CN1224360A - Hydrophobic preparations contg. medium chain monoglycerides - Google Patents
Hydrophobic preparations contg. medium chain monoglycerides Download PDFInfo
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- CN1224360A CN1224360A CN 97196069 CN97196069A CN1224360A CN 1224360 A CN1224360 A CN 1224360A CN 97196069 CN97196069 CN 97196069 CN 97196069 A CN97196069 A CN 97196069A CN 1224360 A CN1224360 A CN 1224360A
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Abstract
Hydrophobic preparations which are useful as, among other things, pharmaceutical delivery systems comprise: (i) an oil phase comprising one or more medium chain monoglycerides, such as Akoline MCM<TM>; (ii) at least one amphiphile, preferably including a phospholipid such as phosphatidyl choline; and (iii) a hydrophilic species, which may be a protein such as insulin or calcitonin or another macromolecule, solubilised or otherwise dispersed in the one or more glycerides. (The hydrophilic species is one that is not normally soluble in the glycerides).
Description
The present invention relates to the preparation of some materials in the hydrophobic solvent that it is insoluble to usually, and the method that obtains this preparation.Specifically, the present invention relates to the preparation of hydroaropic substance in medium chain monoglyceride (MCM) and diglyceride mixt.
The present invention is particularly useful for being generally insoluble hydrophilic macromole in oil or other hydrophobic solvent.
For many application, as at pharmaceutical science, in food manufacturing or the cosmetics industry,, its hydrophilic and polarity highly can act on or mix the degree of fat in mutually because having limited them, problem has been appearred in albumen and similar macromolecular processing.Many natural systems adopt lipid barrier (as skin, cell membrane) to contact with inner compartment to prevent hydrophilic molecule; Protein the ability in the lipid excipient of being dispersed in will be opened a new approach that these macromole is imported in living things systems, thereby contain that proteinic lipid medium can combine with the hydrophobicity composition of barrier rather than repelled by them.
We disclosed the method for preparing hydrophobic formulation in WO-A-9513795, WO-A-9617593 and WO-A-9617594 in the past, and wherein hydroaropic substance is dissolved in the thin aqueous phase that it is insoluble to usually.Especially, this method is suitable for solubilising protein.
Simply and effectively dissolve macromole such as method of protein though above-mentioned preparation provides, we have now found that to send by the macromole that uses specific oil phase and optional specific amphiphile can improve preparation and pass character.Because preparation disclosed herein not only increases the treatment uptake of macromolecules, and good dose reproducibility is provided, therefore when dissolved macromole was protein such as pharmaceutically active albumen, this was especially favourable.
Therefore, first aspect the invention provides a kind of hydrophobic formulation, comprising:
(ⅰ) comprise the oil phase of one or more medium chain monoglycerides; With
(ⅱ) at least a amphiphilic species;
(ⅲ) a kind of dissolving or be dispersed in hydroaropic substance in the glyceride mixture with other method; Wherein this hydroaropic substance is generally insoluble in described one or more monoglycerides.
In the context of the invention, " hydrophobic formulation " is not present in the preparation of aqueous phase for hydroaropic substance wherein.This hydrophobic formulation especially is suitable for being used for oral sending and passs the hydrophilic macromole, as protein.
Prior art comprises many example (Sekine etc., medicine biodynamics magazine (J.Pharmacobiodyn.), the 7:856-63s (1984) of medium chain monoglyceride as penetrating reinforcing agent that use really in intestinal; Higaki etc., medicine biodynamics magazine, 9:532-9 (1986); Unowsky etc., chemotherapy (Chemotherapy), 34:272-6 (1988); Watanabe etc., pharmaceutical science magazine (J.Pharm.Sci.), 77:847-9 (1988); Yeh etc.; Drug research (Pharm.Res.), 11:1148-54 (1994); Constinides etc., drug research, 11:1385-90 (1994)).Yet in each case, active component/medicine all is dissolved in aqueous phase in the disclosed preparation.In fact disclosed method in WO-A-9513795 especially just can obtain wherein hydroaropic substance and really and easily be dissolved in and dredge aqueous phase and kept bioactive preparation.
To not contain a large amount of waters usually and may not contain dissociating water molecule according to preparation of the present invention.
In a preferred specific embodiments, will comprise the mixture of medium chain monoglyceride and diglyceride in the oil phase, aptly, be used for the chain length that medium chain triglycerides of the present invention has 8-10 carbon atom, for example, they can comprise linear saturated fatty acids.In another embodiment, can comprise one or more chain monoglycerides and at least a other composition in the oil phase, as oleic acid, glyceryl monooleate or gelucires.For these two specific embodiments, must composition be the medium chain monoglyceride, no matter be to use the medium chain monoglyceride also to be to use the mixture of glyceride etc. separately, the oil component of use should be big as far as possible in the amount of the monoglyceride of guaranteeing to make when oil component keeps liquid condition under 45 ℃ or lower temperature existence.Especially, monoglyceride can account for the 40-90% of total amount of the oil of existence, preferred 60-70%, and an example of suitable glyceride mixture is Akoline MCM
TM, it comprises medium chain monoglyceride and diglyceride, and it can buy the address from Karlshamns Sweden AB company: Sweden S/374 82Karlshamn.
Preferably, amphiphilic species: macromolecular ratio is 1: 1-20: in 1 the scope (by weight), more preferably 2: 1-8: in 1 the scope (by weight).
The example of suitable amphiphilic species comprises phospholipid, as phosphatidylcholine, phosphatidic acid, phosphatidyl glycerol, PHOSPHATIDYL ETHANOLAMINE and their haemolysis derivant, and octyl glucoside and other glycolipid, tocopherol succinate and Cholesteryl hemisuccinate.Other suitable amphiphilic species comprises Phosphatidylserine, Sodium docusate (sodium docusate) and hydroxypropyl cellulose.Can use more than one amphiphilic species.
