CN108904445A

CN108904445A - Its salts nano suspending liquid

Info

Publication number: CN108904445A
Application number: CN201810884644.XA
Authority: CN
Inventors: 沈媛媛; 陈桔英; 鲍丰祺
Original assignee: JIANGSU ZEYUN PHARMACEUTICAL Co Ltd
Current assignee: JIANGSU ZEYUN PHARMACEUTICAL Co Ltd
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2018-11-30

Abstract

Present invention discloses a kind of suspension of its salts monohydrate of nanocrystal types such as particle size distribution that dynamic light scattering measures for 200-600nm, the suspension includes water phase, and the water phase includes nonionic, polymeric surfactant and the acid isopropyl for being enough to prevent the amount of the formation of its salts monohydrate nanocrystal aggregation and/or crystal growth.Its salts in the suspension has preferable skin-penetrating and biological activity, and has preferable stability characteristic.

Description

Its salts nano suspending liquid

【Invention field】

The present invention relates to its salts of nanocrystal types (calcipotriol) monohydrate suspension, and are related to being included in It is intended for preventing or treating the nanocrystal suspension in the pharmaceutical composition of skin disease and illness.

【Background of invention】

Psoriasis is chronic inflammatory skin disease, shows as erythema shape, drying as caused by hyperkeratosis, squamous spot Block.Patch is most commonly in elbow, knee and scalp, although more lesions possibly are present at the other parts of body, especially waist sacrum Region.Slightly answered to the part that the most common treatment of moderate psoriasis is related to containing corticosteroid as the composition of active constituent With.Although effectively, corticosteroid has the shortcomings that many side effects, such as atrophoderma, striped (striae), acne sample Fash, Perioral Dermatitis, dermatophyte and the undue growth of bacterium, pigmented skin hypochromatism and brandy nose.

However, for many years, the advantageous non-steroidal therapy of psoriasis is raw with the dimension prepared in ointment compositions The local treatment of plain D analog its salts, wherein its salts is present in solvent or antiperspirant cream compositions, wherein its salts Exist with the suspension form of particle.Solvent in ointment compositions is propylene glycol, and there is enhancing active constituent to penetrate into The advantages of skin, leads to the improvement of effect, but known its is also used as skin irritant.Therefore, it was reported that in topical composition Usually will cause patient evolution comprising propylene glycol, (research reports a large amount of thorns of 12.5% propylene glycol at contact dermatitis Swash reaction, referring to M.Hannuksela et al., ContactDermatitis1,1975, the 112-116 page), and ought be with high concentration When using propylene glycol, the quantity of stimulate the reaction increase (such as J.Catanzaro and J.GrahamSmith, J.Am.Acad.Dermatol.24,1991, the 90-95 pages summary).Especially because the presence of propylene glycol and result in calcium pool three Alcohol penetrates into the improvement of skin, it has been found that cream has compared skin irritatin.

Human skin, particularly outer layer cuticula provide antimicrobial pathogen and toxin chemical substance intrusion it is effective Barrier.Although this property of skin is usually advantageous, the dermal administration of drug is complicated, is suffering from skin because applying Even if on the patient skin of skin disease it is a large amount of-be not that most of-active constituent may not penetrate into it to play its active The vibrant layer of skin.In order to ensure active constituent is fully penetrated into corium and epidermis, it is generally preferable to which ground is with dissolved state Comprising active constituent, generally in the presence of the solvent in the form of alcohol such as ethyl alcohol or glycol such as propylene glycol.Propylene glycol is ripe The penetration enhancers known can penetrate cuticula and by the therapeutical active component " pulling in " in low-molecular-weight component such as carrier Substance in epidermis.Propylene glycol itself may cause significant skin irritatin, and it can also be by the low molecule of carrier and can Component can be stimulated " to pull in " in epidermis, causing conventional carrier includes the overall stimulation of propylene glycol.Thus, it is intended to which treatment is scorching Disease skin disease may aggravate inflammatory response as propylene glycol existing for solvent in the composition.

Soft emulsifiable paste is more effective in treatment psoriasis lesion, propylene glycol should not be used as solvent, but has comparable skin Skin penetrability and biological activity characteristic.

Research purpose of the invention is to provide the topical composition comprising its salts as active constituent, has preferable Skin-penetrating and biological activity, and have preferable stability characteristic.

【Summary of the invention】

It has surprisingly been found that the chemically stable (i.e. non-degradable to moor at 24- table calcium of nanocrystal types may be prepared Triol or other catabolites) its salts monohydrate, because unexpectedly, during nanosizing, the not no nothing of significant quantity Setting its salts is formed because of high stress or impact force or high temperature.In addition, nanocrystal is physically stable, because making After standby, the change of aggregation or crystal growth or crystal (polymorphic) form is not observed in the suspension of nanocrystal.It receives Meter Jing Ti can be easily formulated into topical cream and ointment compositions, therefrom its salts (monohydrate) can with come In the vigor layer (i.e. skin corium and epidermis) that a considerable amount penetrated from its salts of ointment penetrates to the skin, and generate class Like or higher levels of biological activity (as measured by the Activated in Vitro of target gene), and without by means of comprising penetrating increasing Strong agent such as propylene glycol (it is potential skin irritant).

Therefore, one aspect of the present invention is related to as particle size distribution that dynamic light scattering measures is 200-600nm The suspension of its salts monohydrate of nanocrystal types, the suspension include water phase, and the water phase includes being enough to prevent Its salts monohydrate nanocrystal aggregation is formed and/or nonionic, the polymeric surfactant of the amount of crystal growth And acid isopropyl, its salts in the suspension have preferable skin-penetrating and biological activity, and have preferable Stability characteristic.

It on the other hand, is the nanocrystalline of 200-600nm the present invention relates to such as particle size distribution that dynamic light scattering measures Its salts monohydrate of body form, the nanocrystal can be obtained by method comprising the following steps：

(a) by comprising the water phase weight about 0.5% to about 5% nonionic, polymeric surfactant and（About 0.5% to About 5%）Crystalline state its salts monohydrate reduction in the water phase of acid isopropyl, leads to the formation of particle, the particle Particle size distribution be about 5-20 μm, about 10 μm of mean particle size；

(b) under about 300-800 bars of pressure, the suspension of step (a) is carried out to the high pressure homogenization one of first circulation Section time, described a period of time are enough to obtain its salts one that the particle size distribution of about 15-40% is 200-600nm and are hydrated The crystal of object；

(c) under about 800-1200 bars of pressure, the suspension of step (b) is carried out to the high pressure homogenization one of second circulation Section time, described a period of time are enough to obtain its salts one that the particle size distribution of about 40-80% is 200-600nm and are hydrated The crystal of object；

(d) under about 1200-1700 bars of pressure, the suspension of step (c) is carried out to the high pressure homogenization of third circulation For a period of time, described a period of time is enough to obtain its salts that about 90% or more particle size distribution is 200-600nm The crystal of monohydrate；With

(e) optionally from water phase separating obtained its salts monohydrate nanocrystal.

On the other hand, the present invention relates to the drugs comprising above-mentioned its salts monohydrate nanocrystal and pharmaceutical acceptable carrier Composition.

It yet still another aspect, the present invention relates to the compositions comprising its salts monohydrate nanocrystal or nano suspending liquid Purposes, for treating skin disease or illness, such as psoriasis, seborrheic psoriasis, palmoplantar pustulosis, dermatitis, xeroderma Ichthyosis, brandy nose or acne.

Detailed description of the invention

Definition

In the context of the present invention, term " nanocrystal " means the crystal grain of its salts monohydrate, is in nanometer Magnitude range, i.e., diameter is between 1-1000nm.Nanocrystal advantageously has such particle size distribution：>=90% receives Meter Jing Ti has the granular size between 100-900nm, particularly between 200-600nm.

Term " nano suspending liquid " means to be suspended in the nanocrystal as defined above in water phase.

