohol/water mixed solution, by this mixed solution and lipid film rotation hydration.Then adopt and suitable filtration and ultrasonic method supersound process identical described in claim 5, obtain the ethosome finally homogenizing.
Described constant temperature is 30～50 ℃, is preferably 40～50 ℃.
Described suitable ultrasonic and filter method comprises: 1) adopt probe type ultrasonic instrument ultrasonic 5min in ice bath to carry out meticulous homogeneity; Use again 0.22 μ m filtering with microporous membrane, obtain final ethosome.2) adopt bath formula Ultrasound Instrument ultrasonic 5-10 minute in ice-water bath, then use 0.22 μ m filtering with microporous membrane, obtain final ethosome.3) adopt extruder at 55 ℃, push continuously by aperture the polycarbonate leaching film of 200nm, 100nm and 80nm respectively 5 times, obtain final ethosome.
As far back as the mid-90 in 20th century, (the TOUITOU E such as Touitou, Compositions for applying activesubstances to or through the skin:US 5540934) a kind of novel lipide that is called ethosome (ethosomes) has been proposed, can be specifically designed to the percutaneous transmission of medicine, because the ethanol that contains relative high concentration in this system is gained the name.It is believed that ethosome is a kind of multilamellar vesicle structure, compare with conventional liposome, its particle diameter is less, Stability Analysis of Structures, envelop rate is high, and bilayer mobility is higher, easily deformable, have better flexibility and permeability, penetrable skin barrier enters deep skin, thereby allegedly can significantly improve percutaneous rate and skin delay dose.Yet, for this macrolides compound with larger molecular weight and structure uniqueness of tacrolimus, form the ethosome form with ideal performance and still have to those skilled in the art challenge.The inventor is surprisingly found out that, by adopting the medicine of suitable proportion and phospholipid, alcohol, optional cholesterol, He Shui, prepared particle diameter less and be evenly distributed, good stability and/or the strong tacrolimus ethosome preparation for skin of skin penetrating power.Tacrolimus ethosome preparation of the present invention is hydrophilic liquid preparation, there is good percutaneous permeability, drug release is very fast, can overcome well viscosity beastly and greasy sensation that tacrolimus ointment exists, be not easy to clean and drug release shortcoming slowly.Especially, the present composition has good horny layer and deep dermal drug retention characteristics.
Accompanying drawing explanation
Fig. 1 is the particle size distribution figure of tacrolimus ethosome.
Fig. 2 is the electron-microscope scanning figure of tacrolimus ethosome.
Fig. 3 is the result of study of the anti-mouse ear atopic dermatitis of Ke Mosi ethosome.
The specific embodiment
By the following examples, can conduct further description the present invention, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and is not deviating under the prerequisite of the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that use in test.Although be well known in the art for realizing many materials and the operational approach that the object of the invention used, the present invention still does to describe in detail as far as possible at this.
a, Preparation Example part
embodiment 1
Take tacrolimus 30mg, soybean lecithin 2.0g, is placed in vial, adds wherein ethanol 40g, under 45 ℃ of constant temperature, airtight condition, by magnetic stirrer, extremely dissolves completely.Then, under identical constant temperature, airtight condition, the above-mentioned alcoholic solution containing medicine and phospholipid is injected to about 60ml water (making system reach 100g) with uniform flow velocity, after having noted, continue to stir, adopt again bath formula Ultrasound Instrument in ice-water bath ultrasonic 5 minutes, finally use 0.22 μ m filtering with microporous membrane, obtain final ethosome.
embodiment 2
Take tacrolimus 0.50g, cephalin 5.0g, is placed in vial, adds wherein ethanol 30g, under 50 ℃ of constant temperature, airtight condition, by magnetic stirrer, extremely dissolves completely.Then, under identical constant temperature, airtight condition, the above-mentioned alcoholic solution containing medicine and phospholipid is injected to about 70ml water (making system reach 100g) with uniform flow velocity, after having noted, continue to stir, then adopt probe type ultrasonic instrument ultrasonic 5min in ice bath to carry out meticulous homogeneity; Use again 0.22 μ m filtering with microporous membrane, obtain final ethosome.
embodiment 3
Take tacrolimus 0.10g, DSPE (DSPE) 1.0g, is placed in vial, adds wherein propylene glycol 45g, under 30 ℃ of constant temperature, airtight condition, by magnetic stirrer, extremely dissolves completely.Then, under identical constant temperature, airtight condition, the above-mentioned propylene glycol solution containing medicine and DSPE is injected to about 55ml water (making system reach 100g) with uniform flow velocity, after having noted, continue to stir, adopt again extruder at 55 ℃, to push continuously by aperture the polycarbonate leaching film of 200nm, 100nm and 80nm each 5 times, obtain final ethosome.