In a preferred specific embodiments, two hydroaropic substances that use are bile salts.In the present invention, should understand, so term bile salts and bile acid are used interchangeably because whether its salt or its coupling acid exist the pH that will depend on surrounding.
Bile salts is the surfactant of natural generation, they be one group be present in all animals and higher plant the chemical compound with common " skeleton " structure of cholic acid.Bile salts can be by monohydroxylated, dihydroxy or trihydroxyization; They always comprise 3 Alpha-hydroxies, and the most common at C
6, C
7Or C
12Other hydroxyl of position can be positioned on the plane of molecule (β) or under (α).
In being described as a compounds of bile salts, comprise having the amphipathic polyhydroxy sterol of carboxyl as an one-level side chain part.Its modal bile salts example is produced by the cholesterol metabolism and exists with the derivatization form in bile and in the whole intestinal in the mammal.
In context, this term is also applicable to the synthetic analogues of the bile salts of the natural generation that shows similar biology effect, or the molecule of microorganisms, as fudidic acid and its derivant.
Bile salts (or multiple salt) can be non-coupling or link coupled.Term " non-coupling " refers to that one-level side chain wherein has a bile salts that is positioned at end not and unsubstituted single carboxyl.The example of non-coupling bile salts comprises cholate, ursodeoxycholate, chenodesoxy cholate and dexycholate.The coupling bile salts has those of carboxyl of a replacement for one-level side chain wherein.Usually substituent group is the amino acid derivativges that links to each other with the bile salts carboxyl by means of nitrogen-atoms.The example of coupling bile salts comprises taurocholate, glycocholate, taurodeoxycholate and sweet dexycholate.
Therefore, among the present invention, the example of suitable bile salts comprises cholate, dexycholate, chenodesoxy cholate and ursodeoxycholate, and ursodeoxycholate is especially preferred.Adoptable other bile salts comprises taurocholate, taurodeoxycholate, TUDCA salt, cattle sulphur chenodesoxy cholate, glycocholate, sweet dexycholate, sweet taurodeoxycholate, sweet chenodesoxy cholate, lithocholate, taurolithocholic acid salt and Calamina cholate.
In the present invention, term " hydroaropic substance " refers to dissolve in usually in the aqueous solvent but is insoluble to any material of hydrophobic solvent.The range that is used for hydroaropic substance of the present invention is various, but the hydrophilic macromole is represented an example of spendable material.
Many macromole are suitable and be used for the present invention.Usually, owing to usually the hydrophobicity macromole is being dissolved in the oil solution seldom difficulty, therefore, macromolecular compound is hydrophilic or possess hydrophilic property zone at least.Suitable macromolecular example comprises protein and glycoprotein, few nucleic acid and Polynucleotide, for example DNA such as plasmid DNA and RNA, and DNA and/or RNA analog, the supramolecular complex of polysaccharide such as heparin (especially low molecular weight heparin) and any of these material.Comprise whole cell or organelle in some cases.With micromolecule such as vitamin molten altogether with macromole especially polysaccharide such as cyclodextrin also may be very easily.Also some molecules such as vitamin B12 and macromole can be carried out chemical coupling, and therefore it is included in the compositions.
Can successfully be comprised insulin by the dissolved concrete proteinic example of the inventive method, calcitonin, hemoglobin, cytochrome C, horseradish peroxidase, aprotinin, the dried mushroom tryrosinase, erythropoietin, growth hormone, growth hormone, somatotropin releasing factor, galanin, urokinase, factor IX, tissue plasminogen activator, superoxide dismutase, catalase, peroxidase, ferritin, interferon, factor VIII and its fragment (top all albumen can from any suitable source).Other protein comprises soybean trypsin inhibitor, GLP1, other thrombin, somatostatin, hirudin and LHPH and all their analog and fragment.
One or more these or other proteinic mixture can be dissolved by the present invention.
Can easily be dissolved by method of the present invention owing to have the glucosan of about 1000000 molecular weight, it seems does not have the upper limit for the macromolecular compound molecular weight.
Except macromole, the inventive method can be used for dissolving less organic molecule and or macromole.The example of little organic molecule comprises glucose, CF 5(6)-Carboxyfluorescein and many medicaments, and as anticarcinogen, but this method can be used for other little organic molecule, for example vitamin or pharmaceutically active agents or bioactivator equally certainly.In addition, use this method also solubilized such as calcium chloride and these chemical compounds of sodium phosphate.Really, changed, for example increase bioavailability, so the present invention is particularly favourable for pharmaceutically active agents and bioactivator owing to using non-aqueous solution can make molecule enter intravital approach.
Can be incorporated into the present invention comprises little organic molecule in the macromolecular preparation and comprises stabilizing agent such as polyglycereol, PEG and glycerol (especially maybe may be for other under proteic situation at insulin), chelating agen such as citric acid, EDTA and EGTA, and antioxidant such as ascorbic acid.
The material that can be included in other type in the hydrophobic composition of the present invention is an inorganic matter, as little inorganic molecule or colloid substance, for example colloidal metal.Method of the present invention makes colloidal metal, and as some character of aurosol, palladium, platinum or rhodium even also kept in hydrophobic solvent, wherein under normal operation, coagulation takes place in hydrophobic solvent these granules.This can be particularly useful for the catalysis of the reaction carried out in organic solvent.
Hydrophobic formulation of the present invention also can randomly comprise other composition.These example comprises antioxidant, metal-chelator, buffer agent and dispersant, the example of suitable dispersant comprises surfactant, contains the surfactant of POE as Tween, Span and Brij class reagent and the ethylating castor oil derivative of polyoxy and other.