Term " particle size distribution " mean according to manufacturers instruction (being available from Malvern Instruments, Britain), Using Zetasizer NanoZS or ZS90, by dynamic light scattering (also referred to as photon correlation spectroscopy) measure minimum and most Span between big its salts monohydrate crystal.Dynamic light scattering by with laser irradiation particle and analyze due in fluid The strength fluctuation in scattering light that the Brownian movement of particle generates measures the size of the solid particle to float on a liquid.Intensity Fluctuation is related to granular size, because the movement of biggish particle smaller particle is slower, i.e., strength fluctuation is slower.

Term " amorphous " means that its molecule does not have the solid matter of ordered arrangement, the i.e. opposite of crystalline state.

" its salts " is formula

Novel vitamin D analogues.

It has been found that its salts exists with two kinds of crystalline form anhydrides and monohydrate.Its salts monohydrate and Its preparation is disclosed in WO94/15912.

Term " chemical stability " or " chemically stable " mean its salts monohydrate nanocrystal in suspension Or it will not significantly be degraded into 24- table its salts or the degradation of other its salts at any time in final drug products Product.In the latter case, " chemical stability " refers to during the shelf-life of product, generally at room temperature 2 years, is no more than 10%, it is preferably no more than 6% its salts monohydrate degradation.By by nanocrystal or containing the composition of nanocrystal 40 DEG C of acceleration for stabilization Journal of Sex Research is carried out, the approximation of chemical stability at room temperature is obtained.If degraded after 3 months at 40 DEG C Substance less than 10%, then it has been generally acknowledged that being equivalent to 2 years shelf-lives of room temperature.

Term " physical stability " or " physically stable " mean that its salts monohydrate nanocrystal has and do not have also Have the essentially identical crystalline form of reference its salts monohydrate for carrying out nanosizing, as measured by Raman spectroscopy, i.e., its Not since nanosizing shows polymorphism.In addition, " physical stability " refers in the requirement patent for being mixed with the nanocrystal In the suspension or pharmaceutical composition of protection, nanocrystal does not show aggregation or crystal growth.

Term " substantially non-aqueous " means freeze-drying or spray drying its salts monohydrate nanocrystal The content of Free water (anti-with the water phase of crystal combination) is less than about the 2% of nanocrystal by weight, preferably few by weight In about the 1% of nanocrystal, such as it is less than about the 0.5% of nanocrystal by weight.Similarly, soft in " substantially anhydrous " The content of Free water is less than about the 3% of composition by weight in composite cream, is preferably less than the pact of composition by weight 2%, such as it is less than about the 1% or 0.5% of composition by weight.

Term " solubilising power " means the ability that solvent or solvent mixture dissolve given substance, is expressed as realizing substance It is completely dissolved required amount.

Term " skin penetration " means that active constituent is diffused into the different layers of skin, i.e. cuticula, epidermis and skin corium In.

Term " Cutaneous permeation " mean active constituent through skin flow into body circulation in, or in vitro research, for example those In the case where the in vitro study reported in embodiment 7 below, active constituent flows into Franz used in experiment through skin The acceptable solution (receptorfluid) of cell device.

Term " biological activity " means when being applied to skin in the compositions of the present invention, vitamin D derivative or class Like the activity of object.As being described in detail in embodiment 6 below, the biological activity of composition measures in measuring method in vitro, measures Activation of the target gene of encoding human marker antibacterial peptide in the reconstruct human epidermal of the human keratinized cell containing culture.Calcium pool The preparation of triol monohydrate nanocrystal

In recent years, the method as the improvement rate of dissolution for providing dissolubility difference drug, the nanocrystal of therapeutic active Or nano suspending liquid has been prepared more and more researchs.When applying drug, the larger surface area of nanocrystal ensures Higher solution rate.So far, which is mainly used for preparing active constituent that is oral or intravenously applying.

If the drying method for preparing medicament nano crystal has been described in document.Generally, this method can be divided into two Class, i.e. grinding and high pressure homogenization.

US5,145,684 is disclosed in surface modifier such as polyvinylpyrrolidone, polyvinyl alcohol, lecithin or other tables In the presence of the activating agent of face, pass through the method for ball milling 4-5 days preparation crystalline state nano particles.

Ball milling its salts monohydrate under these conditions, or to may cause its salts monohydrate directly chemical Degradation, or form a large amount of amorphous its salts, foot of the amorphous its salts for the pharmaceutical composition containing it Enough storage stability/shelf-lives are unfavorable, because amorphous substance is relatively more more sensitive to chemical degradation than amorphous material.

CA2375992, which is disclosed, to be lower than 20 DEG C, is being preferably lower than 0 DEG C of temperature, in anhydrous medium, is split in piston- Preparing granular size by high pressure homogenization in gap (piston-gap) homogenizer is 5 μm or less, preferably 1 μm drugs below The method of grain.By the way that the drug granule of micronized to be carried out to the high pressure homogenization of 10-20 circulation at 1500 bars, implement nanosizing. In the method, we are used to reduce its salts using water-bearing media, because not led using anhydrous medium (atoleine) Any size of its salts monohydrate crystal is caused to reduce.WO2004/054549 discloses the cream in water comprising monoglyceride The topical formulations of spirolactone nano particle in cream base matter.Nano particle is prepared using piston-crack high pressure homogenization.

It has been found that 1500 bars of high pressure homogenizations it is several circulation for reduction its salts monohydrate be it is inappropriate, Because this method leads to the aggregation of its salts monohydrate crystal, even suitable surfactant there are the case where Under.

WO2008/058755 is disclosed through pearl or ball milling and then is prepared cosmetic activity object by high pressure homogenization The nanocrystal of matter.The combination of two methods show it is more advantageous than individual high pressure homogenization because the latter need in high pressure Permitted multicycle homogenizing under (1500 bars).Combined method allows to the homogenizing recycled using the only one under lower pressure, To obtain nanosizing particle.

In advantageous embodiment, the present invention relates to be used to prepare particle size distribution that dynamic light scattering measures such as to be The method of its salts monohydrate nanocrystal of 200-600nm, the method comprising the steps of

(a) by comprising the water phase weight about 0.5% to about 5% nonionic, polymeric surfactant and about 0.5% to about Crystalline state its salts monohydrate reduction in the water phase of 5% acid isopropyl, leads to the formation of particle, of the particle Grain size distribution is about 5-20 μm, about 10 μm of mean particle size；

In final suspension (step (d) is afterwards), one water of its salts of the particle size distribution with 200-600nm The amount of solvate crystal is preferably about 95% or more.

This method is different from oneself disclosed method.It has been found that this method can make its salts monohydrate granular size It is more stable with particle size distribution.

Furthermore, it has been found that in the methods of the invention, its salts chemical stability is higher, because its salts is to light, heat Etc. be it is sensitive, its salts is easy to can by chemical degradation.

Embodiment of the present invention

This suspension can contain nonionic, polymeric surfactant, especially acid isopropyl, be added to more preferable Ground prevents the nanocrystal of its salts from assembling and/or preventing crystal growth.Surfactant should will not be caused The surfactant of any significant solubilising of its salts monohydrate nanocrystal, i.e., its should have poor solubilized energy Power, and poloxamer or polysorbate surfactant and polyoxyethylene C6-24 alkyl ether can be advantageously selected from.Pool Luo Shamu can be selected from Pluronic/Lutrol F 44, PLURONICS F87, poloxamer 237, Pluronic/Lutrol F 108 and poloxamer188.It is special Not, it has been found that, PLURONICS F87 has poor solubilising power for solubilized its salts, therefore as this The current advantageous surfactant of nano suspending liquid.When polysorbate is used as surfactant, poly- mountain can be selected from Pear ester 20, polysorbate 40, polysorbate 60, polysorbate 61, polyoxyethylene sorbitan monoleate and sorbimacrogol oleate 100.Currently preferred polyoxy Ethylene C6-24 alkyl ether is Cetomacrogol 1000 (cetomacrogol1000).

