CN1224360A - Hydrophobic formulations containing medium chain monoglycerides - Google Patents

Abstract

Hydrophobic formulations useful as drug delivery systems and the like include compositions comprising one or more medium chain monoglycerides such as Akoline MCM^TM(ii) at least one amphiphilic substance, preferably comprising a phospholipid such as phosphatidylcholine, and (iii) a hydrophilic molecule, which may be a protein such as insulin or calcitonin or other macromolecule, dissolved or otherwise dispersed in the one or more glycerides. (hydrophilic substances are species that are generally insoluble in glycerides).

Description

Hydrophobic formulations containing medium chain monoglycerides

The present invention relates to formulations of substances in hydrophobic solvents in which they are generally insoluble, and to methods for obtaining such formulations. In particular, the present invention relates to the formulation of hydrophilic substances in mixtures of medium chain monoglycerides (MCM) and diglycerides.

The invention is especially applicable to hydrophilic macromolecules that are generally insoluble in oil or other hydrophobic solvents.

For many applications, such as in pharmaceutical sciences, food manufacturing or the cosmetics industry, the processing of proteins and similar macromolecules arises due to their hydrophilicity and high polarity which limit the extent to which they can act or be incorporated into the lipid phase. problem. Many natural systems employ lipid barriers (e.g. skin, cell membranes) to prevent access of hydrophilic molecules to internal compartments; the ability to disperse proteins in lipid excipients will open a new avenue for introducing these macromolecules into biological systems pathways whereby the protein-containing lipid medium can integrate with the hydrophobic components of the barrier rather than be repelled by them.

We have previously disclosed in WO-A-9513795, WO-A-9617593 and WO-A-9617594 methods for the preparation of hydrophobic formulations in which a hydrophilic substance is dissolved in a hydrophobic phase in which it is normally insoluble. In particular, this method is suitable for solubilizing proteins.

While the formulations described above provide a simple and efficient means of solubilizing macromolecules such as proteins, we have now found that the macromolecule delivery properties of the formulation can be improved by using specific oil phases and optionally specific amphiphiles. This is especially advantageous when the macromolecule being dissolved is a protein, such as a pharmaceutically active protein, since the formulations disclosed herein not only increase the uptake of the therapeutic macromolecule, but also provide good dose reproducibility.

Therefore, in a first aspect, the present invention provides a hydrophobic formulation comprising:

(i) an oil phase comprising one or more medium chain monoglycerides; and

(ii) at least one amphiphilic substance;

(iii) A hydrophilic substance dissolved or otherwise dispersed in the glyceride mixture; wherein the hydrophilic substance is generally insoluble in said one or more monoglycerides.

In the context of the present invention, a "hydrophobic formulation" is a formulation in which hydrophilic substances are not present in the aqueous phase. Such hydrophobic formulations are particularly suitable for oral delivery of hydrophilic macromolecules, such as proteins.

The prior art does contain many examples of the use of medium chain monoglycerides as permeation enhancers in the intestine (Sekine et al., J. Pharmacobiodyn., 7:856-63 (1984); Higaki et al. , Journal of Pharmacobiokinetics, 9:532-9 (1986); Unowsky et al., Chemotherapy, 34:272-6 (1988); Watanabe et al., Journal of Pharmaceutical Sciences (J.Pharm.Sci.), 77 :847-9 (1988); Yeh et al.; Pharm. Res., 11:1148-54 (1994); Constinides et al., Pharm. Res., 11:1385-90 (1994)). However, in each case the active ingredient/drug is dissolved in the aqueous phase in the disclosed formulations. In fact it was not until especially the process disclosed in WO-A-9513795 that formulations in which the hydrophilic substance was truly and easily dissolved in the hydrophobic phase and the biological activity was retained were not available.

Formulations according to the invention will generally not contain a substantial aqueous phase and possibly no free water molecules.

In a preferred embodiment, the oil phase will comprise a mixture of medium chain monoglycerides and diglycerides, suitably the medium chain glycerides used in the present invention have a chain length of 8-10 carbon atoms, For example, they may contain straight chain saturated fatty acids. In another embodiment, the oil phase may comprise one or more chain monoglycerides and at least one other component, such as oleic acid, monoolein or gelucires. For these two specific embodiments, the essential ingredient is medium-chain monoglycerides, whether using medium-chain monoglycerides alone or using a mixture of glycerides, etc., the oil components used should be at a temperature of 45 ° C or more. The low temperature maintains the liquid state while maximizing the amount of monoglyceride present. In particular, monoglycerides may represent 40-90%, preferably 60-70%, of the total amount of oil present, an example of a suitable glyceride mixture is Akoline MCM ^™ , which contains medium chain monoglycerides and glycerol Diesters, which are commercially available from Karlshamns Sweden AB, S/374 82 Karlshamn, Sweden.

