Title:  Hydrophobic preparations containing medium chain monoglycerides

United States Patent:  6,258,377

Assignee:  Provalis UK Limited (GB)

Filed:  December 22, 1998

Foreign Application Priority Data:  Jul 02, 1996[GB] (9613858)

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, solubilized or otherwise dispersed in the one or more glycerides. The hydrophilic species is one that is not normally soluble in the glycerides.

Description of the Invention

The present invention relates to preparations of substances in hydrophobic solvents in which they would normally be soluble and to processes for obtaining these preparations. In particular, the invention relates to preparations of hydrophilic species in mixtures of medium chain monoglycerides (MCMs) and diglycerides.

The invention in particular applies to hydrophilic macromolecules which would not normally be soluble in oils or other hydrophobic solvents.

For many applications, e.g. in the pharmaceutical sciences, in food technology or the cosmetics industry, work with proteins and similar macromolecules presents problems because their hydrophilicity and high degree of polarity limit the extent to which they can interact with or incorporate into lipid phases. Many natural systems employ lipidic barriers (eg skin, cell membranes) to prevent access of hydrophilic molecules to internal compartments; the ability to disperse proteins in lipidic vehicles would open up a new route to introduction of these macromolecules into biological systems, whereby the lipid medium containing the protein can integrate with the hydrophobic constituents of barriers, instead of being excluded by them.

We have previously disclosed, in WO-A-9513795, WO-A-9617593 and WO-A-9617594, methods for preparing hydrophobic preparations where a hydrophilic species is solubilised in a hydrophobic phase in which it would not normally be soluble. In particular, these methods are suitable for solubilising proteins.

Although the above-described preparations provide simple and efficient methods for solubilising macomolecules such as proteins, we have now found that the macromolecule delivery properties of the preparations can be improved by the use of a particular oil phase and, optionally, particular amphiphiles. This is particularly advantageous when the macromolecule to be solubilised is a protein, eg a pharmaceutically active protein, since the preparations disclosed herein provide not only enhanced uptake of the therapeutic macromolecule but also good dose repeatability.

Thus, in a first aspect, the present invention provides a hydrophobic preparation comprising:

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

(ii) at least one amphiphile;

(iii) a hydrophilic species solubilised or otherwise dispersed in the mixture of glycerides;

wherein the hydrophilic species is one that is not normally soluble in the one or more monoglycerides.

In the context of the present invention "hydrophobic preparation" is a preparation in which the hydrophilic species is not present in aqueous phase. Such a hydrophobic preparation is particularly suitable for use in orally delivering a hydrophilic macromolecule such as a protein.

The prior art does contain a number of examples of the use of medium chain monoglycerides as permeation enhancers in the intestine (Sekine et al, J.Pharmacobiodyn., 7:856-63 (1984); Higkai et al, J.Pharmacobiodyn., 9:532-9 (1986); Unowsky et al, Chemotherapy, 34:272-6 (1988); Watanabe et al, 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 every case the formulations disclosed are ones where the active principle/drug is solubilised in an aqueous phase. In fact until the methods disclosed in, inter alia, WO-A-9513795 preparations where a hydrophilic species was truly and readily solubilised in a hydrophobic phase, with retention of biological activity, were not available.

Preparations in accordance with the invention will generally have no bulk aqueous phase and may have no free water molecules.

In a preferred embodiment the oil phase i) will compose a mixture of medium chain mono- and diglycerides. Suitably, medium chain glycerides useful in the present invention have chain lengths of 8 to 10 carbon atoms, for example, they can comprise straight chain saturated fatty acids. In another embodiment the oil phase i) may comprise one or more medium chain monoglycerides together with at least one other component such as oleic acid, glycerol mono-oleate or gelucides. For both these embodiments the essential component will be the medium chain monoglyceride(s). Whether medium chain monoglyceride(s) are used alone or as mixtures of glycerides or the like, the oil component used should be such that the amount of monoglyceride(s) present should be maximised while ensuring that the oil component remains liquid at a temperature of 45^oC. or lower. In particular, monoglyceride(s) can make up 40-90% of the total amount of oil present, preferably 60-70%. An example of a suitable mixture of glycerides is Akoline MCM.TM. which contains both medium chain mono- and diglycerides, available from Karlshamns Sweden AB, S/374 82 Karlsbamn, Sweden.

Preferably, the ratio of amphiphile:macromolecule is in the range 1:1 to 20:1 by weight and more preferably in the range 2:1 to 8:1 by weight.

Examples of suitable amphiphiles include phospholipids such as phosphatidyl choline, phosphatidic acid, phosphatidyl glycerol, phosphatidyl ethanolamine and lyso-derivatives of these, octyl glucoside and other glycolipids, tocopherol succinate and cholesterol hemisuccinate. Other suitable amphiphiles include phosphatidyl serine, sodium docusate and hydroxypropyl cellulose. More than one amphiphile may be used.

In one preferred embodiment the amphiphile used is a bile salt. In the present invention it should be understood that the terms bile salt and bile acid are used interchangeably because whether the salt or its conjugate acid is present will depend on the pH of the surrounding environment.