The amount of surfactant and acid isopropyl can be respectively suspension weight about 0.01% to about in water phase 5%.Generally preferably, in the water phase surfactant amount be suspension weight about 0.6-1.2%.Depending on reduction and Applied condition during high pressure homogenization, its salts monohydrate nanocrystal present in gained suspension can have Such as the mean particle size of 200-350nm, 350-400nm or 400-500nm that dynamic light scattering is measured.

Nano suspending liquid can be freeze-dried or is spray dried to and is nanocrystalline comprising surfactant on the surface Body.Then the nanocrystal of freeze-drying or spray drying can be used for incorporation into anhydrous composition.

It has surprisingly found that：This method for making Nano results in the production of the only amorphous its salts of non-significant amount It is raw.Those skilled in the art are known, and due to lacking molecules align in lattice, the presence of amorphous compound keeps substance unstable It is fixed, make substance be exposed to chemical degradation or make crystal structure be rearranged into different polymorphic forms (referring to Chow et al., J.Pharm.Sci.97 (8), 2008, the 2855-2877 pages).Perkin-ElmerDSC8500 is such as used according to manufacturers instruction The differential scanning calorimetry of instrument is measured, it is impossible to detect the reactive amorphous calcium of the significant quantity of instrument detection limit (about 5%) Moor triol.

In the methods of the invention, the ball for the use of diameter being 1-4mm such as 2-3mm first in water phase by the crystal of its salts Or pearl carries out grinding or pre-grinding.Ball or pearl can be made of glass or similar hard substance such as zirconium oxide.Grinding can close Suitablely about 500-4000 revs/min, for example, about 1000-3000 revs/min, for example, about 2000 revs/min progress 2-5 hours, Such as 3 hours.

Surfactant for grinding can suitably be nonionic, polymeric surfactant.Nonionic, polymer Surfactant and acid isopropyl are respectively with about the 1.5 to about 3% of suspension weight, particularly with the pact of suspension weight 2% amount is added in water phase.Surfactant is preferably chosen from poloxamer or polysorbate surface-active as described above Agent.Suspension is then directly used in high pressure homogenization step, has been obtained using PLURONICS F87 big for particle after high pressure homogenization Particularly advantageous result for small distribution.It should be noted that because being washed with water used in step (a) in this production method Milling apparatus, and high pressure homogenization equipment is washed with water after step (d), thus in final suspension surfactant concentration For the about 0.6-1.2% of suspension weight.

According to manufacturer's explanation, (can be from using piston crack (pistongap) homogenizer, such as EmulsiflexC3 Avestin is obtained) carry out high pressure homogenization step (b)-(d).

For the nanosizing of its salts monohydrate, it has been found that, step is carried out under about 500-650 bars of pressure (b) first circulation high pressure homogenization is advantageous.Obtain one water of its salts that 15-40% has required particle size distribution Closing the time that object nanocrystal needs is 7-15 minutes, such as 8-12 minutes, for example, about 10 minutes.

The high pressure homogenization of second circulation of step (c) can be carried out suitably under about 1000-1100 bars of pressure.It obtains Obtaining 40-80% to have the time required for its salts monohydrate nanocrystal of required particle size distribution is 7-15 points Clock, such as 8-12 minutes, for example, about 10 minutes.The high pressure homogenization of the third circulation of step (d) can be suitably in about 1400- It is carried out under 1500 bars of pressure.Obtain 90% or more its salts monohydrate nanometer with required particle size distribution Time required for crystal is 7-15 minutes, such as 8-12 minutes, for example, about 10 minutes.

If be intended to by its salts monohydrate nanocrystal be included in anhydrous formulation in, can suitably by they into Row freeze-drying or spray drying (are to be less than about 2% based on nanocrystal weight to water content (Free water), for example, less than about 1% or be less than about 0.5%).

Its salts monohydrate nanocrystal or suspension comprising nanocrystal can be included in pharmaceutical composition In object, described pharmaceutical composition includes pharmaceutical acceptable carrier compatible with active constituent.

When mixing with pharmaceutically acceptable excipient to provide composition as described below, non-ionic polymer surfactant Amount is preferably the about 0.03-0.06% based on composition weight.

In one embodiment, this composition is ointment.To be classified according to current FDA, ointment is semisolid dosage form, In can containing by weight up to 20% water and volatile materials and by weight 0.01% to about 5% oleic acid isopropyl Ester, and its contain in the carrier be more than by weight 50% hydrocarbon, wax or polyalcohol.Therefore, according to the present invention, Ointment can be water-in-oil composition, in such a case, it is possible to which nano suspending liquid to be added to the lipophilicity of composition with regard to this In component, so that composition contains the water phase of by weight up to 10%, preferably by weight up to 5%.Alternatively, composition It can be anhydrous ointment, contain the Free water for being less than about 2%, preferably less than 1% based on the weight of composition.

Ointment carrier can suitably contain paraffin, the paraffin that is formed selected from the hydrocarbon by a length of C5-60 of chain and Its mixture.Usually used ointment carrier is vaseline or paraffinum molle alba, by different chain length (maximum value is about C40-44) Hydrocarbon composition or vaseline and atoleine (being made of the hydrocarbon for the different chain length that maximum value is C28-40) Mixture.It is subject to processing the closing of skin surface although vaseline provides, reduces the endermic loss of water, and strengthening group The therapeutic effect for closing active constituent in object, but after application, tend to the quite a while persistently have it is greasy and/or Tacky feeling, and it is not easy to smear.It is preferred, therefore, to be formed using the hydrocarbon by slightly lower chain length Paraffin, such as by with maximum value be C14-16, C18-22, C20-22, C20-26 chain length hydrocarbon or its mix Close the paraffin of object composition.It has been found that such paraffin is that more beauty is acceptable, because they are less tacky in application And/or it is greasy, and be easier to smear.Therefore, it is desirable to which they generate improved patient's compliance.The suitable paraffin of the type It is produced by Sonneborn and with trade (brand) name Sonnecone, such as SonneconeCM, SonneconeDM1, SonneconeDM2 It is sold with SonneconeHV.These paraffin are further disclosed and are characterized in WO2008/141078, and the patent application is at this It is incorporated as referring in text.(being made up of the hydrocarbon that gas chromatography determines paraffin).

It can suitably include lipophilicity viscosity improving components, example to assign required viscosity to the present composition Such as wax.Wax can be mineral wax, by high molecular weight hydrocarbons, for example be saturated C35-70 alkane mixture form, such as Microwax.Alternatively, wax can be plant or animal wax, such as the ester such as beeswax of C14-32 fatty acid and C14-32 fatty alcohol. The amount of viscosity improving components can change according to the thickening power of ingredient, but can the generally about 1- of composition by weight 20%.When viscosity improving components are microwaxes, generally with the about 5-15% of composition by weight, for example, about 10% Amount exist.

In order to maintain the good physical stability of composition, particularly for the separation for avoiding wherein water phase and lipid phase, The water-in-oil emulsifier that HLB value is 3-8 can be advantageously comprised.The example of such emulsifier is polyoxyethylene C8-22 alkyl ether, Such as polyoxyethylene stearyl acyl ether, polyoxyethylene cetyl base ether, polyoxyethylene oleyl ether or polyoxyethylene lauryl ether.Emulsifier Amount be generally the 2-10%w/w of composition.

Composition can extraly include softening agent, can soften the epidermis that plaque psoriasis thickens.Included in this group The suitable softening agent closed in object can be silicon wax (siliconeWax) or volatile silicone oils, because in addition, it is found that silicone Presence its salts can be helped to penetrate into skin.It has also been found that the composition comprising silicone can lead to less skin Stimulation.Cyclomethicone (cyclomethicone), poly- diformazan can be selected from comprising suitable silicone oil in the present compositions Radical siloxane (dimethicone).Amount comprising silicone oil in the present compositions be generally by weight composition about 1% to About 10%, for example, about 5%.