Hydrophobic formulation of the present invention can use following method to prepare, and this method comprises:
(ⅰ) in a kind of liquid medium, hydroaropic substance is combined with amphiphilic species, make in this liquid medium, not have chemical action between amphiphilic species and the hydroaropic substance.
(ⅱ) remove liquid medium, its hydrophilic head group of remaining amphipathic molecule array is towards hydroaropic substance; With
(ⅲ) around hydrophilic substance/amphiphilic species array, provide the mixture of medium chain monoglyceride and diglyceride.
This method is disclosed among the WO-A-9513795.Especially, hydrophobic formulation of the present invention can prepare by the method that is disclosed among the PCT number of patent application PCT/GB97/00749, and this method comprises:
(ⅰ), hydrophilic substance is combined with amphiphilic species there being hydrophobic phase time; With
(ⅱ) remove any hydrophilic solvent of existence; Wherein the step of removing of hydrophilic solvent is being carried out hydrophobic remaining mutually under the solid-state condition.
Preferably, when this method of use, at first hydroaropic substance and amphiphilic species being dissolved in a kind of hydrophilic solvent, as aqueous solvent, only is water usually, then this solution is combined with glyceride mixture.Hydrophilic solvent is removed step and is accomplished by lyophilization easily, finishes guaranteeing that glyceride mixture is retained as under the solid-state temperature like this, is removed until all water.In some cases, because the result that the part of some part (usually at surface and edge) temperature of the solid block that has been removed at all hydrophilic solvents raises may become liquid at the lyophilization medium oil.Here the cooling effect that derives from the hydrophilic solvent distillation no longer exists, and will melt at those regional oil.As long as one occurs, oil promptly is allowed to flow out from the remainder of solid block, and so this situation will cause the generation (if not so, the oil of accumulation will form one deck that the prevention hydrophilic solvent is further removed) of gratifying finished product.
On the other hand, can keep the temperature during the lyophilization, like this even after hydrophilic solvent is removed, it is solid-state that oil still keeps.In this case, after lyophilization, the temperature of rising preparation is to produce monophasic preparation.This realizes that by simply the temperature of freeze-dried preparation being risen to room temperature this temperature will cause glyceride mixture to get back to liquid state.Also can adopt other method of removing hydrophilic solvent, as spray drying.
Hydrophobic formulation of the present invention is polyenergic, has many purposes.They can be used alone or can be mixed with water to form emulsion or the similar two-phase compositions that constitutes second aspect of the present invention.
In this aspect of the invention, provide a kind of aqueous favoring and hydrophobic two-phase compositions mutually of comprising, wherein hydrophobicly comprise a kind of hydrophobic formulation of the present invention that can obtain by said method mutually.
Usually, in this based composition, hydrophobicly be dispersed in the aqueous favoring mutually.
As mentioned in this article, hydrophobic formulation of the present invention has special advantage, because hydrophilic substance is easy to be absorbed into as in the blood behind oral administration.Therefore they are particularly useful for passing as proteinic oral sending.Yet the technical staff will recognize that preparation of the present invention also will facilitate for other route of administration such as part or vagina administration.Therefore, aspect the 3rd of the present invention, provide a kind of pharmaceutical preparation that comprises hydrophobic formulation of the present invention.Pharmaceutical preparation in the scope of the invention comprises capsule, tablet and other system type.In addition, aspect the 4th of the present invention, provide hydrophobic formulation of the present invention to be used for for example purposes of hydrophilic macromole such as proteinic medicine of oral delivery hydroaropic substance in preparation.The present invention also can be used to change immunne response by stimulation or inhibitory action.
The preferred feature of various aspects of the present invention equally also can be used for each others, and vice versa.
With reference to the following examples the present invention is described, wherein these embodiment should not be considered to limit by any way the present invention.Embodiment 1: comprise the preparation of the preparation of calcitonin/PC complex
(ⅰ) preparation of phospholipid dispersion
1. in the steaming and decocting pipe of band ground glass stopper, take by weighing the 1.6g S-PC and add the final weight of distilled water to 8g.Use the nitrogen purge, capping plug is tight, with Parafilm sealing and on orbital shaker, mix gently until disperseing all solids.
2. be transferred in the ultrasonic container of glass round bottom.
3. ultrasonic container is pressed from both sides to Sonics 4 Materials VibraCell VCX 600 ultrasonoscopes that 1 inch diameter probe is housed, and the submergence probe fully is positioned under the liquid meniscus until its base portion.Between probe and pipe top, form a collar with a mucosa bar, with the air space above the nitrogen cleaning liquid.Ultrasonic container is immersed in the ice bath.
4. with 50% amplitude, between emission in 1 second with 4 second chilling room every with the liquid suspension supersound process, until forming milky dispersion (usually total supersound process time is 4 minutes).
5. dispersion is transferred in the conical centrifuge tube of plastics and centrifugal 15 minutes, with any precipitate and separate of supernatant and existence with 1200g.
6. with the phospholipid final concentration of distilled water diluting twice with generation 100mg/ml.
(ⅱ) preparation of protein solution
7. taking by weighing 20mg in 2ml glass screw-cap vial presses down the enzyme peptide and adds the 1ml distilled water.Lid screwed and mix gently until all solids dissolve.
8. in 1ml glass screw-cap vial, take by weighing the 16ml salmon calcitonin see calcimar and add the 0.8ml distilled water.Lid screwed and mix solid dissolving gently until institute.
9. in 1ml glass screw-cap vial, take by weighing 50mg ascorbic acid and 5mg citric acid and add the 1ml distilled water.Lid screwed and mix gently until all solids dissolve.
10. in each of three 10ml glass screw-cap vials, distribute the phospholipid dispersion (200mg solid) of 2ml from step 6,250 μ l press down enzyme peptide solution (5mg solid) from step 7,250 μ l mix the content of each bottle gently from the calcitonin solution (5mg solid) of step 8 and the 200 μ l ascorbic acid/citric acid solution (being respectively 10mg and 1mg solid) from step 9.