embodiment 4
Take tacrolimus 1mg, oleoyl phosphatidylcholine (POPC) 0.50g, cholesterol 0.10g, is placed in vial, adds wherein the PEG400 of 10g, under 40 ℃ of constant temperature, airtight condition, by magnetic stirrer, extremely dissolves completely.Then, under identical constant temperature, airtight condition, the above-mentioned polyglycol solution containing medicine and phospholipid, cholesterol is injected to about 90ml water (making system reach 100g) with uniform flow velocity, after having noted, continue to stir, adopt again bath formula Ultrasound Instrument in ice-water bath ultrasonic 5 minutes, finally use 0.22 μ m filtering with microporous membrane, obtain final ethosome.
embodiment 5
Take tacrolimus 30mg, soybean lecithin 1.0g, cholesterol 0.50g, puts in round-bottomed flask, after adding a small amount of chloroform to dissolve, is placed in Rotary Evaporators, and under room temperature condition, rotary evaporation, except desolventizing, forms uniform lipid film in flask walls.Ethanol 30g is mixed homogeneously with about water 70ml (making system reach 100g), then by this solution and lipid film rotation hydration.Adopt again probe type ultrasonic instrument ultrasonic 5min in ice bath to carry out meticulous homogeneity; Use again 0.22 μ m filtering with microporous membrane, obtain final ethosome.
embodiment 6
Take tacrolimus 0.10g, soybean lecithin 3.0g, is placed in vial, adds wherein ethanol 20g, and propylene glycol 20g, under 50 ℃ of constant temperature, airtight condition, extremely dissolves completely by magnetic stirrer.Then, under identical constant temperature, airtight condition, above-mentioned solution is injected to about 60ml water (making system reach 100g) with uniform flow velocity, after having noted, continue to stir, then adopt probe type ultrasonic instrument ultrasonic 5min in ice bath to carry out meticulous homogeneity; Use again 0.22 μ m filtering with microporous membrane, obtain final ethosome.
embodiment 7
Take tacrolimus 30mg, Ovum Gallus domesticus Flavus lecithin 1.0g, oleoyl phosphatidylcholine (POPC) 0.5g, put in round-bottomed flask, after adding a small amount of chloroform to dissolve, be placed in Rotary Evaporators, under room temperature condition, rotary evaporation, except desolventizing, forms uniform lipid film in flask walls.Ethanol 10g, propylene glycol 20g are mixed homogeneously with about water 70ml (making system reach 100g), then by this solution and lipid film rotation hydration.Adopt again extruder at 55 ℃, to push continuously by aperture the polycarbonate leaching film of 200nm, 100nm and 80nm each 5 times, obtain final ethosome.
b, experimental example part
experimental example 1
The object of this experimental example is to investigate particle diameter and the distribution thereof of tacrolimus ethosome.
Method: adopt dynamic light scattering method, measure the prepared tacrolimus ethosome particle diameter of embodiment 1 with laser particle analyzer, calculate polydispersity index (PDI).
Measurement result is shown in Fig. 1.
Fig. 1 result shows, less according to the mean diameter of the prepared tacrolimus ethosome of embodiment 1, is 91.47nm, and particle size distribution is even, PDI=0.333.
In addition, according to above method, the inventor also finds, the mean diameter of the tacrolimus ethosome that embodiment 2,6,7 is prepared is all between 70-110nm, the mean diameter of the tacrolimus ethosome that embodiment 3,4 is prepared is all between 90-160nm, and the particle diameter that each embodiment obtains is evenly distributed; The PDI value of the tacrolimus ethosome that embodiment 2-7 is prepared is all between 0.2-0.5.
experimental example 2
The object of this experimental example is to measure particle size and the form of tacrolimus ethosome.
Method: adopt transmission electron microscope, measure particle size and the form of the prepared ethosome of embodiment 1.
Measurement result is shown in Fig. 2.
Fig. 2 result shows: the particle according to the prepared tacrolimus ethosome of embodiment 1 is less, and particle shape is elliposoidal, has individually adhesion.Do not present the spherical of rule flexible by force with it, have good morphotropism relevant.Measure in addition the sample of embodiment 2-7, result shows that particle size and the form of ethosome is identical with the sample of embodiment 1 generally.
experimental example 3
This experimental example is that skin is detained experiment, and its object is to measure skin accumulation transit dose and the skin hold-up of tacrolimus ethosome.
Method: get Wistar rat, body weight is 200～250g.Hair is put to death, shaved to its disconnected neck, and separated complete skin of abdomen, carefully peels off subcutaneous fat, standby after cleaning.