Preferably, the amphiphilic substance:macromolecule ratio is in the range of 1:1-20:1 (by weight), more preferably in the range of 2:1-8:1 (by weight).

Examples of suitable amphiphilic substances include phospholipids such as phosphatidylcholine, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine and their lyso-derivatives, octylglucoside and other glycolipids, tocopheryl succinate and cholesterol hemisuccinate. Other suitable amphiphilic substances include phosphatidylserine, sodium docusate and hydroxypropylcellulose. More than one amphiphilic substance may be used.

In a preferred embodiment, the amphiphilic hydrophilic substances used are bile salts. In the present invention, it is understood that the terms bile salt and bile acid are used interchangeably since the presence or absence of a salt thereof or its coupled acid will depend on the pH of the surrounding environment.

Bile salts are naturally occurring surfactants, which are a group of compounds present in all animals and higher plants with a common "backbone" structure of bile acid. Bile salts can be mono-, di- or tri-hydroxylated; they always contain a 3α-hydroxyl group, while the other hydroxyl group, most often at _C6 , _C7 or _C12 , can be positioned above the plane of the molecule (β) or below (α).

Included in the class of compounds described as bile salts are amphiphilic polyhydric sterols having a carboxyl group as part of the primary side chain. The most common examples of bile salts in mammals are produced by the metabolism of cholesterol and exist in derivatized forms in bile and throughout the intestine.

In the context of the specification, the term can also be applied to synthetic analogues of naturally occurring bile salts, or microbially produced molecules, such as carboxoric acid and its derivatives, which exhibit similar biological effects.

The bile salt (or salts) can be unconjugated or conjugated. The term "non-conjugated" refers to bile salts in which the primary side chain has a single carboxyl group that is terminal and unsubstituted. Examples of unconjugated bile salts include cholate, ursodeoxycholate, chenodeoxycholate and deoxycholate. Conjugated bile salts are those in which the primary side chain has a substituted carboxyl group. Usually the substituent is an amino acid derivative linked to the carboxyl group of the bile salt by means of a nitrogen atom. Examples of conjugated bile salts include taurocholate, glycocholate, taurodeoxycholate and glycodeoxycholate.

Accordingly, in the present invention, examples of suitable bile salts include cholate, deoxycholate, chenodeoxycholate and ursodeoxycholate, with ursodeoxycholate being particularly preferred. Other bile salts that may be used include taurocholate, taurodeoxycholate, tauroursodeoxycholate, taurochenodeoxycholate, glycocholate, glycodeoxycholate, ammonium Sulfodeoxycholate, Glycochenodeoxycholate, Lithocholate, Taurolithocholate, and Glycocholate.

In the present invention, the term "hydrophilic substance" refers to any substance that is generally soluble in an aqueous solvent but insoluble in a hydrophobic solvent. The range of hydrophilic substances useful in the present invention varies widely, but hydrophilic macromolecules represent one example of substances that may be used.

Many macromolecules are suitable and useful in the present invention. In general, macromolecular compounds are hydrophilic, or at least have hydrophilic regions, since there is often little difficulty in dissolving hydrophobic macromolecules in oil solutions. Examples of suitable macromolecules include proteins and glycoproteins, oligo- and polynucleic acids, for example DNA such as plasmid DNA and RNA, and DNA and/or RNA analogs, polysaccharides such as heparin (especially low molecular weight heparin) and supersedes of any of these. molecular complex. In some cases whole cells or organelles are involved. It may also be convenient to co-dissolve small molecules such as vitamins with larger molecules, especially polysaccharides such as cyclodextrins. Molecules such as vitamin B12 can also be chemically coupled to macromolecules and thus included in the composition.

Examples of specific proteins that can be successfully solubilized by the method of the present invention include insulin, calcitonin, hemoglobin, cytochrome C, horseradish peroxidase, aprotinin, mushroom tyrosinase, erythropoietin, somatotropin hormone, growth hormone, growth hormone releasing factor, galanin, urokinase, factor IX, tissue plasminogen activator, superoxide dismutase, catalase, peroxidase, ferritin, interferon, factor VIII and fragments thereof (all proteins above may be from any suitable source). Other proteins include soybean trypsin inhibitor, GLP1, other coagulation factors, somatostatin, hirudin and LHPH and all analogs and fragments thereof.

Mixtures of one or more of these or other proteins may be solubilized by the present invention.

Since dextran with a molecular weight of about 1,000,000 can be easily solubilized by the method of the present invention, it appears that there is no upper limit to the molecular weight of macromolecular compounds.