In ointment, it is believed that propylene glycol is the principal element of the skin irritatin of many patient experiences.However, it has been found that Its salts itself may be minimal irritation (A.Fullerton and J.Serup, Br. among the patients J.Dermatol.137,1997,234-240 pages and A.Fullerton et al., Br.J.Dermatol.138,1998, the 259-265 pages).Therefore it is advantageous that in the present compositions include anti-irritation compound, such as glycerol, sorbierite, sucrose, Saccharin, menthol, cineole (eucalyptol) or niacinamide.Glycerol is described as to protect the skin from irritation object The substance (J.Bettinger et al., Dermatology197,1998, the 18-24 pages) of matter, and it has been found that its with agent Amount dependence mode reduces the release of IL-1 α：It has been found, therefore, that：In its salts ointment in the presence of by weight 15% it is sweet Oil leads to IL-1 α release significantly more lower level than the glycerol comprising by weight 10%, in turn, includes by weight 10% Glycerol leads to IL-1 α release significantly more lower level than the glycerol comprising by weight 5%.However, in addition to anti-irritation effect, Surprisingly, it has been found that glycerol can reinforce the biological activity of its salts, because it has been found that, relatively low amount in composition Glycerol increase antibacterial peptide expression (below in measurement described in embodiment 4) (i.e. ought by weight glycerol amount be 5% When express more antibacterial peptides when being respectively 10% or 15% than the amount when glycerol)：It implies that it is for glycerol includes, it is necessary to Balance is found between the effect of advantageous anti-irritation and advantageous synergistic effect.We have found that wrapping in the present compositions Significant anti-irritation effect is produced containing the by weight about glycerol of 5-10%, and significantly enhances the biology of its salts Learn activity.

Known its salts be to acid condition (in Aquo-composition below about 7.0 pH or in anhydrous composition In acid reaction substance) extremely sensitive substance, the acid condition facilitates the fast degradation of its salts.In order to ensure Enough chemical stabilities of substance during the shelf-life of composition, wise is the chemical combination comprising that can neutralize acid impurities Object, the acid impurities are likely to be present in one or more excipient of composition, and to the chemical stabilization of its salts Property is unfavorable.If composition be it is aqueous, acid neutralize compound can be selected from buffer such as phosphate buffer, Can include with the amount of the 0.025-0.065% of composition by weight.On the other hand, if composition be it is anhydrous, Acid, which neutralizes compound, can be advantageously amine, such as triethanolamine, trishydroxymethylaminomethane (trometamol), monoethanol Amine or diethanol amine are contained in composition with the amount of the 0.1-2% of composition by weight.

In another embodiment, this composition is emulsifiable paste, may include with component as ointments, but it is generally Oil-in-water emulsion containing a large amount of water.

In certain embodiments, this composition includes:

Its salts monohydrate of 0.003-0.008%w/w,

And the acid isopropyl of 0.01% to about 5%,

The polyoxyethylene stearyl ether of 2-8%w/w,

The water of 2-10%w/w,

The PLURONICS F87 of 0.001-0.005%w/w,

The paraffin carrier of 70-90%w/w.

This composition is further included in other components usually used in skin preparation, such as antioxidant (such as α- Tocopherol), preservative, sodium ethylene diamine tetracetate, pigment, skin pain relieving agent, skin healing agent and skin conditioner, such as urea, Allantoin or bisabolol, referring to CTFACosmeticIngredientsHandbook, the second edition, 1992.By will be effective Composition of the invention can be used to treat silver-colored bits to the patient for needing the treatment by the composition local application of the invention of amount Disease, seborrheic psoriasis (sebopsoriasis), palmoplantar pustulosis, dermatitis, ichthyosis, brandy nose or acne and relevant Skin disease.The method preferably includes the compositions of topical application treats effective dose once-or twice-a-day.For this purpose, Composition according to the present invention preferably contains about 0.001-0.5mg/g, preferably about 0.002-0.25mg/g, particularly Its salts monohydrate nanocrystal of 0.005-0.05mg/g.Consider that this composition is advantageously used for these skin diseases Maintenance therapy, i.e., continual cure is after Visual symptoms disappearance to postpone the recurrence of symptom.

In order to provide the more effective treatment of acute stage psoriasis He other skin disorders, it can be advantageous to wrap in the composition Containing one or more other treatment active constituents.The example of such other active components include but is not limited to anti-inflammatory medicaments for example Corticosteroid, such as betamethasone and its esters such as valerate or dipropionate, clobetasol or its esters such as propionic ester, hydrogenation Cortisone or its ester such as acetic acid esters；Non-steroid anti-inflammatory medicaments, such as naproxen, Indomethacin, Diclofenac, brufen, the right side Brufen, Ketoprofen, Flurbiprofen, piroxicam, Lornoxicam or Nabumetone, phosphodiesterase 4 inhibitors (such as Compound disclosed in WO2008/077404, WO2008/104175, WO2008/128538 or WO2010/069322) or P 38 map kinase inhibitor (such as compound disclosed in WO2005/009940 or WO2006/063585).

Preferred implementation technical solution of the invention（1-49）It is listed below：

Implement the calcium for the nanocrystal types that such as particle size distribution that dynamic light scattering measures of technical solution 1. is 200-600nm The suspension of triol monohydrate is moored, the suspension also includes water phase, and the water phase includes being enough to prevent one water of its salts Object nanocrystal is closed to assemble to be formed and/or the nonionic of the amount of crystal growth, polymeric surfactant and acid isopropyl.

Implement technical solution 2. according to the suspension for implementing technical solution 1, wherein the surfactant is selected from pool Lip river sand Nurse or polysorbate surfactant and polyoxyethylene C6-24 alkyl ether.

Implement technical solution 3. according to the suspension for implementing technical solution 2, wherein the poloxamer is selected from poloxamer 124, PLURONICS F87, poloxamer 237, Pluronic/Lutrol F 108 and poloxamer188.

Implement technical solution 4. according to the suspension for implementing technical solution 3, wherein the surfactant is poloxamer 188。

Implement technical solution 5. according to the suspension for implementing technical solution 2, wherein the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 61, polyoxyethylene sorbitan monoleate and sorbimacrogol oleate 100.

Implement technical solution 6. according to the suspension for implementing technical solution 2, wherein the polyoxyethylene C6-24 alkyl ether is Cetomacrogol 1000.

Implement technical solution 7. according to the suspension for implementing any one of technical solution 2-6, wherein the table in the water phase The amount and acid isopropyl of face activating agent are respectively based on the weight of suspension about 0.5% to about 5%.

Implement technical solution 8. according to the suspension for implementing technical solution 1, wherein its salts monohydrate nanometer The mean particle size of crystal such as dynamic light scattering is measured as 200-350nm, 350-400nm or 400-500nm.

Implement the nanocrystal types that such as particle size distribution that dynamic light scattering measures of technical solution 9. is 200-600nm Its salts monohydrate, the nanocrystal can be obtained by method comprising the following steps：

(a) by about 0.5% comprising the water phase weight to about 5% nonionic, polymeric surfactant and about 0.5% to Crystalline state its salts monohydrate reduction in the water phase of about 5% acid isopropyl, leads to the formation of particle, the particle Particle size distribution is about 5-20 μm, about 10 μm of mean particle size；

(d) under about 1200-1700 bars of pressure, the suspension of step (c) is carried out to the high pressure homogenization of third circulation For a period of time, described a period of time is enough to obtain its salts that about 90% or more particle size distribution is 200-600nm The crystal of monohydrate；(e) optionally from water phase separating obtained its salts monohydrate nanocrystal.

Implement technical solution 10. according to its salts monohydrate nanocrystal for implementing technical solution 9, wherein the table Face activating agent is selected from poloxamer or polysorbate surfactant and polyoxyethylene C6-24 alkyl ether.

