CN1169114A - Sequestration agents - Google Patents
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- CN1169114A CN1169114A CN95196708.8A CN95196708A CN1169114A CN 1169114 A CN1169114 A CN 1169114A CN 95196708 A CN95196708 A CN 95196708A CN 1169114 A CN1169114 A CN 1169114A
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Abstract
The invention provides the use of an agent to reduce direct interaction between a hydrophobic phase and a hydrophilic phase in which the hydrophobic phase is dispersed. Methods for preparing single phase hydrophobic preparations comprising a hydrophilic species wherein such an agent is added are also provided.
Description
The present invention relates to when hydrophobic when being scattered in the aqueous favoring mutually, can promote generally can not be dissolved in the hydrophilic molecule of dredging aqueous phase purposes at said thin aqueous phase dissolving and certain chemical compound of keeping.The invention particularly relates to this kind reagent promotes the hydrophilic macromole in the ordinary course of things and be insoluble to the purposes that thin aqueous phase wherein keeps.
Because protein has limited they and the lipoid interaction mutually or the degree of infiltration with similar macromolecular hydrophilic and high polarity, they are all having problems such as many applications such as medicine, food or cosmetics industries.Many organisms have lipoid border (as skin, cell membrane) and enter into inside to prevent hydrophilic molecule; If in the lipoid medium, can disperse protein, will open up a new way for these macromole enter into living things system.Containing proteinic lipoid medium can combine with the hydrophobicity formation or the composition on border, rather than is ostracised.
The stability that hydroaropic substance is scattered in oil phase rather than the water-bearing media improving hydroaropic substance has multiple benefit, causes degeneration, hydrolysis and to aspects such as photaesthesia as temperature.Compare with aqueous solution, selected oil phase can keep liquid or have higher viscosity in wider temperature range, can play bigger protective effect, prevents the destruction of rerum natura.In the mixed phase system, in oil phase, add harmful interaction that lyophobic dust can reduce oil phase and aqueous phase and other reagent simultaneously, for example oxidation.
Now existing many examples that contain the combination of macromole and oily substance disclose such example in EP-A-0366277.The compositions of this patent disclosure is one and contains hydrophobic phase and aqueous-favoring emulsion, the wherein hydrophobic lipoid that contains chyle mutually or form chyle.But this macromolecular substances is dissolved in the aqueous favoring rather than is dissolved in thin aqueous phase.
EP-A-0521994 has also related to a kind of macromolecular oral liquid compositions, and it contains the biological active substances of lecithin class or can be used as the chemical compound of lecithin precursor in the body.All compositions examples all comprise aqueous favoring and oleophylic mutually.The same with previous patent, macromolecular substances just is dissolved in the aqueous favoring, rather than be dissolved in oleophylic mutually in.
Though above-mentioned compositions contains macromole and oils really simultaneously, only be only soluble in the aqueous favoring when macromolecular substances begins in all cases, rather than be dissolved in oleophylic mutually in.The effort of the true solutions of preparation macromole in oil phase has only obtained limited success.
Okahata etc. (J.Chem.Soc.Chem.Commun., 1988,1392-1394) a kind of method that is dissolved in the hydrophobic solvent that protein is added is disclosed.But the author points out that in many protein that surrounded by amphiphile, amphiphilic molecule, amphiphile, amphiphilic molecule has formed solid sediment at aqueous phase by hydrogen bond or by electrostatic interaction and protein interaction.
UK Patent Application UK No.9323588.5 discloses a kind of hydrophilic species can being added and has been dissolved in it and is insoluble to method in wherein the hydrophobic solvent generally speaking.This method is based on a beat all discovery, if promptly the hydrophilic species are mixed with amphiphilic substance under given conditions, the compositions that then obtains will be soluble in hydrophobic solvent such as the oils.
But potential problem of this method is to make emulsion with it, promptly for example in the water monophasic hydrophobic formulation is disperseed in aqueous favoring.In this dispersion, will exist by the hydrophilic species of solubilization contrary in the solubilization process and " leakages " tendency in the aqueous favoring is like this under some occasion at least.Therefore even need reduce this effect when guaranteeing that hydrophobic phase will be dispersed in the aqueous favoring subsequently, the hydrophilic species still are retained in thin aqueous phase.
Now unexpectedly find, be scattered in the aqueous favoring mutually as when forming emulsion when hydrophobic, certain chemical compound can reduce add be dissolved in hydrophobic in mutually the hydrophilic species and the degree of the direct interaction between the aqueous favoring.
Therefore, the present invention at first provides a kind of reduction to add to be dissolved in the purposes of the reagent of direct interaction between hydrophobic hydrophilic species in mutually and the hydrophobic aqueous favoring that is scattered in mutually wherein.
" reagent " speech is meant when hydrophobic and itself is scattered in mutually when for example forming emulsion in the aqueous favoring among the present invention, can reduce to add to be dissolved in general and to be insoluble to the hydrophilic species of thin aqueous phase wherein and any species of aqueous favoring direct interaction.
Secondly the present invention provides to contain to be added and is dissolved in single-phase hydrophobic formulation general and that be insoluble to the hydrophilic species in wherein the hydrophobic solvent and can reduce the reagent of direct interaction between hydrophilic species and the hydrophobic aqueous favoring that is scattered in mutually wherein.
Obviously, though the hydrophilic species may contact with the one molecule of aqueous favoring, it does not contact with the aqueous favoring body, has so just reduced " leakage " of hydrophilic species in aqueous favoring.