In addition to macromolecules, the method of the invention can be used to dissolve smaller organic molecules and or macromolecules. Examples of small organic molecules include glucose, carboxyfluorescein and many pharmaceutical agents, such as anticancer agents, but of course the method is equally applicable to other small organic molecules, such as vitamins or pharmaceutical or bioactive agents. In addition, compounds such as calcium chloride and sodium phosphate can be dissolved using this method. Indeed, the present invention is particularly advantageous for pharmaceutically active and biologically active agents since the use of non-aqueous solutions may allow the route of entry of molecules into the body to be altered, eg, to increase bioavailability.

Small organic molecules that may be incorporated into the macromolecule-containing formulations of the present invention include stabilizers such as polyglycerol, PEG and glycerol (especially in the case of insulin or possibly other proteins), chelating agents such as citric acid, EDTA and EGTA , and antioxidants such as ascorbic acid.

Other types of materials that may be included in the hydrophobic compositions of the present invention are inorganic materials such as small inorganic molecules or colloidal materials such as colloidal metals. The method of the present invention allows some of the properties of colloidal metals, such as colloidal gold, palladium, platinum or rhodium, to be preserved even in hydrophobic solvents in which the particles aggregate under normal conditions. This can be used in particular for the catalysis of reactions carried out in organic solvents.

The hydrophobic formulations of the present invention may also optionally contain other ingredients. Examples of these include antioxidants, metal chelating agents, buffers and dispersants. Examples of suitable dispersants include surfactants such as Tween, Span and Brij type agents as well as polyoxyethylated castor oil derivatives and other POE-containing of surfactants.

The hydrophobic formulations of the present invention can be prepared using the following method, which method comprises:

(i) Combining the hydrophilic substance and the amphiphilic substance in a liquid medium such that there is no chemical interaction between the amphiphilic substance and the hydrophilic substance in the liquid medium.

(ii) removal of the liquid medium, leaving an array of amphiphilic molecules with their hydrophilic headgroups facing the hydrophilic material; and

(iii) Provide a mixture of medium chain monoglycerides and diglycerides around the hydrophilic/amphiphilic array.

This method is disclosed in WO-A-9513795. In particular, the hydrophobic formulations of the present invention may be prepared by the method disclosed in PCT Patent Application No. PCT/GB97/00749, which method comprises:

(i) combining a hydrophilic substance with an amphiphilic substance in the presence of a hydrophobic phase; and

(ii) removing any hydrophilic solvent present; wherein the step of removing the hydrophilic solvent is carried out under conditions which maintain the hydrophobic phase in a solid state.

Preferably, when using this method, the hydrophilic and amphiphilic substances are first dissolved in a hydrophilic solvent, such as an aqueous solvent, usually water alone, and this solution is then combined with the glyceride mixture. The hydrophilic solvent removal step is conveniently accomplished by freeze drying at a temperature that ensures that the glyceride mixture remains solid until all water has been removed. In some cases, the oil may become liquid during freeze-drying as a result of a localized increase in temperature in certain parts of the solid mass (usually at the surface and edges) where all the hydrophilic solvent has been removed. Here the cooling effect from the sublimation of the hydrophilic solvent no longer exists and the oil will melt in those regions. As long as the oil is allowed to drain from the remainder of the solid block as soon as it is present, then this situation will lead to a satisfactory end product (if not, the accumulated oil will form a layer that prevents further removal of the hydrophilic solvent ).

On the other hand, the temperature during freeze-drying can be maintained so that the oil remains solid even after the hydrophilic solvent has been removed. In this case, after lyophilization, the temperature of the formulation was raised to produce a single-phase formulation. This is accomplished by simply raising the temperature of the lyophilized formulation to room temperature, which will cause the glyceride mixture to return to a liquid state. Other methods of removing hydrophilic solvents, such as spray drying, can also be used.

The hydrophobic formulations of the invention are multipotent and have many uses. They may be used alone or may be mixed with the aqueous phase to form emulsions or similar two-phase compositions which constitute the second aspect of the invention.

In this aspect of the invention there is provided a biphasic composition comprising a hydrophilic phase and a hydrophobic phase, wherein the hydrophobic phase comprises a hydrophobic formulation of the invention obtainable by the process described above.

Typically, in such compositions, the hydrophobic phase is dispersed within the hydrophilic phase.