Implement technical solution 11. according to its salts monohydrate nanocrystal for implementing technical solution 10, wherein described Poloxamer is selected from Pluronic/Lutrol F 44, PLURONICS F87, poloxamer 237, Pluronic/Lutrol F 108 and poloxamer188.

Implement technical solution 12. according to its salts monohydrate nanocrystal for implementing technical solution 11, wherein described Surfactant is PLURONICS F87.

Implement technical solution 13. according to its salts monohydrate nanocrystal for implementing technical solution 10, wherein described Polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 61, polyoxyethylene sorbitan monoleate and polysorbate 81。

Implement technical solution 14. according to its salts monohydrate nanocrystal for implementing technical solution 10, wherein described Polyoxyethylene C6-24 alkyl ether is Cetomacrogol 1000.

It is nanocrystalline according to its salts monohydrate for implementing any one of technical solution 9-14 to implement technical solution 15. Body, mean particle size such as dynamic light scattering are measured as 200-350nm, 350-400nm or 400-500nm.

It is nanocrystalline according to its salts monohydrate for implementing any one of technical solution 9-15 to implement technical solution 16. Body, for freeze-drying or spray-dried forms.

Implement calcium of the preparation of technical solution 17. if particle size distribution that dynamic light scattering measures is 200-600nm and moors three The method of alcohol monohydrate nanocrystal, the method includes the steps：

(d) under about 1200-1700 bars of pressure, the suspension of step (c) is carried out to the high pressure homogenization of third circulation For a period of time, described a period of time is enough to obtain its salts that about 90% or more particle size distribution is 200-600nm The crystal of monohydrate；

Implement technical solution 18. according to implement technical solution 17 method, wherein the reduction of the step (a) by using Diameter is that the ball of 1-4mm such as 1.5-2.5mm or 2-3mm or the wet ball mill of pearl carry out.

Implement technical solution 19. according to the method for implementing technical solution 17, wherein the surface-active used in step (a) Agent is selected from poloxamer or polysorbate surfactant and polyoxyethylene C6-24 alkyl ether.

Implement technical solution 20. according to the method for implementing technical solution 19, wherein the poloxamer is selected from poloxamer 124, PLURONICS F87, poloxamer 237, Pluronic/Lutrol F 108 and poloxamer188.

Implement technical solution 21. according to the method for implementing technical solution 20, wherein the surfactant is poloxamer 188。

Implement technical solution 22. according to the method for implementing technical solution 19, wherein the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 61, polyoxyethylene sorbitan monoleate and sorbimacrogol oleate 100.

Implement technical solution 23. according to the method for implementing technical solution 19, wherein the polyoxyethylene C6-24 alkyl ether is Cetomacrogol 1000.

Implement technical solution 24. according to the method for implementing any one of technical solution 17-23, wherein the surfactant And acid isopropyl is added with about the 1.5 to about 3% of suspension weight, particularly with about 2% amount of suspension weight respectively Into step (a).

Implement technical solution 25. according to the method for implementing technical solution 17, wherein first of the step (b) recycles High pressure homogenization carries out under about 500-650 bars of pressure.

Implement technical solution 26. according to the method for implementing technical solution 17, wherein second of the step (c) recycles High pressure homogenization carries out under about 1000-1100 bars of pressure.

Implement technical solution 27. according to the method for implementing technical solution 17, wherein what the third of the step (d) recycled High pressure homogenization carries out under about 1400-1500 bars of pressure.

Implement technical solution 28. according to the method for implementing any one of technical solution 17-27, wherein the high pressure homogenization walks Suddenly (b)-(d) is carried out using piston crack homogenizer.3

Implement technical solution 29. according to the method for implementing any one of technical solution 17-28, further comprise freeze-drying or It is spray-dried its salts monohydrate nanocrystal.

Implement 30. pharmaceutical composition of technical solution, it includes the calcium according to any one of implementation technical solution 9-16 Moor triol monohydrate nanocrystal and pharmaceutical acceptable carrier.

Implement technical solution 31. according to the composition for implementing technical solution 30, wherein the ionic polymeric surface is living The amount of property agent and acid isopropyl is respectively the about 0.03-0.6% of composition weight.

Implement technical solution 32. according to the composition for implementing technical solution 30, wherein the carrier includes at least one stone Wax, selected from by chain length C5-60, the carbon that chain length maximum value is C14-16, C18-22, C20-22, C20-26, C28-40 and C40-44 The paraffin of or mixtures thereof hydrogen compound composition.

Implement technical solution 33. according to the composition for implementing any one of technical solution 30-32, further includes oily packet Water emulsifier is selected from polyoxyethylene C8-22 alkyl ether, such as polyoxyethylene stearyl ether, polyoxyethylene cetyl base ether or polyoxy second Alkene lauryl ether.

Implement technical solution 34. according to the composition for implementing any one of technical solution 30-33, further includes viscosity Increase ingredient.

Implement technical solution 35. according to the composition for implementing technical solution 34, wherein the viscosity improving components are wax.

Implement technical solution 36. according to the composition for implementing any one of technical solution 30-35, further includes silicon wax Or volatile silicone oils.

Implement technical solution 37. according to the composition for implementing technical solution 36, wherein the volatile silicone oils are ring diformazans Base silicone or dimethyl silicone polymer.

Implement technical solution 38. according to the composition for implementing any one of technical solution 30-37, further includes anti-thorn Swash property compound.

Implement technical solution 39. according to the composition for implementing technical solution 38, wherein the anti-irritation compound is sweet Oil, sorbierite, sucrose, saccharin, menthol, cineole or niacinamide.

Implement technical solution 40. according to the composition for implementing any one of technical solution 30-39, further includes in energy The compound of unfavorable acid impurities with the chemical stability to its salts monohydrate in composition.

Implement technical solution 41. according to the composition for implementing technical solution 40, wherein the compound is amine, such as three Ethanol amine, trishydroxymethylaminomethane, monoethanolamine or diethanol amine.

Implement technical solution 42. according to the composition for implementing any one of technical solution 30-41, the composition is soft Cream.

Implement technical solution 43. according to the composition for implementing technical solution 42, the composition is substantially anhydrous soft Cream.

Implement technical solution 44. according to the composition for implementing any one of technical solution 30-41, the composition is cream Cream.

Implement technical solution 45. according to the composition for implementing any one of technical solution 30-44, it includes about 0.001- 0.5mg/g, preferably about 0.002-0.25mg/g, particularly 0.005-0.05mg/g its salts monohydrate nanocrystal.

Implement technical solution 46. according to the composition for implementing any one of technical solution 30-45, further includes one kind Or various other therapeutic activity ingredients.

Implement technical solution 47. according to the composition for implementing technical solution 46, resists wherein the other active components are selected from Anti-inflammatory drugs such as corticosteroid such as betamethasone and its esters such as valerate or dipropionate, clobetasol or its esters such as third Acid esters, hydrocortisone or its ester such as acetic acid esters；Non-steroid anti-inflammatory medicaments such as naproxen, Indomethacin, Diclofenac, Bu Luo Sweet smell, Dexibuprofen, Ketoprofen, Flurbiprofen, piroxicam, Lornoxicam or Nabumetone, phosphodiesterase 4 inhibitors Or p 38 map kinase inhibitor.

Implement technical solution 48. according to the composition for implementing any one of technical solution 30-47, for treating skin disease Or illness.

Implement technical solution 49. according to the composition for implementing technical solution 48, wherein the skin disease and illness are silver Consider disease, seborrheic psoriasis, palmoplantar pustulosis, dermatitis, ichthyosis, brandy nose or acne to be worth doing.

The present invention is further illustrated by the examples that follow, and it is special that the embodiment is not intended to limit requirement The scope of the present invention of benefit protection.

【Embodiment】

Embodiment 1

The preparation of its salts monohydrate nanocrystal

4g PLURONICS F87 and 4g acid isopropyl are dissolved in the water of the laboratory 196ml, stirred simultaneously, and appropriate amount is added NaOH pH is adjusted to 8.5.