" hydrophilic species " speech is meant and generally dissolves in the aqueous solvent but be insoluble to any species in the hydrophobic solvent among the present invention.Suitably, reagent can be:
(I) acid lipoid, for example CHEMS (Chems) or phosphate acid ester; Or
(ii) can not infiltrate the emulsion stabilizer of dredging aqueous phase, for example, the chemical compound of casein and so on.
The 3rd, the present invention also be provided for reducing add be dissolved in general and be insoluble in wherein the hydrophobic solvent the hydrophilic species with hydrophobic be scattered in mutually wherein as the aqueous favoring of formation emulsion between a kind of reagent of direct interaction.
Reagent described herein is suitable for adding in the dissolution method described in the UK Patent Application UK No.9323588.5.Therefore, the present invention further also is provided at the preparation method of the single-phase hydrophobic formulation that contains the hydrophilic species in the hydrophobic solvent, and this method comprises:
(i) in liquid medium, hydrophilic species and amphiphile are associated, in this liquid medium, do not have chemical interaction between amphiphile and the hydrophilic species;
(ii) liquid medium is removed, the hydrophilic end group of remaining amphiphile, amphiphilic molecule is towards hydrophilic species orientations; And
(iii) around hydrophilic species/amphiphile arrangement, add hydrophobic solvent;
Wherein, in above-mentioned one or more steps, add to reduce the reagent of direct interaction between hydrophilic species and the hydrophobic aqueous favoring that is scattered in mutually wherein.
This reagent preferably adds with amphiphile in (i) step simultaneously in this method, and preferably a kind of emulsion stabilizer that can not infiltrate thin aqueous phase, as CHEMS (Chems) or phosphatidic acid (PA).
" chemical interaction " is meant the interaction as covalent bond or ionic bond or hydrogen bond among the present invention.Do not comprise the interaction of Van der Waals force or other this order of magnitude.
The present invention provides a kind of single-phase hydrophobic formulation that will contain the hydrophilic species in hydrophobic solvent to be scattered in method in the aqueous favoring on the other hand, and it comprises in aqueous favoring and adds a kind of step that reduces the reagent of direct interaction between hydrophilic species and the aqueous favoring.
In the method, reagent preferably can not infiltrate the emulsion stabilizer of dredging aqueous phase, for example, and the chemical compound of casein and so on.
The macromolecular substances of numerous species can be suitable for method of the present invention and carry out solubilization.This macromolecular compound generally is hydrophilic, or has hydrophilic position at least, and solubilization hydrophobicity macromole is generally seldom had any problem in oily solution.Suitable macromolecular example comprises: protein and glycoprotein, and oligomerization and Polynucleotide be DNA and RNA for example, and the polysaccharide and the supermolecule assembly of these molecules arbitrarily comprise whole cell, organellae or virus (whole or part wherein) sometimes.Also micromolecule such as vitamin and macromole, particularly polysaccharide such as cyclodextrin can be associated and cosolubilization.Micromolecule such as vitamin B
12Also can carry out chemical bond-linking, therefore be also included within the compositions with macromole.
Especially, when being protein or polypeptide by the macromolecular substances of solubilization, reagent is preferably acid lipoid.
The concrete proteinic example that can successfully carry out solubilization with method of the present invention comprises insulin, calcitonin, hemoglobin, cytochrome C, horseradish peroxidase, aprotinin, Mushroom Tyrosinase, erythropoietin, growth hormone (somatotropin), growth hormone (growth hormone), somatotropin releasing factor, galanin, urokinase, factors IX, organize plasminogen activator, superoxide dismutase, catalase, peroxidase, ferritin, interferon, Factor IX, melanocyte and their fragment (above all protein can have various sources).The zymic RNA extract of glucosan that other operable protein is the FITC labelling and Torula.
Except macromolecular substances, method of the present invention also can be used for the solubilization organic molecule.The example of organic molecule comprises for example anticarcinogen of glucose, ascorbic acid, carboxyl resorcinolphthalin. and many pharmaceutical agents, and this method is equally applicable to other organic molecule certainly, for example other vitamin or pharmacology or biological active substances.In addition, carry out solubilization such as the also available method of the present invention of the molecule of calcium chloride and calcium phosphate.In fact, owing to use non-aqueous solution, improved molecule and entered intravital approach, for example improved biological effectiveness, the present invention can be particularly advantageous for pharmacology and biological active substances.
The another kind of material that may comprise in hydrophobic composition of the present invention is an inorganic material, for example inorganic molecules or colloidal substance, for example colloidal metal.Method of the present invention can make colloidal metal such as aurosol, palladium, platinum or rhodium even keep some character of its colloidal metal in hydrophobic solvent, and under normal operation, these granules are agglomerative in hydrophobic solvent.This catalysis for the reaction of carrying out in organic solvent is particularly useful.
Have considerable amphiphile to can be used among the present invention, wherein amphion amphiphile such as phospholipid are found to be particularly suit a kind of.The application of phospholipid that contains phosphatidylcholine (PC) end group is particularly successful, the example of this type of phospholipid comprises phosphatidylcholine itself (PC), LYSO-PHOSPHATIDYLCHOLINE LYSOPC (lyso-pc), sphingomyelin, in these phospholipid any one derivant such as HEXADECYL PHOSPHOCHOLINE or contain the amphipathic polymer of Phosphorylcholine and the halogenation amphiphile as fluoridizing phospholipid.In should using, phosphatidylcholine (PC) and lecithin are equal to.The natural phosphatidyl choline that is suitable for can be any source, for example egg and particularly Semen sojae atricolor.Under most situation, preferably chemically close amphiphile with used hydrophobic solvent, this will give more detailed discussion below.