(ⅲ) lyophilization of water
11. the content of rapid freezing each bottle of sound in liquid nitrogen.
12. be lower than-40 ℃ condenser temperature and less than lyophilized overnight under 0.1 millibar the vacuum.
(ⅳ) preparation of oil phase
13. 0.8g PS and 7.2g Akoline MCM are claimed to B8 glass screw-cap vial.Clean bottle with nitrogen, lid is screwed and seal, on the pulley type blender, mix gently until obtaining homogeneous solution with Parafilm.
(ⅴ) in oil phase, dissolve
14. sample behind the bone dry, adds the oil phase of 1.779g from step 13 in every bottle in freeze dryer.Clean each bottle with nitrogen, lid is screwed and seal with Parafilm.
15. allowed solid dissolve in 2 hours by in the pulley type blender, mixing under the room temperature, then under 37 ℃, shake until forming clear solution.
16. be stored in+when needs, use under 4 ℃.
(ⅵ) vivo medicine-feeding
17. in the water-bath each bottle heated to 37 ℃ to melt oil and to form a clear solution.
18. in each bottle, add the PBS of 4ml temperature, vortex 30 seconds, as mentioned above, to 1 pig administration 1.2ml in jugular vein.
Embodiment 2, comprise the preparation of the preparation of insulin/chenodeoxy cholic acid salt composite
(ⅰ) preparation of chenodeoxy cholic acid saline solution
1. in the glass conical flask, take by weighing 6.0g chenodeoxy cholic acid sodium, add the final weight that is distilled to 60g.Clean with nitrogen, jam-pack and with the Parafilm sealing mixes gently until all solids at orbital shaker and to dissolve.
(ⅱ) preparation of protein solution
2. taking by weighing 120mg in 2ml glass screw-cap vial presses down the enzyme peptide and adds the 60ml distilled water.Lid screwed and mix gently until all solids dissolve.
3. will be independently three parts of 106.5mg islets of langerhans be called conical flask usually, (B) 25ml conical flask and (C) in the 10ml screw-cap vial to (A) 100ml.In flask (A), add the chenodeoxy cholic acid saline solution (1g solid) of 30ml from step 3.1.1.Add 15ml chenodeoxy cholic acid saline solution (1.5g solid) to flask (B), and in bottle (C), add 7.5ml (0.75g solid).Cover flask and bottle with Parafilm, and under 37 ℃, on orbital shaker, mix gently and dissolve until all solids.
4. in 2ml glass screw-cap vial, take by weighing the 54mg citric acid and add the 6.0ml distilled water.Lid screwed and mix gently until all solids dissolve.
5. distribute the insulin/chenodeoxy cholic acid saline solution (A) of 2ml from step 3 in each of the young bottle of 12 7ml glass, 100 μ l press down enzyme peptide solution and the 100 μ l citric acid solution from step 4 from step 2.Mix the content in each bottle gently.
6. distribute the insulin/chenodeoxy cholic acid saline solution (B) of 1ml from step 3 in each of 12 7ml glass screw-cap vials, 100 μ l press down enzyme peptide solution and the 100 μ l citric acid solution from step 4 from step 2.Mix the content in each bottle gently.
7. distribute the insulin/chenodeoxy cholic acid saline solution of 0.5ml from step 3 in each of 12 7ml glass spiral shell bottles, 100 μ l press down enzyme peptide solution and the 100 μ l citric acid solution from step 4 from step 2.Mix the content in the bottle gently.
(ⅲ) lyophilization of water
8. in liquid nitrogen that the content of each bottle is freezing rapidly and be lower than-40 ℃ condenser temperature and crossing liquid less than lyophilization under 0.1 millibar the vacuum.
(ⅳ) preparation of oil phase
9. 6.0g PS and 54g Akoline MCM are claimed to the 100ml vial.Clean bottle with nitrogen, lid is screwed, with Parafilm sealing and on the pulley type blender, mix gently until obtaining homogeneous solution.
(ⅴ) in oleic acid, dissolve
10. sample behind the bone dry, adds the 0.79g oil phase formulation (A) from step 10 in each bottle in freeze dryer.Clean each bottle with nitrogen, lid is screwed and seal with Parafilm.
11. sample behind the bone dry, adds the 0.8g oil phase formulation (B) from step 10 in each bottle in freeze dryer.Clean each bottle with nitrogen, lid is screwed and seal with Parafilm.
12. product behind the bone dry, add the 0.94g oil phase formulation (C) from step 10 in each bottle in freeze dryer mutually.Clean each bottle with nitrogen, lid is screwed and seal with Parafilm.
13. allowed the solid dissolving in 2 hours by in the pulley type blender, mixing under the room temperature, then shake until forming a clear solution at 37 ℃, be stored in+when needs, use under 4 ℃.
(ⅵ) vivo medicine-feeding
14. in the water-bath bottle heated to 37 ℃ to melt oil and to form a clear solution.
15. in each bottle, add the PBS of 2ml temperature, vortex 30 seconds, and as mentioned above, to the content of pig administration one whole bottle in jugular vein.
Embodiment 3. comprises the preparation of the preparation of insulin/PC complex
(ⅰ) preparation of phospholipid dispersion
1. take by weighing 2g soybean phospholipid acyl phatidylcholine in the pipe and add distilled water boiling of band ground glass stopper to produce the final weight of 8g.Use nitrogen washing, jam-pack is with Parafilm sealing and mix gently on orbital shaker until disperseing all solids.
2. be transferred in the ultrasonic container of glass round bottom.