As mentioned herein, the hydrophobic formulations of the present invention have particular advantages because hydrophilic substances are readily absorbed, eg into the blood, after oral administration. They are therefore especially suitable for oral delivery of eg proteins. However, the skilled artisan will recognize that the formulations of the present invention will also facilitate other routes of administration, such as topical or vaginal administration. Accordingly, in a third aspect of the invention there is provided a pharmaceutical formulation comprising a hydrophobic formulation of the invention. Pharmaceutical formulations within the scope of this invention include capsules, tablets and other forms. Furthermore, in the fourth aspect of the present invention, there is provided the use of the hydrophobic formulation of the present invention in the preparation of a medicament for the oral delivery of a hydrophilic substance such as a hydrophilic macromolecule such as a protein. The invention can also be used to alter the immune response through stimulation or inhibition.

Preferred features of the various aspects of the invention can also be used in the various other aspects, and vice versa.

The invention will now be illustrated with reference to the following examples, which should not be construed as limiting the invention in any way. Example 1: Preparation of Formulations Comprising Calcitonin/PC Complex

(i) Preparation of Phospholipid Dispersion

1. In a cooking tube with a ground glass stopper, 1.6 g of soybean phosphatidylcholine was weighed and distilled water was added to a final weight of 8 g. Flush with nitrogen, stopper tightly, seal with parafilm and mix gently on an orbital shaker until all solids are dispersed.

2. Transfer to a glass round bottom sonication vessel.

3. Clamp the ultrasound vessel into a Sonics 4 Materials VibraCell VCX 600 sonicator fitted with a 1 inch diameter probe and immerse the probe until its base is well below the meniscus of the liquid. A mucous strip is used to form a collar between the probe and the top of the tube, and the air space above the liquid is purged with nitrogen. Submerge the sonication vessel in an ice bath.

4. The liquid suspension was sonicated at 50% amplitude, with 4 second cooling intervals between 1 second shots, until a milky white dispersion formed (typically 4 minutes total sonication time).

5. The dispersion was transferred to a plastic conical centrifuge tube and centrifuged at 1200 g for 15 minutes, and the supernatant was separated from any precipitate present.

6. Dilute two-fold with distilled water to yield a final phospholipid concentration of 100 mg/ml.

(ii) Preparation of protein solution

7. Weigh 20 mg of aprotinin into a 2 ml glass screw vial and add 1 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

8. Weigh 16ml of salmon calcitonin into a 1ml glass screw vial and add 0.8ml of distilled water. Tighten the cap and mix gently until the solids dissolve.

9. Weigh 50 mg of ascorbic acid and 5 mg of citric acid in a 1 ml glass screw vial and add 1 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

10. Dispense 2 ml of the phospholipid dispersion from step 6 (200 mg solid), 250 μl of the aprotinin solution from step 7 (5 mg solid), 250 μl of the calcitonin solution from step 8 ( 5 mg solid) and 200 μl of the ascorbic acid/citric acid solution from step 9 (10 mg and 1 mg solid, respectively), and gently mix the contents of each vial.

(iii) Freeze-drying of the aqueous phase

11. The contents of each vial were sonically snap frozen in liquid nitrogen.

12. Freeze dry overnight at a condenser temperature below -40°C and a vacuum of less than 0.1 mbar.

(ⅳ) Preparation of oil phase

13. 0.8g Polysorbate 80 and 7.2g Akoline MCM were weighed into a B8 glass screw cap vial. The vial was flushed with nitrogen, capped tightly and sealed with parafilm, and mixed gently on a roller mixer until a homogeneous solution was obtained.

(v) Soluble in the oil phase

14. After the samples were completely dried in the lyophilizer, 1.779 g of the oil phase from step 13 was added to each vial. Each vial was purged with nitrogen, capped tightly and sealed with parafilm.

15. The solids were dissolved by mixing in a roller mixer at room temperature for 2 hours, followed by shaking at 37°C until a clear solution formed.

16. Store at +4°C until use when required.

(ⅵ) In vivo administration

17. Each vial was warmed to 37°C in a water bath to melt the oil and form a clear solution.

18. 4 ml of warm PBS was added to each vial, vortexed for 30 seconds, and 1.2 ml was administered via the jugular vein to 1 pig as described above.

Example 2, preparation of formulations comprising insulin/chenodeoxycholate complex

(i) Preparation of chenodeoxycholate solution

1. Weigh 6.0 g of sodium chenodeoxycholate in a glass Erlenmeyer flask, add and distill to a final weight of 60 g. Flush with nitrogen, stopper tightly and seal with parafilm, and mix gently on an orbital shaker until all solids are dissolved.

(ii) Preparation of protein solution

2. Weigh 120 mg of aprotinin into a 2 ml glass screw vial and add 60 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

3. Three separate portions of 106.5 mg of insulin were weighed into (A) 100 ml Erlenmeyer flasks, (B) 25 ml Erlenmeyer flasks and (C) 10 ml screw top vials. Add 30 ml of chenodeoxycholate solution (1 g solid) from step 3.1.1 to flask (A). 15ml of chenodeoxycholate solution (1.5g solid) was added to flask (B) and 7.5ml (0.75g solid) was added to vial (C). Cover flasks and vials with parafilm and mix gently on an orbital shaker at 37 °C until all solids are dissolved.