The inventor finds that amphion amphiphile such as phospholipid are specially adapted to the inventive method, and it is obviously different with the method for Okahata etc. that this further demonstrates the present invention.The author of aforementioned patent sums up and thinks that anion and amphoteric ion type lipoid are not suitable for their method fully, and the productive rate of complex is zero when pointing out to adopt these lipoids.
The selection of hydrophobic solvent will depend on the final use of compositions, by the type of solubilization species and amphiphile.The solvent that is suitable for comprises: nonpolar oils, as mineral oil, squalane and Squalene; Long-chain fatty acid, wherein preferred unsaturated fatty acid is as oleic acid and linoleic acid; The long-chain alcohol of the alcohol of alcohols, particularly medium chain such as capryl alcohol and branching such as phytol, isoprenoid are as spending pure and mild geraniol, terpinol; Monoglyceride is as glyceryl monooleate (GMO); Other esters is as ethyl acetate, pentyl acetate and borneol acetate; The triglyceride of Diglyceride and triglyceride, particularly medium chain and their mixture, the halogenation congener of aforementioned substances comprises halogenation oils, for example long-chain fluorine carbon hydrocarbon or iodate triglyceride, for example class lipidol arbitrarily.
When hydrophobic solvent and amphiphile approximate match, generally can reach optimal results.For example, when oleic acid was made solvent, LYSO-PHOSPHATIDYLCHOLINE LYSOPC (lyso-PC) was than the more efficiently amphiphile of phosphatidylcholine (PC), and when hydrophobic solvent was triglyceride, situation was then just in time opposite.
Find in addition, in some cases, hydrophobic and hydrophilic species/amphiphile arrange contact before, it is more beneficial to add a certain amount of amphiphile in hydrophobic solvent.This guarantees that amphiphile, amphiphilic molecule can be owing to amphiphile breaks away from from the position around the hydrophilic species the high affinity of hydrophobic solvent.
Preparation very preferably of the present invention is optically transparent, can be by measuring turbidity or detect through detecting its sedimentation situation after a while sometimes in visible wavelength.
Can realize that amphipathic molecule aligns towards the hydrophilic species position with its terminal hydrophyllic group by several approach, the example of the method for particularly suitable is discussed below in more detail.
First method, similar (the biological engineering (Biotechnology) of starting point of the method that its starting point and Kirby etc. describe, in November, 1984,979-984, and LiposomeTechnology, I volume, 19-27 page or leaf, Gregoriadis, Ed, CMC Press, Inc., Boca Raton, Florida, USA), the hydrophilic species are mixed with the dispersion bulk phase of amphiphile in hydrophilic solvent, amphipathic molecule is just arranged like this, and its hydrophilic end group is to the outside to the aqueous favoring that contains the hydrophilic species.Remove hydrophilic solvent then to stay dried compositions, wherein the hydrophilic end group of amphipathic molecule is just directed towards the hydrophilic species.
In described methods such as Okahata, also be that protein solvent is mixed with the dispersion bulk phase of amphiphile in water.But obviously, these authors believe, need obtain a kind of precipitate that dissolved in afterwards in the hydrophobic solvent.Because the preferred amphiphile of many present invention does not generate precipitate, they are otiose for identifications such as Okahata.This does not require in the method for the invention to generate precipitation, and it is generally acknowledged that in fact sedimentary generation is undesirable, because can reduce the productive rate of expection product.
In first method, though also can use other polar solvent, preferred water is as hydrophilic solvent.
The set form that amphiphile is taked can be micelle, unilamellar vesicle (the preferred general diameter of understanding is about the little unilamellar vesicle of 25 nanometers), multilamellar vesicle or tubular-shaped structures (for example helical form is cylindrical), hexagon, cube or myelin type structure.The set form depends on selected amphiphile, and the amphiphile that for example resembles phosphatidylcholine (PC) tends to generate unilamellar vesicle, and LYSO-PHOSPHATIDYLCHOLINE LYSOPC then generates micelle.But in all these structures, the hydrophobicity tail of amphiphile, amphiphilic molecule inwardly stretches to the center of structure, and the hydrophilic end group stretches to the solvent that is dispersed with the hydrophilic species outwardly.
The weight ratio of amphiphile and hydrophilic species generally in 1: 1 to 100: 1 scope, is preferably 2: 1 to 20: 1, for particularly preferred about 8: 1 of PC, and for lyso-PC, particularly preferred 4: 1.
These ratios are preferred ratio only, special needs to be pointed out is that its upper limit is for economically consideration and fixed, promptly preferably adopt a small amount of as far as possible amphiphile.Lower limit is comparatively crucial, and it seems that 2: 1 or lower ratio only be applicable to that the hydrophilic species have very big hydrophobic part or unusual big situation itself.
Remove the effect desolvate and can obtain down quickly, a kind of to remove the facilitated method of desolvating be lyophilization, but also can adopt other method.
Containing salt in some cases in hydrophilic solution may be helpful, if when particularly the hydrophilic species are macromolecular compound such as big protein.But, because therefore the appearance that the existence of a large amount of inorganic salts is tended to cause crystalline generation and therefore caused turbid solution may preferably adopt organic salt rather than inorganic salt such as sodium chloride.Ammonium acetate is owing to there being extra advantage especially to meet this needs, because it more easily is removed by lyophilization.