3.5g 2mm glass marble is weighed in the bottle for being equipped with nut.0.035g its salts monohydrate is added to In each bottle, then the solution of 2% PLURONICS F87 of 1.05g and 2% acid isopropyl is added in each bottle.? 2000 revs/min of oscillations on orbital shaker, to grind its salts monohydrate.

After grinding, the bottle and glass marble for grinding are washed with the laboratory 24.0g water pH8.5, and by its salts one Hydrate suspension pours into blue lid bottle.Suspension is transferred in EmulsiflexC3 (Avestin) high-pressure homogenizer, it will be blue Lid bottle is washed with the laboratory 4.9g water pH8.5.500 bars progress high pressure homogenization 10 minutes, 1000 bars carry out 10 minutes and 1400 bars carry out 10 minutes.After high pressure homogenization, the cylinder of Emulsiflex device is washed with the laboratory 4.9g water pH8.5, then Particle size distribution is measured using ZetasizerNanoZS90, by dynamic light scattering, is 200-600nm, mean particle size For 350-400nm.By Raman spectroscopy, by the Raman spectrum of nanocrystal and one water of its salts without carrying out nanosizing The Raman spectrum for closing object compares, and determines that obtained nanocrystal is its salts monohydrate.

Embodiment 2

The ointment of the nanocrystal of monohydrate containing its salts

The ointment of composition shown in the following table 1-1 is prepared as follows：Be heated to 80-85 DEG C and be slowly stirred it is lower mixing lipid phase at Divide (hydrocarbon+polyoxyethylene -2- stearyl ether+alpha-tocopherol+acid isopropyl).By disodium ethylene diamine tetraacetate and phosphoric acid Disodium hydrogen dihydrate is dissolved in proper amount of adjust to one water of aqueous its salts for containing 50 μ g/g its salts monohydrates Object nano suspending liquid (preparation as described in Example 1) is closed to prepare water phase.It, will in the case where being stirred and heated to 35-40 DEG C Glycerol is added in suspension, and as needed adjusts the pH of mixture to 8.5 with 1N HCl or NaOH.

Water phase is added in lipid phase and is stirred 30 minutes.Later, obtained ointment is slowly cooled to 32 DEG C hereinafter, simultaneously It is packed into aluminum pipe neutralization and is stored in room temperature.

Table 1-1

Ingredient（mg/g）	Composition A	Composition B	Composition C	Composition D
					Its salts monohydrate nanocrystal	0.05	0.05	0.05	0.05
Disodium hydrogen phosphate dihydrate	0.26	0.26	0.26	0.26
					Atoleine	50	50	50	50
Polyoxyethylene -2- stearyl ether	50	50	50	50
					Disodium ethylene diamine tetraacetate	0.065	0.065	0.065	0.065
Entirely-racemic .a- tocopherol	0.02	0.02	0.02	0.02
					Glycerol	100	100	100	100
Purified water	50	50	50	50
					PLURONICS F87	0.03	0.03	0.03	0.03
Paraffinum molle alba	699.6	699.6	699.6	699.6
					Acid isopropyl	50	-	-	-
Methyl oleate	-	50	-	-
					Rou Dou guan isopropyl propionate	-	-	50	-

Evaluation to suspension

After emulsification, immediately by the suspension prepared in composition A-D use up scattering particle size distribution analysis instrument (FPAR-1000, by Otsuka Electronics Co., Ltd. production) the median diameter record of scatter intensity distribution that is obtained by CONTIN method For partial size, result is shown in table 1-2-1.

The part 2mL is respectively taken out from the suspension prepared in composition A-D, and introduces silication bottle (silicoated Vial, CS-10 are produced by Fuji Glass Co., Ltd.) in, by the bottled standby rubber stopper, and use alumiseal.These are hanged Supernatant liquid is stored 30 days or 60 days at 40 DEG C, then carries out grain diameter measurement as described above.It is evaluated and is prepared according to following standards Variation caused by the partial size measured immediately afterwards, and its result is shown in table 1-2-2.

A：Do not observe that partial size increases or increases but is less than 5nm.

B：Partial size increases as greater than 10nm but less than 20nm.

C：Partial size increases as greater than 20nm but less than 50nm.

D：Partial size increases for 50nm or bigger.

The part 2mL is respectively taken out from the suspension prepared in composition A-D, and introduces silication bottle (CS-10, by Fuji The production of Glass Co., Ltd.) in, by the bottled standby rubber stopper, and use alumiseal.These dispersion liquids are stored 30 at 40 DEG C It or 60 days, and the pH of its salts and amount and suspension in relation to substance is then detected by high performance liquid chromatography, As a result it is shown in table 1-2-1, table 1-2-2.

Table 1-2-1 embodiment 2 stores the evaluation of 0 day stability at 40 DEG C

	Median diameter	Change of size	Its salts content	Related substance	pH
						Composition A	473nm	-	99.5%	0.7%	8.5
Composition B	480nm	-	99.8%	0.3%	8.5
						Composition C	458nm	-	99.9%	0.3%	8.5
Composition D	479nm	-	99.6%	0.6%	8.5

Table 1-2-2 embodiment 2 stores the evaluation of 60 days stability at 40 DEG C

	Median diameter	Change of size	Its salts content	Related substance	pH
						Composition A	-	A	98.7%	1.8%	8.5
Composition B	-	B	95.8%	5.3%	8.5
						Composition C	-	B	92.6%	8.5%	8.4
Composition D	-	C	90.8%	11.1%	8.2

As the result is shown：Under test conditions, its salts in composition A has more satisfying stability.

Embodiment 3

The emulsifiable paste of the nanocrystal of monohydrate containing its salts

By in 80 DEG C of fusing Cetomacrogol 1000s, cetostearyl alcohol, atoleines, oleic acid isopropyl The emulsifiable paste that composition is shown in the following table 2-1 is prepared with paraffinum molle alba.By at 80 DEG C by disodium phosphate dihydrate and chlorination chlorine Three ammonium of acrylic hexamethylene (chloroallylhexaminiumchloride) is dissolved in purified water to prepare water phase.It is stirring Mix it is lower glycerol is added in solution, and as needed, the pH of mixture is adjusted with 1NHCl or NaOH to 8.5.

Water phase and lipid are mixed under homogenizing, and are cooled to 55 DEG C.It is added remaining water under vigorous stirring, and It stirs at low speed and lower gained emulsifiable paste is cooled to 25 DEG C.

At 30 DEG C of <, adjust proper amount of to its salts monohydrate containing 50 μ g/g its salts monohydrates Nano suspending liquid (preparation as described in Example 1) is added in emulsifiable paste and stirs 30 minutes.By in obtained emulsifiable paste loading tube simultaneously Storage is stand-by.

Table 2-1

Ingredient（mg/g）	Composition E	Composition F	Composition G	Composition H
					Its salts monohydrate nanocrystal	0.05	0.05	0.05	0.05
Disodium hydrogen phosphate dihydrate	2	2	2	2
					Cetostearyl alcohol	60	60	60	60
Cetomacrogol 1000	30	30	30	30
					Three ammonium of chlorination Chloroallyl hexamethylene	0.5	0.5	0.5	0.5
Glycerol	30	30	30	30
					PLURONICS F87	50	50	50	50
Atoleine	50	50	50	50
					Paraffinum molle alba	170	170	170	170
Acid isopropyl	50	-	-	-
					Methyl oleate	-	50	-	-
Rou Dou guan isopropyl propionate	-	-	50	-
					Purified water	Add to 1g	Add to 1g	Add to 1g	Add to 1g

Evaluation to suspension

After emulsification, immediately by the suspension prepared in composition E-H use up scattering particle size distribution analysis instrument (FPAR-1000, by Otsuka Electronics Co., Ltd. production) the median diameter record of scatter intensity distribution that is obtained by CONTIN method For partial size, result is shown in table 2-2-1.