4. Weigh 54 mg of citric acid in a 2 ml glass screw vial and add 6.0 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

5. Dispense 2 ml of the insulin/chenodeoxycholate solution from step 3 (A), 100 μl of the aprotinin solution from step 2 and 100 μl of the citric acid solution from step 4 into each of twelve 7 ml glass mouth vials. Gently mix the contents of each vial.

6. Dispense 1 ml of the insulin/chenodeoxycholate solution from step 3 (B), 100 μl of the aprotinin solution from step 2 and 100 μl of the citric acid solution from step 4 into each of twelve 7 ml glass screw vials. Gently mix the contents of each vial.

7. Dispense 0.5 ml of the insulin/chenodeoxycholate solution from step 3, 100 μl of the aprotinin solution from step 2 and 100 μl of the citric acid solution from step 4 into each of twelve 7 ml glass screw vials. Gently mix the contents of the vial.

(iii) Freeze-drying of the aqueous phase

8. The contents of each vial were snap frozen in liquid nitrogen and lyophilized overnight at a condenser temperature below -40°C and a vacuum of less than 0.1 mbar.

(ⅳ) Preparation of oil phase

9. Weigh 6.0g Polysorbate 80 and 54g Akoline MCM into a 100ml glass bottle. The vial was purged with nitrogen, capped tightly, sealed with parafilm and mixed gently on a roller mixer until a homogeneous solution was obtained.

(v) Soluble in oleic acid

10. After the samples were completely dried in the lyophilizer, 0.79 g of the oil phase formulation (A) from step 10 was added to each vial. Each vial was purged with nitrogen, capped tightly and sealed with parafilm.

11. After the samples were completely dried in the lyophilizer, 0.8 g of the oil phase formulation (B) from step 10 was added to each vial. Each vial was purged with nitrogen, capped tightly and sealed with parafilm.

12. After the phase product was completely dried in the lyophilizer, 0.94 g of the oil phase formulation (C) from step 10 was added to each vial. Each vial was purged with nitrogen, capped tightly and sealed with parafilm.

13. The solids were dissolved by mixing in a roller mixer at room temperature for 2 hours, followed by shaking at 37°C until a clear solution formed and stored at +4°C until required.

(ⅵ) In vivo administration

14. The vial was warmed to 37°C in a water bath to melt the oil and form a clear solution.

15. 2 ml of warm PBS was added to each vial, vortexed for 30 seconds, and one pig was administered the entire contents of the vial via the jugular vein as described above.

Embodiment 3. Preparation of Formulations Containing Insulin/PC Complexes

(i) Preparation of Phospholipid Dispersion

1. In a cooking tube with a ground glass stopper, 2 g of soybean phosphatidylcholine was weighed and distilled water was added to give a final weight of 8 g. Wash with nitrogen, stopper tightly, parafilm seal and mix gently on an orbital shaker until all solids are dispersed.

2. Transfer to a glass round bottom sonication vessel.

3. Clamp the ultrasound vessel into a Sonics Material VibraCell VC x 60 sonicator fitted with a 1 inch diameter probe and immerse the probe until its base is 1 cm below the meniscus of the liquid. Using an adhesive strip to form a loop between the probe and the top of the tube, purge the air space above the liquid with nitrogen, and immerse the ultrasound vessel in an ice bath.

4. The liquid suspension was sonicated at 50% amplitude with 4 second cooling intervals between 1 second pulses until a milky white dispersion formed (typically 4 minutes total sonication time).

5. The dispersion was transferred to a plastic conical centrifuge tube and centrifuged at 1200g for 15 minutes. Any precipitate present was separated from the supernatant.

(ii) Preparation of protein solution

6. Weigh 30 mg of aprotinin into a 2 ml glass screw vial and add 1.5 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

7. 110 mg of insulin was weighed into a 25 ml Erlenmeyer flask and 15 ml of distilled water to which 150 μl of glacial acetic acid was added was added. Cover the flask with parafilm and mix gently on an orbital shaker at 37 °C until all solids are dissolved.

8. Weigh 10 mg of sodium citrate into a 2 ml glass screw vial and add 1.5 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

9. Dispense 1 ml of insulin solution from step 7 (7.33 mg solid, 200 iu), 100 μl of sodium citrate (0.67 mg solid) from step 8, 100 μl of protinin from step 6 into each of twelve 7 ml glass screw cap vials Peptide solution (2 mg solid) and 400 μl of phospholipid dispersion from step 5 (100 mg solid). Gently mix the contents of each vial.