3. ultrasonic container is pressed from both sides to Sonics Material VibraCellVC * 60 ultrasonoscopes that 1 inch diameter probe is housed, and the submergence probe is positioned at 1cm under the liquid meniscus until its base portion.Use a mucosa bar to form a collar between probe and pipe top, the air space with above the nitrogen cleaning liquid is immersed in ultrasonic container in the ice bath.
4. with 50% amplitude, 1 second interpulse with 4 second chilling room every with the liquid suspension supersound process, until forming milky dispersion (usually total supersound process time is 4 minutes).
5. dispersion is transferred in the conical centrifuge tube of plastics and centrifugal 15 minutes with 1200g.Any precipitation that exists is separated with supernatant.
(ⅱ) preparation of protein solution
6. taking by weighing 30mg in 2ml glass screw-cap vial presses down the enzyme peptide and adds the 1.5ml distilled water.Lid screwed and mix gently until all solids dissolve.
7. the 110mg islets of langerhans is called usually to the 25ml conical flask, and adds the distilled water that 15ml has wherein added 150 μ l glacial acetic acid.Cover flask with Parafilm, and under 37 ℃, on orbital shaker, mix gently and dissolve until all solids.
8. in 2ml glass screw-cap vial, take by weighing the 10mg sodium citrate and add the 1.5ml distilled water.Lid screwed and mix gently until all solids dissolve.
9. in each of 12 7ml glass screw-cap vials, distribute insulin solutions (the 7.33mg solid of 1ml from step 7,200iu), 100 μ l are from the sodium citrate (0.67mg solid) of step 8, and 100 μ l press down enzyme peptide solution (2mg solid) and the 400 μ l phospholipid dispersion (100mg solid) from step 5 from step 6.The content that mixes each bottle gently.
(ⅲ) preparation of oil phase
10. 1.5g PS and 13.5g Akoline MCM are claimed to the 20ml vial.Clean bottle with nitrogen, lid is screwed and seal, on the pulley type blender, mix gently until obtaining homogeneous solution with Parafilm.
(ⅳ) lyophilization of water
11. use and add the akoline/ PS mixture of 1ml in large volume positive displacement allotter each bottle in step 9 from step 10.
12. it is, then freezing in liquid nitrogen immediately with the rapid vortex of each bottle 10 seconds.
13. be lower than-40 ℃ condenser temperature and less than two nights of lyophilization under 0.1 millibar the vacuum.
(ⅴ) preparation of oil solution
14. from freeze dryer, take out each bottle, clean with nitrogen, lid tightly and with Parafilm seals.37 ℃ are shaken down insulation gently until obtaining clear solution, are stored in+4 ℃ of uses when needs.
(ⅵ) vivo medicine-feeding
15. in the water-bath each bottle heated to 37 ℃ to melt oil and to form clear solution.
16. in each bottle, add 2ml temperature PBS, vortex 30 seconds, and as mentioned above, to the content of a pig through jugular vein administration one whole bottle.
Embodiment 4. comprises the preparation of the preparation of insulin/ursodeoxycholate complex
(ⅳ) preparation of ursodeoxycholate solution
1. in the glass conical flask, take by weighing 525mg ursodesoxycholic acid sodium and add distilled water to the final weight that produces 15g.Clean with nitrogen, lid is tight, seals and mixes gently until all solids on orbital shaker with Parafilm and dissolve.
(ⅱ) preparation of protein solution
2. taking by weighing 30mg in 2ml glass screw-cap vial presses down the enzyme peptide and adds the 1.5ml distilled water.Lid is screwed and mix gently until all solids and fuse.
3. be called usually the 110mg islets of langerhans to the 25ml conical flask and add the ursodeoxycholate solution (525g solid) of 15ml from step 1.Cover flask with Parafilm, and mix gently until solid on orbital shaker at 37 ℃ and to dissolve.
4. in 2ml glass screw-cap vial, take by weighing the 13.5mg sodium citrate and add the 1.5ml distilled water.Lid screwed and mix gently until all solids dissolve.
5. distribute 1ml from step 3 insulin/chenodeoxy cholic acid saline solution (7.3mg insulin/35mg bile salts) in each of 12 7ml glass screw-cap vials, 100 μ l press down enzyme peptide solution (2mg solid) from the sodium citrate (0.9mg solid) of step 4 and 100 μ l from step 2.The content that mixes each bottle gently.
(ⅲ) preparation of oil phase
6. 1.5g PS and 13.5g akoline MCM are claimed to the 20ml vial.Clean bottle with nitrogen, lid is screwed and seal, on the pulley type blender, mix gently until obtaining homogeneous solution with Parafilm.
(ⅳ) lyophilization of water
7. use and add the akoline/ PS mixture of 1ml in large volume positive displacement allotter each bottle in step 5 from step 6.
8. with the rapid vortex of each bottle 10 seconds, then freezing in liquid nitrogen immediately.
9. be lower than-40 ℃ condenser temperature and less than two nights of lyophilization under 0.1 millibar the vacuum.
(ⅴ) preparation of oil solution
10. take out each bottle from freeze dryer, clean with nitrogen, lid tightly and with Parafilm seals.37 ℃ are incubated 10 minutes down until obtaining clear solution.
11. be stored in+4 ℃ of uses when needs.
(ⅵ) vivo medicine-feeding
12. in the water-bath each bottle heated to 37 ℃ to melt oil and to form clear solution.
13. in each bottle, add 2ml temperature PBS, vortex 30 seconds, and as mentioned above, to the content of a pig through jugular vein administration one whole bottle.
Embodiment 5: be used for intestinal and send the preparation of passing calcitonin
Be prepared as follows preparation:
1) mixture of salmon calcitonin see calcimar and akoline MCM
As embodiment 1, just (a) saves the proteinaceous lyophilization thing of phospholipid dispersion (b) and is not dissolved in the oil phase, (c) before to animals administer, protein lyophilization thing is dissolved in the 4ml phosphate buffered saline(PBS), and (d) solution that produces is added in the 2ml oil phase (Akoline MCM/Tween 80) and disperse by vortex.