The part 2mL is respectively taken out from the suspension prepared in composition E-H, and introduces silication bottle (silicoated Vial, CS-10 are produced by Fuji Glass Co., Ltd.) in, by the bottled standby rubber stopper, and use alumiseal.These are hanged Supernatant liquid is stored 30 days or 60 days at 40 DEG C, then carries out grain diameter measurement as described above.It is evaluated and is prepared according to following standards Variation caused by the partial size measured immediately afterwards, and its result is shown in table 2-2-2.

B：Partial size increases as greater than 10nm but less than 20nm.

C：Partial size increases as greater than 20nm but less than 50nm.

D：Partial size increases for 50nm or bigger.

The part 2mL is respectively taken out from the suspension prepared in composition E-H, and introduces silication bottle (CS-10, by Fuji The production of Glass Co., Ltd.) in, by the bottled standby rubber stopper, and use alumiseal.These dispersion liquids are stored 30 at 40 DEG C It or 60 days, and the pH of its salts and amount and suspension in relation to substance is then detected by high performance liquid chromatography, As a result it is shown in table 2-2-1, table 2-2-2.

Table 2-2-1 embodiment 3 stores the evaluation of 0 day stability at 40 DEG C

	Median diameter	Change of size	Its salts content	Related substance	pH
						Composition E	279nm	-	99.4%	0.7%	8.5
Composition F	257nm	-	99.3%	0.8%	8.5
						Composition G	267nm	-	99.6%	0.5%	8.5
Composition H	273nm	-	99.2%	0.9%	8.5

Table 2-2-2 embodiment 3 stores the evaluation of 60 days stability at 40 DEG C

	Median diameter	Change of size	Its salts content	Related substance	pH
						Composition E	-	A	98.8%	1.5%	8.5
Composition F	-	B	96.8%	3.9%	8.4
						Composition G	-	B	94.4%	6.2%	8.3
Composition H	-	C	91.8%	9.4%	8.1

As the result is shown：Under test conditions, its salts in composition E has more satisfying stability.

Embodiment 4

The non-aqueous ointment of the nanocrystal of monohydrate containing its salts

Its salts monohydrate nano suspending liquid lyophilized overnight that will be prepared as described in Example 1.By will be nanocrystalline Body is dispersed in atoleine and paraffinum molle alba is added in dispersion, will be freeze-drying, substantially anhydrous nanocrystalline Body is used to prepare ointment.

The composition of non-aqueous ointment is shown in the following table 3-1.

Table 3-1

Ingredient（mg/g）	Composition I	Composition J
			Its salts monohydrate nanocrystal	50μg	50μg
PLURONICS F87	0.05mg	0.05mg
			Atoleine	50mg	50mg
Paraffinum molle alba	750mg	750mg

Explanation：Acid isopropyl in embodiment 1 is removed, other with it is identical, prepare composition J with method by composition I later.

Evaluation to suspension

After emulsification, immediately by the suspension prepared in composition I-J use up scattering particle size distribution analysis instrument (FPAR-1000, by Otsuka Electronics Co., Ltd. production) the median diameter record of scatter intensity distribution that is obtained by CONTIN method For partial size, result is shown in table 3-2-1.

The part 2mL is respectively taken out from the suspension prepared in composition I-J, and introduces silication bottle (silicoated Vial, CS-10 are produced by Fuji Glass Co., Ltd.) in, by the bottled standby rubber stopper, and use alumiseal.These are hanged Supernatant liquid is stored 30 days or 60 days at 40 DEG C, then carries out grain diameter measurement as described above.It is evaluated and is prepared according to following standards Variation caused by the partial size measured immediately afterwards, and its result is shown in table 3-2-2.

B：Partial size increases as greater than 10nm but less than 20nm.

C：Partial size increases as greater than 20nm but less than 50nm.

D：Partial size increases for 50nm or bigger.

The part 2mL is respectively taken out from the suspension prepared in composition I-J, and introduces silication bottle (CS-10, by Fuji The production of Glass Co., Ltd.) in, by the bottled standby rubber stopper, and use alumiseal.These dispersion liquids are strong in 25 DEG C and illumination It is stored 30 days or 60 days under degree 4500LX, and then by high performance liquid chromatography detection its salts and in relation to the amount of substance And the pH of suspension, result are shown in table 3-2-1,3-2-2.

Table 3-2-1 embodiment 4 stores the evaluation of 0 day stability under 25 DEG C and illumination

	Median diameter	Change of size	Its salts content	Related substance	pH
						Composition I	577nm	-	99.3%	0.9%	8.5
Composition J	562nm	-	99.4%	0.6%	8.5

Table 3-2-2 embodiment 4 stores the evaluation of 60 days stability under 25 DEG C and illumination

	Median diameter	Change of size	Its salts content	Related substance	pH
						Composition I	-	A	98.4%	1.8%	8.5
Composition J	-	C	91.4%	9.3%	8.0

As the result is shown：Under test conditions, its salts in composition I has satisfactory stability.

Vitro skin in 5 monkey abdomen cadaver skin of embodiment is penetrated/is permeated

The preparation of monkey skin

Before experiment starts, vitrification -20 DEG C about 4 months.The skin samples of defrosting Aesculap dermatotome (Aesculap AG, Tuttlingen, Germany) is cut to 500 μm of thickness, is sheared to be allowed to be suitble to diffusion zone, and will Between its supply chamber and receiving chamber for being assembled to the diffusion pond Franz (diffusion Franz cells) (Franz 1975).

The measurement of skin integrity

The integrality of skin by penetrate through skin tritium-labeled water (³H₂O it) is evaluated and is determined；By 400 μ L³H₂O (0.1MBq/mL) is applied to skin surface.By 30 minutes balances, with cotton swab handle³H₂O is removed from skin；2mL is taken to connect By phase (composition of acceptable solution described below), skin is penetrated with measurement³H₂The score of O.The radioactivity received in phase is strong Degree is in liquid scintillation system 2500TR (Liquid Scintillation Systems 2500TR) (Packard Instr.Co., Meriden, CT, USA) in use liquid scintillation count measurement.Quenching correction is carried out using external standard method.With sealing Calibration curve is quenched in standard items (Packard Instr) foundation.

External percutaneous Cutaneous permeation and the determination penetrated into skin

Skin is used as and separates the supply chamber in the static state pond Franz (volume of about 7.3mL, the inside diameter of 16mm) manually and connects By the film of room.Receiving chamber is full of acceptable solution (aqueous solution of 0.5% Brij78 [Volpo20]), to simulate the physiological condition of the mankind With drug from the systematic removal of skin.The acceptable solution also other penicillin/streptomycin mixture containing 100U/mL1%, with Prevent microbial contamination.

For effective skin area of diffusion and the volume of receiving chamber respectively 1.78 to 2.14cm²(average 2.01cm²) model In enclosing and in the range of 6.98 to 7.54mL.The temperature in pond is kept constant using 34 ± 1 DEG C of circulator bath.Entirely testing Period persistently uses magnetic stirring bar, to guarantee the homogeneity received.

The collection and processing of sample

The skin being mounted on the diffusion pond Franz is applied to using the composition A-D preparation of the amount of 243-305mg as single dose (correspond to sampling time=0h, each preparation corresponds to 3-4 pond) on sample.Preparation is left on skin 48 hours.In order to avoid The evaporation and drying of preparation, the parafilm (M) of the supply chamber apertures in the pond Franz is semiclosed.For penetrating through skin 4,7,20,25.5,28,31,44 and 48 hours after application, the acceptable solution of 200 μ L is collected from receiving chamber for the measurement of activating agent Aliquot.The volume taken out from receiving chamber uses the fresh acceptable solution of same volume to replace every time, to be maintained at entire measurement The constant volume of period total acceptable solution.