(iii) Preparation of oil phase

10. Weigh 1.5g Polysorbate 80 and 13.5g Akoline MCM into a 20ml glass bottle. The vial was flushed with nitrogen, capped tightly and sealed with parafilm, and mixed gently on a roller mixer until a homogeneous solution was obtained.

(ⅳ) Freeze-drying of aqueous phase

11. Add 1 ml of the akoline/polysorbate 80 mixture from step 10 to each vial in step 9 using a large volume positive displacement dispenser.

12. Vials were vortexed rapidly for 10 seconds, then immediately frozen in liquid nitrogen.

13. Freeze-drying at a condenser temperature below -40°C and a vacuum of less than 0.1 mbar for two nights.

(v) Preparation of oil solution

14. Vials were removed from the lyophilizer, flushed with nitrogen, capped and sealed with parafilm. Incubate at 37°C with gentle shaking until a clear solution is obtained, store at +4°C until use when needed.

(ⅵ) In vivo administration

15. Each vial was warmed to 37°C in a water bath to melt the oil and form a clear solution.

16. 2 ml of warm PBS was added to each vial, vortexed for 30 seconds, and one pig was administered the entire contents of the vial via the jugular vein as described above.

Embodiment 4. Preparation of Formulations Comprising Insulin/Ursodeoxycholate Complex

(iv) Preparation of ursodeoxycholate solution

1. Weigh 525 mg sodium ursodeoxycholate in a glass Erlenmeyer flask and add distilled water to give a final weight of 15 g. Flush with nitrogen, cap tightly, seal with parafilm and mix gently on an orbital shaker until all solids are dissolved.

(ii) Preparation of protein solution

2. Weigh 30 mg of aprotinin into a 2 ml glass screw vial and add 1.5 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

3. 110 mg of insulin was weighed into a 25 ml Erlenmeyer flask and 15 ml of ursodeoxycholate solution from step 1 (525 g solids) was added. Cover the flask with parafilm and mix gently on an orbital shaker at 37 °C until the solids dissolve.

4. Weigh 13.5 mg of sodium citrate into a 2 ml glass screw vial and add 1.5 ml of distilled water. Tighten cap and mix gently until all solids are dissolved.

5. Dispense 1 ml of the insulin/chenodeoxycholate solution from step 3 (7.3 mg insulin/35 mg bile salts), 100 μl of sodium citrate (0.9 mg solid) from step 4, and 100 μl of aprotinin solution from step 2 (2 mg solid). Gently mix the contents of each vial.

(iii) Preparation of oil phase

6. Weigh 1.5g polysorbate 80 and 13.5g akoline MCM into a 20ml glass bottle. The vial was flushed with nitrogen, capped tightly and sealed with parafilm, and mixed gently on a roller mixer until a homogeneous solution was obtained.

(ⅳ) Freeze-drying of aqueous phase

7. Add 1 ml of the akoline/polysorbate 80 mixture from step 6 to each vial in step 5 using a large volume positive displacement dispenser.

8. Vials were vortexed rapidly for 10 seconds, then immediately frozen in liquid nitrogen.

9. Freeze-dry over two nights at a condenser temperature below -40°C and a vacuum of less than 0.1 mbar.

(v) Preparation of oil solution

10. Vials were removed from the lyophilizer, flushed with nitrogen, capped and sealed with parafilm. Incubate at 37°C for 10 minutes until a clear solution is obtained.

11. Store at +4°C until use when needed.

(ⅵ) In vivo administration

12. Each vial was warmed to 37°C in a water bath to melt the oil and form a clear solution.

13. 2 ml of warm PBS was added to each vial, vortexed for 30 seconds, and one pig was administered the entire contents of the vial via the jugular vein as described above.

Example 5: Formulations for Enteral Delivery of Calcitonin

The formulations were prepared as follows:

1) A mixture of salmon calcitonin and akoline MCM

As in Example 1, except that (a) the phospholipid dispersion is omitted (b) the protein-containing lyophilizate is not dissolved in the oil phase, (c) the protein lyophilizate is dissolved in 4 ml of phosphoric acid before administration to the animal buffered saline solution, and (d) add the resulting solution to 2 ml of the oil phase (Akoline MCM/Tween 80) and disperse by vortexing.

2) Mixture of salmon calcitonin and diolein

As in formulation (1), only glyceryl dioleate was used instead of Akoline MCM.

3) A mixture of salmon tantalum calcitonin and oleic acid.

Like formulation (1), only oleic acid is used instead of Akoline MCM.