Adopt improved Franz diffusing cells method method, the above-mentioned skin of handling well is placed between supply chamber and receiving chamber, stratum corneum side is to supply chamber; Acceptable solution is PBS (pH7.4), in supply chamber, add 1.0ml tacrolimus ethosome (embodiment 1 preparation) or commercially available product (Pu Bite ointment, 0.03%, Astellas pharmacy (China) company limited product), after (37 ± 1) ℃ constant temperature water bath 24h, get receiving liquid, adopt RP-HPLC method to measure wherein drug level, and calculate accumulation transit dose.After experiment finishes, with tape stripping method SCD, will be stained with cuticular adhesive tape and be placed in 3.0ml methanol, vortex mixed 5min, this solution, after 0.22 μ m microporous filter membrane filters, suitably dilutes, get 20 μ L injection liquid chromatographies, measure the medicament contg in large stratum corneum.Smash and be placed in the skin histology after tape stripping to pieces 10ml methanol, vortex mixed 10min, ultrasonic 30min circulates after 3 times, and solution filters through 0.45 μ m microporous filter membrane, gets 20 μ L injection liquid chromatographies, measures the tacrolimus content in the skin of rat deep.
Result of the test is in Table 1.
The skin permeability test result (n=3) of table 1, tacrolimus ethosome and commercially available product (those ointment of Pood)
| Sample | Accumulation transit dose (%) | Horny layer hold-up (ug) | Deep skin hold-up (ug) |
| Those ointment of Pood | Do not detect | 2.18±0.46 | 1.62±0.33 |
| Tacrolimus ethosome | Do not detect | 22.98±1.94 | 36.60±0.43 |
Result: 24 hours transdermal amounts of two kinds of preparations all, below detection limit, illustrate that transit dose is few, and therefore, medicine is difficult for entering blood circulation, avoids bringing systemic adverse reactions.In addition, from result, ethosome type compositions prepared by the present invention all exceeds a lot of times than commercially available product (those ointment of Pood) in the hold-up of horny layer and deep skin, and the treatment target site of atopic dermatitis is at horny layer and deep skin part.
In addition, the sample of test implementation example 2-7 in the same way, result shows that these samples are substantially the same with the sample of embodiment 1 aspect accumulation transit dose (%), horny layer hold-up (ug), deep skin hold-up (ug).For example, the result of the accumulation transit dose (%) of these samples is all not detect, and the average of the horny layer hold-up (ug) of these samples and deep skin hold-up (ug) and embodiment 1 sample accordingly result differs and is all less than 5%.For example the horny layer hold-up (ug) of the sample of embodiment 3 and deep skin hold-up (ug) are respectively 22.66 (ug) and 38.21 (ug).
the research of experimental example 4, the anti-mouse ear atopic dermatitis of Ke Mosi ethosome
1. reagent: DNF (DNFB), be dissolved in acetone-olive oil (3: 1, v/v) in mixed solvent, be mixed with the solution of 0.15%w/v; 0.1% tacrolimus ethosome (pressing the formula preparation of embodiment 3); Commercially available 0.1% tacrolimus ointment (Astellas Toyama company, Japan); Dexamethasone ointment (Shenzhen three nine-day periods after the winter solstice 10g:5mg of company).
2. the preparation of animal model: get male BALB/c mouse in 7 week age (body weight 18～22g), be divided at random sensitization group and group of solvents, 30 of sensitization groups, 6 of group of solvents.Allergen is coated with 0.15%DNFB 25 μ l and brings out dermatitis in mouse right ear both sides, group of solvents is smeared the acetone-olive oil of same volume, and (3: 1, v/v) mixed solvent (did not cause inflammation) in contrast.After one week, observe Mus ear and whether obvious tumefaction occurs, the sign whether being successfully prepared as model.
3. experiment grouping and administration: the mice of successful sensitization is divided into 4 groups at random, 6 every group.Attack the last time first 8 hours, every group gives respectively tacrolimus ethosome (embodiment 3), blank ethosome (formula of embodiment 3 is prepared but do not contained tacrolimus), the commercially available ointment of tacrolimus (0.1%) and the commercially available ointment of dexamethasone.The dosage of two kinds of tacrolimus formulations is identical, and is about its ointment people with 6 times of dosage (with body weight conversions), and dexamethasone is approximately also 6 times of people's use dosage.
4. the mensuration of observation index one Mus ear thickness: test each group after last attack 6h with digital display micrometer caliper (Kenta company, Singapore) measure mouse right ear thickness and with attack before Mus ear Thickness Ratio.
5. the results are shown in Fig. 3.In figure, data rod 1 is not for causing scorching solvent control group, and data rod 1 is blank ethosome group, and data rod 3 is the commercially available ointment group of tacrolimus, and data rod 4 is the commercially available ointment group of dexamethasone, and data rod 5 is tacrolimus ethosome group of the present invention.