Second kind of method for compositions for preparing the amphiphile arrangement that contains its head group sensing hydrophilic species hydrophilic site is that hydrophilic species and amphiphile are carried out cosolubilization in common solvent, and then removes and desolvate.
The product of this method of the present invention is new, thereby also is one aspect of the present invention, and it provides the single-phase hydrophobic formulation of the hydrophilic species that contain in hydrophobic solvent.
In single-phase hydrophobic formulation, except that the hydrophilic species, also can comprise other composition.This point is suitable especially when the hydrophilic species are macromole, and this moment, preparation may contain and macromole bonding or associating molecule, for example bile salts, vitamin or other micromolecule.
Although Kirby etc. disclose some macromole/amphiphile and arranged thing, disclosed arrangement thing all is the intermediate that generates liposome, and as preceding discussion, they do not recognize non-liposome or the hydrophobic composition that contains this arrangement thing.Therefore, amphiphile does not form unilamellar vesicle in arrangement thing of the present invention, therefore can not generated liposome by expection yet, and this arrangement thing is new.
An advantage of preparation of the present invention is that they are anhydrous, thereby is more stable for hydrolysis.Under proteinic situation, they also are stable to freezing molten, and high temperature is also had bigger stability, may be because for making the cause that protein launches and degeneration must have water to exist.This means that the hydrophilic species can have longer storage period than their water soluble preparation.
Solution of the present invention has outstanding many-sided suitability, can be used in many application.They can use separately, but preferably with they with contain water and mix to form emulsion or similar two-phase compositions, this has also constituted one aspect of the present invention.
Of the present invention this on the one hand in, provide and contained aqueous favoring and hydrophobic two-phase compositions mutually, thin aqueous phase contains the preparation of hydrophilic species in lipophilic solvent that can obtain by method as herein described.
Usually, in this compositions, hydrophobicly will be scattered in the aqueous favoring mutually.
This two-phase compositions can be an emulsion, and it can be can be stable emulsion also temporarily, depends on the purposes that they are required.
The average-size of emulsion particle depends on hydrophobic phase and water-soluble both characteristics mutually own.But it can be about 2 microns.
With hydrophobic formulation water-soluble mutually in dispersion can realize by mixing, for example can pass through intensive eddy current of short time, 10 to 60 seconds according to appointment, usually at about 15 seconds, also can mix slowly, as using the orbital oscillation device by several hours.
The present invention provides a kind of preparation to add the dispersion of the hydrophobic phase that is dissolved with the hydrophilic species such as the method for emulsion on the other hand, comprise being dispersed in mutually in the aqueous favoring, wherein add a kind of the reduction by the reagent of direct interaction between the hydrophilic species of so solubilization and the hydrophobic aqueous favoring that is scattered in mutually wherein with hydrophobic.
The present invention will obtain explanation by the following examples, and these embodiment should not be counted as any qualification that the present invention is carried out.Embodiment 1
60 milligrams of sodium tetraborates are dissolved in 100 ml distilled waters, and pH value is transferred to 8.00, obtain the borate buffer solution that concentration is 0.0015 mol.In having the B9 glass vial of cap nut, take by weighing 5 milligrams of BAPNA, add the dissolving of 3 ml methanol.Take by weighing 10 milligrams of trypsin in 15 milliliters of plastic centrifuge tubes, eddy current stirs and adds 10 milliliters of borate buffer solutions down.Mix suspending body in the roll forming blender is then with insoluble matter centrifugal sedimentation, with supernatant decant.
Put aprotinin diluent (every pan 50 microlitre aprotiniies) along the sequence branch of shallow bid, concentration is respectively 0 to 30 mcg/ml.
The buffer that adds 1 milliliter to 20 milliliters in the superincumbent BAPNA solution dilutes 20 times, adds 100 microlitre BAPNA working solutions and thorough the mixing then in every pan.Shallow bid was cultivated 40 minutes 37 ℃ of following joltings, on the disc type reader of 405 nanometers, carried out reading then.
The optical density that will produce owing to the variation of measured object can be observed a flex point to the concentration mapping of aprotinin in the pan, and under this concentration, aprotinin enough neutralizes tryptic activity just.Move with the addition that joins the aprotinin in the specimen in pan the position of this flex point, by relatively knowing the concentration of flex point by inference with standard sample.The aprotinin that can obtain from oil, discharging and enter into the ratio of the aprotinin of aqueous phase thus.
S-PC dispersion (100 mg/ml that 100 microlitres are obtained by ultrasonicization of scheme of embodiment 4, in the distilled water) and 25 microlitre aprotinin solution (20 Bo grams per milliliters, in the distilled water) the mixture lyophilization, add 100 microlitre Miglyol 818 then, aprotinin is added be dissolved among the Miglgol 818.The concentration of aprotinin is 5 mg/ml in oil phase.Do not add aprotinin by last method and prepare comparative solution.Every kind of oil of 10 microlitres and 1 milliliter of borate buffer solution were stirred for 10 seconds, obtain the dispersion of these oil in water.The ultimate density of aprotinin is 0 to 50 mcg/ml in these secondary dispersions.Be added in the pan of shallow bid by the described branch of preceding method after dispersion diluted twice, wherein the concentration of diluent is respectively 0,5,10,15 and 25 mcg/ml.Normalized optical density be listed in the table below and accompanying drawing in.The contrast standard specimen of 12.5 mcg/ml concentration relatively shows to have at least 50% aprotinin to discharge into aqueous phase from oil.