2) mixture of salmon calcitonin see calcimar and glyceryl dioleate
As preparation (1), just adopt glyceryl dioleate to replace Akoline MCM.
3) salmon tantalum calcitonin and oleic mixture.
As preparation (1), just adopt oleic acid to replace Akoline MCM.
4) in Akoline MCM, comprise the oil of salmon calcitonin see calcimar/PC complex
As described in example 1 above.
5) in Akoline MCM, comprise the oil of salmon tantalum calcitonin/chenodeoxy cholic acid salt composite
Preparation identical with present embodiment preparation (4) on forming just adopts the chenodesoxy cholate of 100mg/g concentration to replace PC in oil phase.
6) in Akoline MCM, comprise the oil of salmon calcitonin see calcimar/chenodeoxy cholic acid salt composite
Preparation identical with present embodiment preparation (4) on forming just saved the chenodeoxy cholic acid sodium that presses down the enzyme peptide and adopt 17.5mg/g concentration in oil phase and replaced PC.
7) in Akoline MCM, comprise the oil of salmon calcitonin see calcimar/ursodeoxycholate complex
Preparation on forming with top (4) in identical, just save the ursodesoxycholic acid sodium that presses down the enzyme peptide and in oil phase, adopt 17.5mg/g concentration and replace PC.
8) in Akoline, comprise the oil of salmon calcitonin see calcimar/tocopheryl succinate complex
Preparation on forming with top (4) in identical, just save the alpha tocopheryl succinate that presses down the enzyme peptide and in oil phase, adopt 25mg/g concentration and replace PC.
9) in Akoline MCM, comprise the oil of salmon calcitonin see calcimar/Sodium docusate complex.
Preparation on forming with top (4) in identical, just save the Sodium docusate that presses down the enzyme peptide and in oil phase, adopt 25mg/g concentration and replace PC.Embodiment 6: be used for intestinal and send the preparation of passing insulin
Be prepared as follows preparation:
10) mixture of insulin and Akoline MCM
As preparation (1), just adopt 7.3mg insulin (200iu), 2mg presses down enzyme peptide and 1ml Akoline/Tween 80 oil phases.
11) in Akoline MCM, comprise the oil of insulin/PC complex
As embodiment 2.
12) in Akoline MCM, comprise insulin/chenodeoxy cholic acid salt composite and be the oil of high bile salt concentration
As embodiment 3.
13) in Akoline MCM, comprise insulin/chenodeoxy cholic acid salt composite and be the oil of medium bile salt concentration
As embodiment 3.
14) in Akoline MCM, comprise insulin/chenodeoxy cholic acid salt composite and be the oil of low bile salt concentration
As embodiment 3.
15) in Akoline MCM, comprise the oil (contain press down enzyme peptide) of insulin/ursodeoxycholate complex
As embodiment 4.
16) in Akoline MCM, comprise the oil (do not contain press down enzyme peptide) of insulin/ursodeoxycholate complex
With embodiment 4, just from preparation, save and press down the enzyme peptide fully.
17) phosphate buffered saline(PBS) of insulin/ursodeoxycholate
Freeze-drying solution as embodiment 4 steps (3) preparation insulin.Before the administration, be dissolved in again in the phosphate buffered saline(PBS) (the 7.3mg insulin/2ml).
Embodiment 7: zoopery
Zoopery
The preparation of animal model
The animal model that is used for detecting the calcitonin oral formulations is young pig.Why select pig to be because it has the body weight that is similar to the people, and the structure of small intestinal, function are similar to people's small intestinal with size.For eliminate with people and pig between the relevant worry of difference, to the animal operation that undergos surgery, material directly can be imported in the jejunum by means of burying intubate like this.This also reduced usually with oral administration dosage arrival intestinal in time relevant uncertainty, and cause the raising of the statistical quality that obtains.
Before first administration at least 10 days, a thin plastic cannula underwent operative is inserted in the jejunum of pig and then under skin, is drawn at the back of pig, can not make like this animal pain as and if substances is injected jejunum.The conduit that buries that will also pass skin of back and draw inserts in the aorta by middle arteria saphena, can obtain multiple blood sample like this.Second conduit also is imported in the carotid artery.The administration of preparation
Under the clear-headed fully fasting state of consciousness, pig is tested, after taking out three baseline blood samples, given peptide formulations by means of oral route.Single experiment takies 8-9 hour time usually, and the pig that is carried out operation technique reaches 3 times weekly participating in test in the time all around.After test finishes pig is killed, check the naked eyes of small intestinal and the variation of microscopically.
Experimental session provides water quantity-unlimitingly, at 8:00 and 15:30 pig is carried out feeding.Any food that after 16:30 removes the 15:30 feeding, is left.During handling, remove the food of 08:00, after extracting last blood sample, provide every day whole requirements 3/4.
During handling, offer drug solns by instiling by means of the intubate of burying of leading in the jejunum.Intubate is fully cleaned with the aseptic phosphate buffered saline(PBS) of 1ml temperature (25-30 ℃) before administration, and under uniform temp after the administration, fully clean with the 5ml same substance.Provided by the form of the dispersion of vortex in the phosphate buffered saline(PBS) of 4ml temperature with 2ml oil wherein to be blended in lipid and to send calcitonin in the delivery system, and the dispersion that every animal is produced to 1.3ml.The calcitonin of every animals received 5000iu.The form that is dispersed in the dispersion in the phosphate solution of 2ml temperature at whole 3 milliliters of quilts also contained the 200iu insulin with 1ml wherein before jugular vein is administered to a single animal oil provides insulin preparation.Sample collection in the animal
The conduit that will be used to collect blood with the saline solution (500iu/ml) of heparinization every day cleans once.Between sampling date, in the conduit of sampling, adopt the heparin solution (50iu/ml) of low concentration.