Result from figure, group of solvents does not have a significant effect to mouse ear is thick; Compare with blank ethosome group, the commercially available ointment of the commercially available ointment machin dexamethasone of tacrolimus all can reduce Mus ear thickness, and tacrolimus ethosome group can obviously reduce the thickness of mouse ear, illustrates that said preparation has the effect of significant inhibition mouse ear dermatitis.
Therefore, local skin of the present invention not only can overcome with tacrolimus ethosome preparation that the greasing base of commercially available ointment produces makes us the shortcoming of unjoyful viscosity and greasy, non-easy cleaning, and can improve the rate of release of medicine, increase the hold-up of medicine in skin, thereby heighten the effect of a treatment.There is feasibility.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (11)
1. an ethosome compositions for tacrolimus, it is composed of the following components: the water of 0.001～0.5wt% tacrolimus, 0.2～10wt% phospholipid, 10～60wt% alcohol, 0～0.5wt% cholesterol and surplus; And
Described phospholipid is to be selected from one or more in following: soybean lecithin, Ovum Gallus domesticus Flavus lecithin, cephalin, creatinine, lipositol, lysophosphatide, phosphatidic acid, phosphatidyl glycerol, stearoyl/palmityl/oleoyl phosphatidylcholine, or stearoyl/palmityl/oleoyl PHOSPHATIDYL ETHANOLAMINE, phosphatidylcholine, HSPC, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, phosphatidyl glycerol and phosphatidylinositols, DSPE, oleoyl phosphatidylcholine;
Described alcohol is that molecular weight is the Polyethylene Glycol of 200-600;
Said composition is examined under a microscope, and wherein contains the vesicle particle that is elliposoidal.
2. the compositions of claim 1, wherein comprises 0.5～5wt% phospholipid.
3. the compositions of claim 1, wherein comprises 10～45wt% alcohol.
4. the compositions of claim 1, wherein said Polyethylene Glycol is selected from Macrogol 200, Liquid Macrogol, PEG400, Macrogol 600.
5. the compositions of claim 1, it is composed of the following components: the water of 0.001～0.5wt% tacrolimus, 0.5～5wt% phospholipid, 10～45wt% alcohol, 0～0.5wt% cholesterol and surplus.
6. the compositions of claim 1-5 any one, examines under a microscope, and wherein containing mean diameter is the vesicle particle of 50-200nm.
7. the compositions of claim 1, it consists of: tacrolimus 1mg, oleoyl phosphatidylcholine 0.50g, cholesterol 0.10g, 10g PEG400, water are to 100g.
8. the method for preparing compositions described in claim 1-7 any one, it is injection method, described injection method comprises the steps:
I) tacrolimus, phospholipid and optional cholesterol are dissolved in alcohol;
Ii) make step I) in gained mixture injected water; With
Iii) making step I i) gained mixture carries out homogeneous dispersion, obtains compositions.
9. the method for claim 8, at described step I ii) by mixture by homogeneous dispersion after, resulting composition is examined under a microscope, wherein containing mean diameter is the vesicle particle of 50-200nm.
10. the method for preparing compositions described in claim 1-7 any one, it is film dispersion method, described film dispersion method comprises the steps:
A) in suitable container, tacrolimus, phospholipid and optional cholesterol are dissolved in organic solvent, then gained solution evaporation is removed organic solvent, so that form uniform lipid film on chamber wall;
B) to the mixture that adds alcohol and water in the step container that comprises lipid film a), make described lipid film hydration;
C) making step b) gained mixture carries out homogeneous dispersion, obtains compositions.
The method of 11. claim 10, at described step c) by mixture by homogeneous dispersion after, resulting composition is examined under a microscope, wherein containing mean diameter is the vesicle particle of 50-200nm.
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| CN103142468A (en) * | 2012-12-28 | 2013-06-12 | 中山大学 | Tacrolimus eye ointment for cornea transplantation and preparation method thereof |
| CN103271826B (en) * | 2013-05-15 | 2014-08-06 | 广东恒健制药有限公司 | Industrial production method for Tacrolimus ointments |
| CN108158979A (en) * | 2018-03-27 | 2018-06-15 | 浙江日升昌药业有限公司 | A kind of Tacrolimus paste composition |
| CN112920554B (en) * | 2021-04-07 | 2023-03-17 | 国网内蒙古东部电力有限公司呼伦贝尔供电公司 | Toughened epoxy resin sound insulation material and preparation method thereof |
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| CN1342075A (en) * | 1999-03-11 | 2002-03-27 | 藤泽药品工业株式会社 | Liposome preparations |
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| 祝伟伟等.醇质体的研究进展.《食品与药品》.2007,第9卷(第1期),参见第46页摘要，第46页左栏第1段—第47页左栏第4段. |
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