Embodiment 2
| Oil+/-aprotinin | Aprotinin concentration | |||||
| ???0 | ???5 | ??10 | ??15 | ???20 | ????25 | |
| ????-/PC/M818 | ?0.373 | ?0.358 | ?0.343 | ??0.3 | ??0.055 | ????0 |
| Aprotinin/PC/M818 | ?0.269 | ?-0.004 | ?0.05 | ??0.058 | ??-0.03 | ????0 |
| 12.5 μ g/ml aprotinin | ?0.337 | ?0.299 | ?0.135 | ??-0.008 | ??-0.017 | ????0 |
| Aprotinin/PC/M818 doubling dilution | ?0.332 | ?0.264 | ?0.201 | ??-0.036 | ??-0.055 | ????0 |
Be dissolved among the Miglyol 818 by a last embodiment described aprotinin is added, but contain the phosphatidic acid of phosphatidylcholine and 10% (weight) in the phospholipid dispersion.Each dispersion of sequential testing, and compare with contrast standard specimen 25 mcg/ml and 12.5 mcg/ml.Through and these standard specimens relatively, show that being no more than 25% aprotinin is released to aqueous phase.
Embodiment 3
| Aprotinin concentration (mcg/ml) | 20 | 15 | 10 | 5 | 0 |
| Buffer | 0.234 | 0.26 | 0.273 | 0.283 | 0.277 |
| 25 μ g/ml aprotiniies | 0 | 0.003 | 0.005 | 0.068 | 0.167 |
| ????12.5μg/ml | 0 | -0.009 | 0.202 | 0.258 | 0.258 |
| ????PC：PA/m818-0 | 0.099 | 0.182 | 0.164 | 0.194 | 0.216 |
| ??PC：PA/M818-50μg/ml | 0 | 0.059 | 0.182 | 0.219 | 0.245 |
Be dissolved among the Miglyol 818 by front embodiment described aprotinin is added, but contain the CHEMS (chems) and the phosphatidylcholine of 10% (weight) in the phospholipid dispersion.Each dispersion of sequential testing, and compare with contrast standard specimen 25,12.5 and 6.25 mcg/ml.Through and these standard specimens relatively, show that being no more than 12.5% aprotinin is released to aqueous phase.
Embodiment 4
| 60 minutes readings | Aprotinin concentration (mcg/ml) | |||||||||
| Sample | ????0 | ????6 | ??11 | ??15 | ??18 | ???20 | ???22 | ???24 | ???25 | ??30 |
| ??0μg/ml | ?0.272 | ?0.192 | ??0 | |||||||
| ??25μg/ml | ?0.265 | ?0.102 | ?0.002 | ?0.008 | ?0.015 | ?0.015 | ?0.008 | ?0.001 | ????0 | |
| ?12.5μg/ml | ?0.267 | ?0.266 | ?0.241 | ?0.044 | ?0.007 | |||||
| ?6.25μg/ml | ?0.285 | ?0.269 | ?0.195 | ?0.236 | ?-0.003 | |||||
| ?PC：Chems/ ??M818/- | ?0.157 | ?0.165 | ?0.156 | ?0.165 | ?0.152 | ?0.167 | ?0.166 | ?0.114 | ?0.138 | ??0 |
| PC:Chems/ M818/ aprotinin (50 μ g/ml) | ?0.192 | ?0.213 | ?0.195 | ?0.22 | ?0.203 | ?0.197 | ?0.14 | ?0.143 | ?0.062 | ??0 |
The aqueous dispersion of preparation S-PC (soy PC) contains 100 milligrams/gram suspended substance, with the thorough purge of nitrogen, and carry out ultrasonicization with the ripple of 8 microns amplitudes.Each aliquot is carried out 4 minutes supersound process altogether, pauses 30 seconds therebetween with the 30 second time of ice bath cooling.Then the milky dispersion of the monolayer vesicles (SUV) of gained is carried out 15 minutes centrifugal treating to remove the titanium granule.
The colloidality aurosol is pressed the method preparation.15 microlitres, 25 mMs/rise potassium carbonate, 15 microlitres, 1% tannin and 50 milligrams of trisodium citrates and distilled water constitute the solution that gross weight is 22.5 grams jointly.10 milliliters of these solution are joined in the glass conical flask (A) of 25 milliliters of band plugs and in water-bath, be heated to 60 ℃.25 milligrams of auric chloride trihydrates and distilled water are constituted 250 milligrams solution, and this solution of getting 50 microlitres joins in the 50 milliliters of band plug glass conical flasks (B) that fill 40 ml distilled waters, with same heating in water bath to 60 ℃.Thing among the bottle A is mixed with thing among the bottle B, and heating was kept 75 minutes, generated peony colloidality aurosol during this period.After being cooled to room temperature, in wherein 10 milliliters, sneak into 2 milligrams of bovine serum albumins to play a Stabilization.
1 milliliter of stabilized aurosol is mixed with 0.6 milliliter of SUV, the frozen overnight drying, and disperse with 300 milligrams of Miglyol 818 under the lyophilization thing eddy current stirring with gained.One hour with the clarifying red sub prose style free from parallelism of interior generation aurosol in Miglyol.The aliquot of three 50 milligrams of these dispersions is joined in the cuvette, and in each pipe, add the glucose solution (300 mMs/rise glucose contains 1 mM/rise sodium phosphate, and pH is 7.4) and the SUV of 500 milligrams of water, phosphate-buffered.The mixed liquor eddy current was stirred for 10 seconds carry out emulsifying, observe then.