During studying, during 9:00,, extract the blood sample that does not exceed 5ml taking out 2ml blood with after from conduit, removing any residual anticoagulant, follow with 50iu/ml heparin solution cleaning pipe to prevent condensing in the conduit.Then animal is carried out administration according to processing scheme.Blood sampling and about medicine intersected carry out, as after 9:00 (zero-time) is carried out the sample administration 0.25 so that can handle many animals simultaneously; 0.5; 1.0; 1.5; 2.0; 3.0; 4.0, extract the blood sample of 4ml altogether at each time point with 6.0 hours.Analytical method
Following processing blood sample:
After discarding first part of blood, first 2ml is put to by in the plastic containers of heparinization.In refrigerated centrifuger with before centrifugal 20 minutes of the 3000rpm, sample is stored in+4 ℃ reach 30 minutes, then the blood plasma that will produce is divided into two parts and be transferred in two suitable containers, and, be refrigerated to-20 ℃ using before the Kone automatic analyzer carries out colorimetric analysis to plasma calcium or concentration of glucose.A sample is analyzed, and kept another sample.The result:
Below the peak of the AUC that reduces by calcium or glucose level of table 1 and 2 and calcium or glucose level reduce zooperal result be provided.
Table 1: as the result of embodiment 5 described calcitonin preparations
| The agent that preparation describes in detail with 5000iu/ animal gives calcitonin | The AUC (4 hours) that Ca reduces | The peak of Ca reduces (mmol/L) |
| 1.sCT/ press down enzyme peptide+Akoline/tween 80 mixture in PBS | -2.19±0.97 | -0.74±0.31 |
| 2.sCT/ press down enzyme peptide+GDO/Tween 80 mixture in PBS | -0.6±0.39 | -0.33±0.26 |
| 3.sCT/ press down enzyme peptide+oleic acid/Tween 80 mixture in PBS | -0.5±0.39 | -0.44±0.04 |
| 4.sCT/ press down enzyme peptide ‖ PC ‖ Akoline ‖ Tween 80 | -0.82±0.89 | -0.39±0.10 |
| 5.sCT/ press down enzyme peptide ‖ Fel Anseris domestica hydrochlorate ‖ Akoline ‖ Tween 80 | -1.85±1.48 | -0.63±0.06 |
| 6.sCT/ press down enzyme peptide ‖ Fel Anseris domestica hydrochlorate ‖ oleic acid ‖ Tween 80 | -0.63±0.38 | -0.24±0.18 |
| 7.sCT ‖ Fel Anseris domestica hydrochlorate ‖ Akline ‖ Tween 80 | -1.73±1.20 | -0.60±0.36 |
| 8.sCT ‖ ursol cholate ‖ Akoline ‖ Tween 80 9.sCT ‖ α TS ‖ Akoline ‖ Tween 80 10.sCT ‖ dioctylsulfosuccinat salt ‖ Akoline ‖ Tween 80 | -2.26±1.31 -2.22±1.04 -1.55±0.83 | -0.73±0.44 -0.73±0.32 -0.54±0.25 |
Table 2: as the result of embodiment 6 described insulin preparations
| The dosage that preparation describes in detail with 200iu/ animal gives insulin | The AUC (4 hours) that glucose reduces | The peak of glucose reduces (mmol/L) |
| 11. insulin/press down enzyme peptide+Akoline/Tween 80 mixture in PBS | -3.78±2.65 | -2.34±1.79 |
| 12. insulin/but enzyme peptide ‖ PC ‖ Akoline ‖ Tween 80 | -2.84±2.97 | -1.45±1.54 |
| 13. insulin/but enzyme peptide ‖ Fel Anseris domestica hydrochlorate (height) ‖ Akoline ‖ Tween 80 | -2.02±1.44 | -1.94±1.17 |
| 14. insulin/but enzyme peptide ‖ Fel Anseris domestica hydrochlorate (in) ‖ Akoline ‖ Tween 80 | -2.93±1.58 | -1.96±1.17 |
| 15. insulin/but enzyme peptide ‖ Fel Anseris domestica hydrochlorate (low) ‖ Akoline ‖ Tween 80 | -2.98±2.88 | -2.18±1.68 |
| 16. insulin/but enzyme peptide ‖ ursol cholate (low) ‖ Akoline ‖ Tween 80 | -3.66±2.17 | -1.85±1.29 |
| 17. insulin ‖ ursol cholate (low) ‖ Akoline ‖ Tween 80 is only arranged | -5.69±2.32 | -2.75±0.59 |
| 18. the PBS solution of insulin/ursol cholate | -2.09±1.23 | -1.03±0.93 |
| 19. negative control (no insulin) | -1.58±1.03 | N/A |
| 11-18. | -1.69 | - |
| 17-18 | -3.60 |
In table 2, the hydroholic solution (17) that insulin is mixed with in the medium chain monoglyceride produces than the bigger effect of the simple two-phase mixture of each composition (11).The AUC that glucose reduces but not the reduction of peak glucose is more remarkable, this explanation long action period in improving effect is a key factor.The AUC value that from (11) and (17), deducts group (18) provide by send pass effect that excipient produces greater than with the effect contribution that is higher than with the insulin of free form administration.As can be seen, the insulin that is mixed with the solution in the medium chain monoglyceride produces greater than the duple effect by the potentiation that simple mixtures provided of each composition.
Claims (24)
1. a hydrophobic formulation comprises
(ⅰ) a kind of oil phase that comprises one or more medium chain monoglycerides;
(ⅱ) at least a amphiphilic species; With
(ⅲ) a kind of dissolving or otherwise be dispersed in hydroaropic substance in described one or more glyceride; Wherein hydroaropic substance is generally insoluble in described one or more glyceride.