At the end for the treatment of phase (applying rear 48h), the residual preparation on each skin samples surface is carefully removed with cot ton tip applicator, Application region is washed with aqueous cotton, and new cotton applicator is softly dried.The process is in triplicate.Then with commercially available viscous Adhesive tape (Scotch550,3M) separates cuticula through 21 adhesive tape peel strip bands from skin.Discard first peel strip Remaining 20 peel strip band is put into bottle (number 2-6,7-11,12- to avoid the potential pollution for carrying out self-preparing agent by band 16, the peel strip band of 17-21 pools together).The living tissue specimen of the treatment region of the skin of removing is punched with 1.2cm diameter Device is acquired and is weighed.Before analysis by the skin samples freezing of acceptable solution, adhesive tape peel strip band and removing and in -20 DEG C of preservations. The concentration of tacrolimus in sample is used the LC-MS/MS by verifying to analyze and is determined；Lower limit of quantitation is 0.5ng/mL (acceptable solution And peel strip band) or 5ng/g (skin samples).

Table 4-1 result (mean ± SD [range], n=1-4)

Preparation/its salts	Composition A	Composition B	1% in propylene glycol/ethyl alcohol 7:In 3 (v/v)
				Concentration in cuticula：The 2-21 articles peel strip band (ng/cm²)	2235±1342	1453±1235	46±24
Concentration (ng/cm in skin after 48hr²)	53±33	34±27	2163±1322
				(Lag time) (hr) is rushed when lag	13.6	16.6	22.6

Table 4-2 result (mean ± SD [range], n=1-4)

Preparation/its salts	Composition A	Composition C	1% in propylene glycol/ethyl alcohol 7:In 3 (v/v)
				Concentration in cuticula：The 2-21 articles peel strip band (ng/cm²)	2343±1355	1164±1053	45±26
Concentration (ng/cm in skin after 48hr²)	58±32	35±25	2535±1276
				(Lag time) (hr) is rushed when lag	12.7	18.3	21.8

The results show that composition A vitro skin penetrate/osmotic effect is significantly higher than composition B/C.

Embodiment 6

The biological activity of composition

Antibacterial peptide is the antimicrobial peptide expressed in human keratinized cell.On the skin of infection or the skin barrier of destruction, The expression of antibacterial peptide is by induced strong.In psoriasis, the level of antibacterial peptide is to increase in the affected skin of psoriatic 's.It has been found that vitamine D3 or novel vitamin D analogues such as its salts are by can induce coding in conjunction with vitamin D receptor The gene of antibacterial peptide expression (referring to TTWang et al., J.Immunol.173 (5), 2004, the 2909-2912 pages；J Schauber et al., Immunology 118 (4), 2006, the 509-519 pages；Schauber and Gallo, J. Allergy Clin Immunol122,2008, the 261-266 pages；M.Peric et al., PloSOne4 (7), on July 22nd, 2009, e6340).The discovery is applied to develop a kind of measuring method, wherein encoding the induction water of the gene of anti-mattress peptide by measurement It is flat, it has been determined that intake and biological activity of its salts from test composition in human keratinized cell.

In the measuring method, by its salts monohydrate nanocrystal emulsifiable paste (group of the preparation as described in example 3 above Close object G) it is applied topically to reconstruct people's epidermis in triplicate with the amount of 10 μ l, the reconstruct people epidermis is by 0.5em2 polycarbonate 12 days normal human keratinized cell compositions are cultivated on filter (being available from Laboratories, Nice, France).In cell factor The tissue 1 or 2 days are handled in the presence of IL-17A (20ng/ml), IL-22 (20ng/ml) and TNF-α (5ng/ml), then from poly- Epidermis is separated in carbonic ester filter, and quick-frozen in liquid nitrogen.RNA is extracted from cell by conventional method and synthesizes cDNA.So Afterwards using the test CAMPHs0018038_m1 and GAPDHHs99999905_m1 from Applied Biosystems below into Row real-time quantitative PCR (qPCR).The expression of antibacterial peptide is normalized through GAPDH, and by carrying out compared with composition H Relative quantification.As the result is shown in the following table 5.

Table 5

	It activates the 1st day	It activates the 2nd day
			Composition E	3.3	6.8
Composition F	2.1	4.3
			Composition G	1.6	3.1
Composition H	1.0	2.3

As the result is shown：The application of composition E produces and about 1.5-3 times of the activation effect with other emulsifiable pastes.

Composition A, B, C and D of the preparation as described in example 2 above are used for the triplicate topical application of amount of 10 μ l On people's epidermis of reconstruct, people's epidermis of the reconstruct is by 0.5cm²Polycarbonate filter (it is available from Laboratories, Nice, France) on cultivate 12 days normal human keratinized cell form.The tissue 1 or 2 days are handled, then from polycarbonate filter Middle separation epidermis, and it is quick-frozen in liquid nitrogen.RNA is extracted from cell by conventional method and synthesizes cDNA.Then using following Test CAMPHs0018038_m1 and GAPDHHs99999905_m1 from AppliedBiosystems carry out qPCR.It will resist The expression of bacterium peptide is normalized through GAPDH, and by carrying out relative quantification compared with composition D.

As the result is shown in the following table 6.Table 6

	It activates the 1st day	It activates the 2nd day
			Composition A	3.4	6.6
Composition B	2.2	4.3
			Composition C	1.5	3.1
Composition D	1.0	2.2

As the result is shown：The application of composition A produces and about 1.5-3 times of the activation effect with other emulsifiable pastes.

Embodiment 7

Local tolerance research in miniature pig

By dermal application, daily administration assessed the local tolerance of composition A, B, C and D of embodiment 2 to miniature pig 4 weeks. Ointment is used to compare.Animal is daily exposure to test article 8 hours.The research is carried out in SPF miniature pig.Every animal has 6 practical sites receive the test formulation of 250mg amount in 10 each practical sites of female.Before administration starts and On the day of ptomatopsia, clinical indication is recorded daily, and score once a day the dermoreaction of practical site in relation to erythema and oedema. Record food consumption daily, and weight is recorded weekly.In process phase finally, carry out total ptomatopsia to all animals, and receive Collect skin samples, is used for histopathological examination.

As the result is shown：Composition A, B are not observed bad treatment relevant clinical indication during research, but composition C, D observes 1-2 grades of dermoreaction（Erythema）.As the result is shown：Composition A, B ointment can be more preferable resistance in human patients By.

Claims

1. as its salts one for the nanocrystal types that particle size distribution that dynamic light scattering measures is 200-600nm is hydrated The suspension of object, the suspension also include water phase, and the water phase includes being enough to prevent its salts monohydrate nanocrystal Aggregation is formed and/or nonionic, polymeric surfactant and the acid isopropyl of the amount of crystal growth.

2. the suspension of claim 1, wherein the surfactant is selected from poloxamer or polysorbate surfactant, with And polyoxyethylene C6-24 alkyl ether.

3. the suspension of any one of claim 1-2, wherein in the water phase surfactant amount and acid isopropyl About 0.6% to about 5% respectively based on the weight of suspension.

4. as its salts one for the nanocrystal types that particle size distribution that dynamic light scattering measures is 200-600nm is hydrated Object, the nanocrystal can be obtained by method comprising the following steps：

5. preparing its salts monohydrate nanocrystal such as particle size distribution that dynamic light scattering measures for 200-600nm Method, the method includes the steps：

(d) under about 1200-1700 bars of pressure, the suspension of step (c) is carried out to the high pressure homogenization of third circulation For a period of time, described a period of time is enough to obtain its salts that about 90% or more particle size distribution is 200-600nm The crystal of monohydrate.

6. pharmaceutical composition, it includes its salts monohydrate nanocrystals and pharmaceutical acceptable carrier described in claim 5.

7. the composition of claim 6, the composition is ointment.

8. the composition of claim 6 further includes one or more other treatment active constituents.

9. the composition of any one of claim 6, for treating skin disease or illness.

10. the composition of claim 9, wherein the skin disease and illness are psoriasis, seborrheic psoriasis, palm plantar warts Disease, dermatitis, ichthyosis, brandy nose or acne.