In 1 hour, this emulsion begins to take place the oil phase layering, and layering has occurred to very high degree after the standing over night.The color of all observing lower floor's water in all cases becomes pink, shows that the part aurosol is released from oil phase.But, in the presence of SUV, carry out emulsive preparation, the degree that its upper strata oil emulsion phase color keeps is quite high, and lower floor's water is correspondingly lower.Therefore the existence of phospholipid SUV dispersion is obviously played and is reduced the effect that aurosol runs off from oil phase.Embodiment 5
With 1 milliliter 1% insulin solutions (containing 2% acetic acid hydrotropy) and 1 micromicrocurie I
125The insulin of labelling mixes, and adds the made CHEMS that contains SUV of 3 gram embodiment 3 then.With this mixture lyophilization, and lyophilization thing and 3 gram Miglyol 818 disperseed, on an orbital oscillation device, mix the clarification dispersion of insulin in oil obtained labelling in 4 hours.200 milligrams of two five equilibriums these dispersion samples, each all mixes with 800 milligrams of phosphate buffers (PBS), and difference eddy current 10 seconds of stirring and emulsifying and 30 seconds.9 milliliters of PBS dilutions of the every kind of oil that obtains/aqueous emulsion reuse, again under 80000g centrifugal 40 minutes so that creaming of emulsion.The top layer of oil phase is carefully transferred in the phial, because remaining I is arranged
125-labelling insulin can be measured its gamma activity.To contain d/d I
125The supernatant of-labelling insulin is transferred in the independent container, more remaining solids that may generate.The representative part and the oil phase residue of these solids, supernatant are all carried out radioactivity determination, oil phase is done suitable correction to any pollution that supernatant forms.
In two kinds of emulsions, eddy current disperses to remain with 45.4% radio-labeled in its oil phase of emulsion in 30 seconds, centrifugal solids had 3.0% (it can regard liposome as in essence), and disperseed the emulsion in 10 seconds for eddy current, and corresponding numerical value is respectively 43.3% and 1.3%.As a comparison, prepare in the independent experiment of oil dispersion at other two employing SUV, adopt pure Soy PC rather than Soy PC/Chems, the amount of the label that oil phase keeps is 31% and 28%, and each has all increased by 1% in the solid.This shows and adopts the amphiphilic system contain Chems to prepare the dispersion of protein in oil, when oil phase emulsified and when forming water/oily secondary dispersion, can improve the reservation amount of protein in oil phase.Embodiment 6
Preparation I
125The insulin of-labelling, and by embodiment 3 described joining in the oil phase, but following prescription adopted.
| The preparation sequence number | The amphiphilic system | Oil phase |
| ????1 | ?????Soy?PC?SUV | ??Miglyol?818 |
| ????2 | Soy PC/ phosphatidic acid (PA) SUV * | ??Miglyol?818 |
*PC/PA SUV presses embodiment 2 preparations
Preparation 2 samples of one 100 milligrams preparation 1 samples and an equivalent join respectively in 10 milliliters of centrifuge tubes.In each sample, add 1 milliliter of PBS.Two all up hill and dale eddy current stirred for 10 seconds, the latter dilutes with 10 milliliters of PBS then.Described with the emulsion centrifugal treating and be classified into their component phase by embodiment 2 then.
| The preparation sequence number | Dispersant | Retention % in every kind of fraction | ||
| Oil phase | Solid | Supernatant | ||
| ????1 | ????PBS | ????27.6 | ????1.5 | ????70.9 |
| ????2 | ????PBS | ????26.1 | ????20.9 | ????53.0 |
The gained solid is considered to be made of the oil institute of containing phospholipid at high proportion substantially.In containing the preparation of phosphatidic acid, be released to the want much less of the quantity of the labelling insulin in the water solublity supernatant than the preparation that only contains phosphatidylcholine.Embodiment 7
Preparation I
125-labelling insulin also joins in the oil phase by embodiment 2, but the composition below adopting:
| The preparation sequence number | The amphiphilic system | Oil phase |
| ????1 | ????Soy?PC?SUV | Oleic acid |
*PC/PA SUV presses embodiment 2 preparations.
One 100 milligrams preparation 1 samples take by weighing in 10 milliliters of centrifuge tubes, add 1 milliliter of 0.5% casein solution and up hill and dale eddy current stirred for 10 seconds.Use 10 milliliter of 0.5% material in the casein dilution tube then.It is described with the emulsion centrifugal treating to press embodiment 2 then, and it is classified into their component phase.
| The preparation sequence number | Dispersant | Retention % in every kind of fraction | ||
| Oil phase | Solid | Supernatant | ||
| ????1 | 0.5% casein | ????63.3 | ????4.7 | ????32 |
Can see that quite a high proportion of radiolabeling insulin is retained in the oil phase.Embodiment 8
With 0.8 milliliter of 25 mM/liter calcium chloride solution with mix mutually by 0.8 milliliter of prepared Soy PC SUV of embodiment 1, frozen overnight is dry and gained lyophilization thing and 0.5 is restrained Miglyol 818 mix mutually.Obtain complete clarifying dispersion after the standing over night.150 milligrams of dispersions are mixed with about 0.17 milligram Sudan 4 dyestuffs with to this part dispersion dyeing, obtain clarifying dark red solution.2 milliliter of 1% solution of sodium alginate is transferred in the teat glass, with Pasteur's pipet coloured oil dispersion added wherein when eddy current stirs.With the opaque emulsion of gained under 500g centrifugal 5 minutes, and the peach rich oil in upper strata come out from the clarifying aqueous phase decant of lower floor, and under optical microscope, observe.All oil exist with a large amount of discrete bead pearls this moment.Do not observe the coacervation phenomenon.Part oil droplet looks and surrounded by one deck outer wall, this it is believed that be since the calcium ion that discharges in alginic acid root and the oil droplet due to the coordination at interface.Place after 12 days, on still being macroscopic view from microcosmic, this emulsion does not still have ruined sign.