4) Oil containing salmon calcitonin/PC complex in Akoline MCM

As described in Example 1.

5) Oil containing salmon tantalum calcitonin/chenodeoxycholate complex in Akoline MCM

The composition of the preparation is the same as that of the preparation (4) of this example, except that chenodeoxycholate at a concentration of 100 mg/g is used instead of PC in the oil phase.

6) Oil containing salmon calcitonin/chenodeoxycholate complex in Akoline MCM

The composition of the preparation is the same as that of the preparation (4) of this example, except that aprotinin is omitted and PC is replaced by sodium chenodeoxycholate at a concentration of 17.5 mg/g in the oil phase.

7) Oil containing salmon calcitonin/ursodeoxycholate complex in Akoline MCM

The formulation was the same in composition as in (4) above, except that aprotinin was omitted and sodium ursodeoxycholate at a concentration of 17.5 mg/g was used in the oil phase instead of PC.

8) Oil containing salmon calcitonin/tocopheryl succinate complex in Akoline

The formulation was identical in composition to that in (4) above, except that aprotinin was omitted and alpha-tocopheryl succinate at a concentration of 25 mg/g was used instead of PC in the oil phase.

9) Oil containing salmon calcitonin/dioctyl sodium sulfosuccinate complex in Akoline MCM.

The formulation was identical in composition to that in (4) above, except that aprotinin was omitted and dioctyl sodium sulfosuccinate at a concentration of 25 mg/g was used instead of PC in the oil phase. Example 6: Formulations for Enteral Delivery of Insulin

The formulations were prepared as follows:

10) Mixture of Insulin and Akoline MCM

Such as preparation (1), just use 7.3mg insulin (200iu), 2mg aprotinin and 1ml Akoline/Tween 80 oil phase.

11) Oil containing insulin/PC complex in Akoline MCM

As in Example 2.

12) Oil containing insulin/chenodeoxycholate complex in Akoline MCM with high bile salt concentration

As in Example 3.

13) Oil containing insulin/chenodeoxycholate complex and medium bile salt concentration in Akoline MCM

As in Example 3.

14) Oil containing insulin/chenodeoxycholate complex in Akoline MCM and low bile salt concentration

As in Example 3.

15) Oil containing insulin/ursodeoxycholate complex in Akoline MCM (with aprotinin)

As in Example 4.

16) Oil containing insulin/ursodeoxycholate complex in Akoline MCM (without aprotinin)

It is exactly the same as Example 4, except that aprotinin is omitted from the preparation.

17) Insulin/ursodeoxycholate in phosphate buffered saline

A freeze-dried solution of insulin was prepared as in step (3) of Example 4. Before administration, redissolve in phosphate-buffered saline (7.3 mg insulin/2 ml).

Embodiment 7: animal experiments

Animal experiment

Preparation of animal models

The animal model used to test oral formulations of calcitonin is young pigs. The pig was chosen because it has a body weight similar to that of a human, and its small intestine is similar in structure, function and size to the human small intestine. To address concerns related to differences between humans and pigs, the animals were surgically manipulated so that material could be introduced directly into the jejunum by means of a buried cannula. This also reduces the uncertainty usually associated with the time of arrival of an orally administered dose in the intestine and leads to an improvement in the statistical quality obtained.

At least 10 days prior to the first dose, a thin plastic cannula was surgically inserted into the jejunum of the pig and then brought out under the skin on the pig's back, allowing the test substance to be injected into the jejunum without pain to the animal. A buried catheter, also brought out through the dorsal skin, was inserted through the middle saphenous artery into the aorta so that repeated blood samples could be obtained. A second catheter is also introduced into the carotid artery. Administration of preparations

Pigs were tested in a fully conscious fasted state and the peptide preparation was administered by oral route after three baseline blood samples were taken. A single experiment usually takes 8-9 hours, and the operated pigs participate in the experiment up to 3 times a week over a four-week period. After the experiment, the pigs were killed, and the macroscopic and microscopic changes of the small intestine were checked.

Water was provided ad libitum during the experiment and pigs were fed at 8:00 and 15:30. Any food left over after the 15:30 feeding was removed at 16:30. During treatment, food was withdrawn at 08:00, providing 3/4 of the full daily requirement after the last blood sample was drawn.