2. the described hydrophobic formulation of claim 1, wherein oil phase comprises the mixture of medium chain monoglyceride and diglyceride.
3. the described hydrophobic formulation of claim 2, wherein medium chain triglycerides has the chain length of 8-10 carbon atom.
4. claim 2 or 3 described hydrophobic formulations, wherein oil phase further comprises other chemical compound, as oleic acid, glyceryl monooleate or gelucires.
5. the described hydrophobic formulation of claim 4, wherein oil phase comprises the 40-90% monoglyceride.
6. the described hydrophobic formulation of claim 5, wherein oil phase comprises the 60-70% monoglyceride.
7. each described hydrophobic formulation of claim 1-6, wherein oil phase comprises Akoline MCM.
8. each described hydrophobic formulation, wherein amphiphilic species of claim 1-7: the ratio of hydrophilic substance is (by weight) in 1: 1 to 20: 1 scope.
9. the described hydrophobic formulation of claim 8, wherein amphiphilic species: the ratio of hydrophilic substance is 1.2: 1 to 8: 1 (by weight).
10. each described hydrophobic formulation of claim 1-9, wherein amphiphilic species is phosphatidylcholine, phosphatidic acid, phosphatidyl glycerol, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine or its haemolysis derivant, octyl glucoside or other glycolipid, tocopherol succinate and Cholesteryl hemisuccinate, Sodium docusate or hydroxypropyl cellulose.
11. each described hydrophobic formulation of claim 1-9, wherein amphiphilic species is a bile salts.
12. the described hydrophobic formulation of claim 11; wherein amphiphilic species bile salts, comprise cholate, dexycholate, chenodesoxy cholate, ursodeoxycholate, taurocholate, taurodeoxycholate, TUDCA salt, cattle sulphur chenodesoxy cholate, glycocholate, sweet dexycholate, sweet ursodeoxycholate, sweet chenodesoxy cholate, lithocholate, taurolithocholic acid salt or Calamina cholate.
13. each described hydrophobic formulation of claim 1-12, wherein hydroaropic substance is the hydrophilic macromole.
14. the described hydrophobic formulation of claim 13, wherein the hydrophilic macromole comprises protein, glycoprotein, few nucleic acid and/or Polynucleotide such as DNA or RNA, with and analog, polysaccharide such as heparin or its supramolecular complex.
15. claim 13 or 14 described hydrophobic formulations, wherein micromolecule and hydrophilic macromole are molten altogether.
16. the described hydrophobic formulation of claim 15, wherein micromolecule is polysaccharide such as cyclodextrin or vitamin B12.
17. each described hydrophobic formulation of claim 13-16, wherein macromole is a protein.
18. the described hydrophobic formulation of claim 17, wherein protein is insulin, calcitonin, hemoglobin, soybean trypsin inhibitor, presses down enzyme peptide, GLP1, erythropoietin, growth hormone, growth hormone, somatotropin releasing factor, galanin, urokinase, blood factor such as factor VIII and factor IX, tissue plasminogen activator, superoxide dismutase, catalase, peroxidase, interferon, somatostatin, hirudin, LHRU or its analog or fragment.
19. each described hydrophobic formulation of claim 1-12, wherein hydroaropic substance is little organic molecule, little inorganic molecule or colloidal materials.
20. the described hydrophobic formulation of claim 19, wherein hydroaropic substance is glucose, CF 5(6)-Carboxyfluorescein, anticarcinogen, vitamin, calcium chloride, sodium phosphate or colloidal metal, as aurosol, palladium, platinum or rhodium.
21. each described hydrophobic formulation of claim 1-20, it further comprises one or more other compositions that is selected from antioxidant, metal-chelator, buffer agent and dispersant.
22. an Emulsion, it comprises each defined hydrophobic formulation as claim 1-21.
23. a medicament, it comprises each defined hydrophobic formulation as claim 1-21.
24. each defined hydrophobic formulation of claim 1-21 is used for the oral purposes of sending the medicine of passing hydroaropic substance in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97196069 CN1224360A (en) | 1996-07-02 | 1997-07-02 | Hydrophobic preparations contg. medium chain monoglycerides |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9613858.1 | 1996-07-02 | ||
| CN 97196069 CN1224360A (en) | 1996-07-02 | 1997-07-02 | Hydrophobic preparations contg. medium chain monoglycerides |
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| Publication Number | Publication Date |
|---|---|
| CN1224360A true CN1224360A (en) | 1999-07-28 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 97196069 Pending CN1224360A (en) | 1996-07-02 | 1997-07-02 | Hydrophobic preparations contg. medium chain monoglycerides |
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| CN (1) | CN1224360A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002085408A1 (en) * | 2001-04-20 | 2002-10-31 | Tsinghua University | Method of production of insulin-containing oil-based preparation for oral |
| CN103826613A (en) * | 2011-05-06 | 2014-05-28 | 瓦辛内有限公司 | Hydrophobic preparations |
| CN107952063A (en) * | 2017-12-27 | 2018-04-24 | 北京四环生物制药有限公司 | Calcitonin piece and preparation method thereof |
-
1997
- 1997-07-02 CN CN 97196069 patent/CN1224360A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002085408A1 (en) * | 2001-04-20 | 2002-10-31 | Tsinghua University | Method of production of insulin-containing oil-based preparation for oral |
| CN103826613A (en) * | 2011-05-06 | 2014-05-28 | 瓦辛内有限公司 | Hydrophobic preparations |
| CN103826613B (en) * | 2011-05-06 | 2016-08-24 | 瓦辛内有限公司 | Hydrophobic formulation |
| CN107952063A (en) * | 2017-12-27 | 2018-04-24 | 北京四环生物制药有限公司 | Calcitonin piece and preparation method thereof |
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