Claims (31)
1. a reduction adds the purposes that is dissolved in the reagent of direct interaction between hydrophobic hydrophilic species in mutually and the hydrophobic aqueous favoring that is scattered in mutually wherein.
2. the desired purposes of claim 1, wherein, described reagent is the acid lipoid or the emulsion stabilizer of the hydrophobic phase of porous not.
3. the desired purposes of claim 2, wherein, described reagent is CHEMS (Chems), phosphate acid ester (PA) or casein.
4. single-phase hydrophobic formulation, said preparation contains and is added the hydrophilic species that are dissolved in the hydrophobic solvent and reduce the reagent that hydrophilic species and hydrophobic formulation are scattered in direct interaction between wherein the aqueous favoring, and these hydrophilic species generally and be insoluble in this hydrophobic solvent.
5. comprise the preparation method of the single-phase hydrophobic formulation of hydrophilic species in hydrophobic solvent, this method comprises:
I) hydrophilic species and amphiphile are associated, making does not have chemical interaction between the amphiphile and hydrophilic species in this liquid medium;
Ii) remove liquid medium, the arrangement of remaining amphiphile molecules, wherein the hydrophilic end group of amphiphile, amphiphilic molecule is orientated towards the hydrophilic species; With
Iii) around hydrophilic species/amphiphile arrangement, introduce hydrophobic solvent; Wherein, in above-mentioned one or more steps, add to reduce the reagent of direct interaction between hydrophilic species and the hydrophobic aqueous favoring that is scattered in mutually wherein.
6. the desired method of claim 5, wherein, described reagent is acid lipoid.
7. the desired method of claim 6, wherein, described reagent is CHEMS (Chems) or phosphatidic acid (PA).
8. one kind will contain the single-phase hydrophobic formulation that adds the hydrophilic species that are dissolved in the hydrophobic solvent and be scattered in method in the aqueous favoring, and this method comprises add the step that reduces the reagent of direct interaction between hydrophilic species and the aqueous favoring in aqueous favoring.
9. the desired method of claim 8, wherein, described reagent is the emulsion stabilizer of the hydrophobic phase of porous not.
10. the desired method of claim 9, wherein, described reagent is casein.
11. each desired purposes in the claim 1 to 3, each desired method in desired preparation of claim 4 or the claim 5 to 10, wherein, described hydrophilic species are selected from the supermolecule assembly of protein, glycoprotein, oligomerization and Polynucleotide, polysaccharide and these molecules.
12. the desired purposes of claim 11, preparation or method, wherein, described hydroaropic substance is an insulin, calcitonin, hemoglobin, cytochrome C, horseradish peroxidase, aprotinin, Mushroom Tyrosinase, erythropoietin, growth hormone, growth hormone, somatotropin releasing factor, galanin, urokinase, factors IX, organize plasminogen activator, superoxide dismutase, catalase, peroxidase, ferritin, interferon, Factor IX, melanocyte and their any fragment, DNA, RNA, the glucosan of FITC labelling or vitamin B12.
13. each desired method in the claim 5 to 7, wherein amphiphile is a phospholipid.
14. the desired method of claim 13, wherein phospholipid has the phosphatidylcholine end group.
15. the desired method of claim 14, wherein, phospholipid is the derivant such as the HEXADECYL PHOSPHOCHOLINE of phosphatidylcholine (PC), LYSO-PHOSPHATIDYLCHOLINE LYSOPC (lyso-PC), sphingomyelin, aforementioned phospholipid or the amphipathic polymer that contains phosphatidylcholine.
16. each desired method in claim 5 to 7 or 13 to 15, wherein hydrophobic solvent comprises alcohol, branching long-chain alcohol, monoglyceride, the Diglyceride of long-chain fatty acid, medium chain, the triglyceride or the long chain triglycerides of medium chain.
17. each desired method in claim 5 to 7 or 13 to 16, wherein phospholipid comprises that PC and hydrophobic solvent are triglyceride, and perhaps wherein phospholipid is that lyso-PC and hydrophobic solvent are oleic acid.
18. each desired method in claim 5 to 7 or 13 to 16 wherein, is mixed the hydrophilic species and is removed hydrophilic solvent with the dispersion of amphiphile in hydrophilic solvent, form hydrophilic species/amphiphile and arrange.
19. the desired method of claim 18, wherein, described hydrophilic solvent is a water.
20. claim 18 or 19 desired methods, wherein, the aggregation of amphiphile comprises micelle, unilamellar vesicle, multilamellar vesicle or tubular-shaped structures such as helical form is cylindrical, hexagon, cube or myelin type structure.
21. each desired method in the claim 18 to 20, wherein hydrophilic solvent is removed by lyophilization.