During treatment, the dosing solution was provided by instillation with the aid of a buried cannula leading into the jejunum. The cannula was thoroughly rinsed with 1 ml of warm (25-30° C.) sterile phosphate-buffered saline solution before administration and with 5 ml of the same substance at the same temperature after administration. Calcitonin blended in the lipid delivery system was provided as a dispersion in which 2 ml of oil was vortexed in 4 ml of warm phosphate buffered saline and 1.3 ml of the resulting dispersion was given to each animal. Each animal received 5000 iu of calcitonin. The insulin preparation was also provided as a dispersion in which 1 ml of oil containing 200 iu of insulin was dispersed in 2 ml of warm phosphate solution before the entire 3 ml was administered via the jugular vein to a single animal. Sample Collection in Animals

Catheters used for blood collection were washed once daily with heparinized saline solution (500 iu/ml). During sampling, a lower concentration of heparin solution (50 iu/ml) was used in the sampling catheter.

During the study, at 9:00, no more than 5 ml of blood was drawn after 2 ml of blood was withdrawn to remove any residual anticoagulant from the catheter, followed by rinsing the catheter with 50 iu/ml heparin solution to prevent clotting in the catheter. Animals are then dosed according to the treatment protocol. Blood samples and appointments were crossed to allow many animals to be treated simultaneously at 0.25; 0.5; 1.0; 1.5; 2.0; 3.0; A total of 4 ml blood samples were drawn at each time point. Analytical method

Blood samples were processed as follows:

After discarding the first blood, place the first 2ml into a heparinized plastic container. Samples were stored at +4°C for 30 minutes before centrifugation at 3000 rpm in a refrigerated centrifuge for 20 minutes. The resulting plasma was then split in two and transferred to two suitable containers, and the plasma was analyzed using the Kone Autoanalyzer. Freeze to -20°C prior to colorimetric analysis of calcium or glucose concentrations. One sample is analyzed while the other is retained. result:

Tables 1 and 2 below provide the results of the animal experiments by AUC of calcium or glucose level reduction and peak reduction of calcium or glucose level.

Table 1: Results of calcitonin formulations as described in Example 5 Formulation Details Calcitonin was administered at a dose of 5000 iu/animal AUC for Ca reduction (4 hours) Ca peak decrease (mmol/L) 1. sCT/aprotinin+Akoline/tween 80 mix in PBS -2.19±0.97 -0.74±0.31 2. sCT/Aprotinin+GDO/Tween 80 mix in PBS -0.6±0.39 -0.33±0.26 3. sCT/aprotinin+oleic acid/Tween 80 mix in PBS -0.5±0.39 -0.44±0.04 4. sCT/Aprotinin‖PC‖Akoline‖Tween 80 -0.82±0.89 -0.39±0.10 5. sCT/Aprotinin‖Cicholic acid salt‖Akoline‖Tween 80 -1.85±1.48 -0.63±0.06 6. sCT/Aprotinin‖Cicholic acid‖Oleic acid‖Tween 80 -0.63±0.38 -0.24±0.18 7. sCT‖cheicholic acid salt‖Akline‖Tween 80 -1.73±1.20 -0.60±0.36 8. sCT‖Usocholic acid salt‖Akoline‖Tween 809． sCT‖αTS‖Akoline‖Tween 8010． sCT‖dioctyl sulfosuccinate‖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: Results for insulin preparations as described in Example 6 Formulation Details Insulin was administered at a dose of 200iu/animal Glucose lowered AUC (4 hours) Glucose peak reduction (mmol/L) 11. Insulin/Aprotinin + Akoline/Tween 80 mix in PBS -3.78±2.65 -2.34±1.79 12. Insulin/Aprotinin‖PC‖Akoline‖Tween 80 -2.84±2.97 -1.45±1.54 13. Insulin/Aprotinin‖Cicholic acid salt (high)‖Akoline‖Tween 80 -2.02±1.44 -1.94±1.17 14. Insulin/Aprotinin‖Cicholic acid salt (medium)‖Akoline‖Tween 80 -2.93±1.58 -1.96±1.17 15. Insulin/Aprotinin‖Cicholic acid (low)‖Akoline‖Tween 80 -2.98±2.88 -2.18±1.68 16. Insulin/Aprotinin‖Usocholate (Low)‖Akoline‖Tween 80 -3.66±2.17 -1.85±1.29 17. Insulin only‖Usocholate (Low)‖Akoline‖Tween 80 -5.69±2.32 -2.75±0.59 18. Insulin/ursocholate in PBS -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, formulation of insulin as a hydrophobic solution in medium chain monoglycerides (17) yielded greater efficacy than a simple biphasic mixture of components (11). The reduction in AUC for glucose but not peak glucose was more pronounced, suggesting that longer duration of action is an important factor in improving efficacy. Subtracting the AUC value of group (18) from (11) and (17) gives the effect produced by the delivery vehicle to be greater than and above the effect contribution of insulin administered in free form. It can be seen that insulin formulated as a solution in medium chain monoglycerides produced more than double the potentiation provided by the simple mixture of the ingredients.

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.