22. each desired method in claim 5 to 7 or 13 to 17 wherein, in common solvent, and is removed this solvent with hydrophilic species and amphiphile cosolubilization subsequently, forms hydrophilic species/amphiphile and arranges.
23. each desired method in claim 5 to 7 or 13 to 17, wherein, with solution and hydrophilic species the emulsifying soln in hydrophilic solvent of amphiphile in hydrophobic solvent, form emulsion, and remove hydrophobic solvent, form hydrophilic species/amphiphile and arrange.
24. the method for claim 22 or 23, wherein the weight ratio of amphiphile and hydrophilic species is about 1: 1 to 50: 1.
25. the desired method of claim 23, wherein, described emulsion is the emulsion of water in oil.
26. claim 23 or 24 desired methods, wherein hydrophobic solvent is the low boiling organic ether, as ether.
27. the single-phase hydrophobic formulation of the available hydrophilic species of each desired method in hydrophobic solvent in claim 5 to 7 or 13 to 25.
28. comprise aqueous favoring and hydrophobic two-phase compositions mutually, wherein hydrophobic claim 4 or the 26 desired preparations of comprising mutually.
29. the desired compositions of claim 27, wherein hydrophobic being scattered in mutually in the successive aqueous favoring.
30. claim 27 or 28 desired compositionss, it is an emulsion.
31. claim 4 or 12 desired preparations, the purposes of each desired compositions in hydrophilic species oral in desired preparation of claim 27 or the claim 28 to 30.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9424901.8A GB9424901D0 (en) | 1994-12-09 | 1994-12-09 | Sequestration Agents |
| GB9424901.8 | 1994-12-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1169114A true CN1169114A (en) | 1997-12-31 |
Family
ID=10765693
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95196708.8A Pending CN1169114A (en) | 1994-12-09 | 1995-12-08 | Sequestration agents |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0796086A1 (en) |
| JP (1) | JPH10510207A (en) |
| CN (1) | CN1169114A (en) |
| AU (1) | AU4183496A (en) |
| CA (1) | CA2207274A1 (en) |
| FI (1) | FI972411A0 (en) |
| GB (1) | GB9424901D0 (en) |
| IL (1) | IL116311A0 (en) |
| NO (1) | NO972607L (en) |
| WO (1) | WO1996017594A1 (en) |
| ZA (1) | ZA9510459B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9323588D0 (en) | 1993-11-16 | 1994-01-05 | Cortecs Ltd | Hydrophobic preparation |
| GB9521806D0 (en) * | 1995-10-25 | 1996-01-03 | Cortecs Ltd | Preservation methods |
| US6165773A (en) * | 1995-10-25 | 2000-12-26 | Provalis Uk Limited | Methods of preserving viruses |
| GB9613858D0 (en) | 1996-07-02 | 1996-09-04 | Cortecs Ltd | Hydrophobic preparations |
| US6458373B1 (en) | 1997-01-07 | 2002-10-01 | Sonus Pharmaceuticals, Inc. | Emulsion vehicle for poorly soluble drugs |
| US7030155B2 (en) | 1998-06-05 | 2006-04-18 | Sonus Pharmaceuticals, Inc. | Emulsion vehicle for poorly soluble drugs |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS53107408A (en) * | 1977-02-28 | 1978-09-19 | Yamanouchi Pharmaceut Co Ltd | Micellar preparation for rectal infusion |
| JPS574913A (en) * | 1980-06-11 | 1982-01-11 | Green Cross Corp:The | Urokinase preparation for oral administration |
| US4578391A (en) * | 1982-01-20 | 1986-03-25 | Yamanouchi Pharmaceutical Co., Ltd. | Oily compositions of antitumor drugs |
| GB9323588D0 (en) * | 1993-11-16 | 1994-01-05 | Cortecs Ltd | Hydrophobic preparation |
-
1994
- 1994-12-09 GB GBGB9424901.8A patent/GB9424901D0/en active Pending
-
1995
- 1995-12-08 JP JP8517441A patent/JPH10510207A/en active Pending
- 1995-12-08 EP EP95940365A patent/EP0796086A1/en not_active Withdrawn
- 1995-12-08 CN CN95196708.8A patent/CN1169114A/en active Pending
- 1995-12-08 ZA ZA9510459A patent/ZA9510459B/en unknown
- 1995-12-08 CA CA002207274A patent/CA2207274A1/en not_active Abandoned
- 1995-12-08 AU AU41834/96A patent/AU4183496A/en not_active Abandoned
- 1995-12-08 WO PCT/GB1995/002908 patent/WO1996017594A1/en not_active Application Discontinuation
- 1995-12-10 IL IL11631195A patent/IL116311A0/en unknown
-
1997
- 1997-06-06 FI FI972411A patent/FI972411A0/en unknown
- 1997-06-06 NO NO972607A patent/NO972607L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| IL116311A0 (en) | 1996-03-31 |
| ZA9510459B (en) | 1997-06-09 |
| JPH10510207A (en) | 1998-10-06 |
| AU4183496A (en) | 1996-06-26 |
| GB9424901D0 (en) | 1995-02-08 |
| FI972411A (en) | 1997-06-06 |
| MX9704272A (en) | 1998-07-31 |
| NO972607L (en) | 1997-08-07 |
| EP0796086A1 (en) | 1997-09-24 |
| CA2207274A1 (en) | 1996-06-13 |
| FI972411A0 (en) | 1997-06-06 |
| WO1996017594A1 (en) | 1996-06-13 |
| NO972607D0 (en) | 1997-06-06 |
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