Bile salts are naturally occurring surfactants. They are a group of compounds with a common "backbone" structure based on cholanic acid found in all mammals and higher vegetables. Bile salts may be mono-, di- or tri-hydroxylated; they always contain a 3.alpha.-hydroxyl group whereas the other hydroxyl groups, most commonly found at C₆, C₇ or C₁₂, may be positioned either above (.beta.) or below (.alpha.) the plane of the molecule.

Within the class of compounds described as bile salts are included amphiphilic polyhydric sterols bearing carboxyl groups as part of the primary side chain. The most common examples of these in mammals result from cholesterol metabolism and are found in the bile and, in derivatised form, throughout the intestine.

In the context of this specification, the term may also apply to synthetic analogues of naturally occurring bile salts which display similar biological effects, or to microbially derived molecules such as fusidic acid and its derivatives.

The bile salt (or salts) may be either unconjugated or conjugated. The term "unconjugated" refers to a bile salt in which the primary side chain has a single carboxyl group which is at the terminal position and which is unsubstituted. Examples of unconjugated bile salts include cholate, ursodeoxycholate, chenodeoxycholate and deoxycholate. A conjugated bile salt is one in which the primary side chain has a carboxyl group which is substituted. Often the substituent will be an amino acid derivative which is inked via its nitrogen atom to the carboxyl group of the bile salt. Examples of conjugated bile salts include taurocholate, glycocholate, taurodeoxycholate and glycodeoxycholate.

Thus, in the present invention examples of suitable bile salts include cholate, deoxycholate, chenodeoxycholate and ursodeoxycholate, with ursodeoxycholate being particularly preferred. Other bile salts which may be employed include taurocholate, taurodeoxycholate, taurouodeoxycholate, taurochenodeoxycholate, glycholate, glycodeoxycholate, glycoursodeoxycholate, glycochenodeoxycholate, lithocholate, taurolithocholate and, glycolithocholate.

In the present invention the term "hydrophilic species" relates to any species which is generally soluble in aqueous solvents but insoluble in hydrophobic solvents. The range of hydrophilic species of use in the present invention is diverse but hydrophilic macromolecules represent an example of a species which may be used.

A wide variety of macromolecules is suitable for use in the present invention. In general, the macromolecular compound will be hydrophilic or will at least have hydrophilic regions since there is usually little difficulty in solubilising a hydrophobic macromolecule in oily solutions. Examples of suitable macromolecules include proteins and glycoproteins, oligo and polynucleic acids, for example DNA, eg plasmid DNA, and RNA, as well as DNA and/or RNA analogues, polysaccharides such as heparin (particularly low molecular weight heparin) and supramolecular assemblies of any of these including, in some cases, whole cells or organelles. It may also be convenient to co-solubilise a small molecule such as a vitamin in association with a macromolecule, particularly a polysaccharide such as a cyclodextrin. Small molecules such as vitamin B12 may also be chemically conjugated with macromolecules and may thus be included in the compositions.

Examples of particular proteins which may be successfully solubilised by the method of the present invention include insulin, calcitonin, haemoglobin, cytochrome C, horseradish peroxidase, aprotinin, mushroom tyrosinase, erythropoietin, somatotropin, 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 of the above proteins can be from any suitable source). Other proteins include soy bean trypsin inhibitor, GLP1, other blood coagulation factors, somatostatin, hirudin, and LHRH and analogues and fragments of all of them.

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

It seems that there is no upper limit of molecular weight for the macromolecular compound since dextran having a molecular weight of about 1,000,000 can easily be solubilised by the process of the present invention.

In addition to macromolecules, the process of the present invention is of use in solubilising smaller organic molecules as well as or instead of macromolecules. Examples of small organic molecules include glucose, carboxyfluorescin and many pharmaceutical agents, for example anticancer agents, but, of course, the process could equally be applied to other small organic molecules, for example vitamins or pharmaceutically or biologically active agents. In addition, compounds such as calcium chloride and so phosphate can also be solubilised using this process. Indeed, the present invention would be particularly advantageous for pharmaceutically and biologically active agents since the use of non aqueous solutions may enable the route by which the molecule enters the body to be varied, for example to increase bioavailability.

Small organic molecules which may be incorporated into macromolecule-containing preparations of the invention include stabilising agents such as polyglycerols, PEGs and glycerol (particularly in the case of insulin or, possibly, other proteins), chelating agents, such as citric acid, EDTA and EGTA, and antioxidants such as ascorbate.

Another type of species which may be included in the hydrophobic compositions of the invention is an inorganic material such as a small inorganic molecule or a colloidal substance, for example a colloidal metal. The process of the present invention enables some of the pries of a colloidal metal such as colloidal gold, palladium, platinum or rhodium, to be retained even in hydrophobic solvents in which the particles would, under normal circumstances, aggregate. This could be particularly useful for catalysis of reactions carried out in organic solvents.

The hydrophobic preparations of the invention may also optionally comprise further components. Examples of these include antioxidant, metal chelating agents, buffering agents and dispersion agents. Examples of suitable dispersion agents include surface active agents such as the Tween, Span and Brij classes of agent, as well as polyoxyethylated castor oil derivatives, and other POE-containing surfactants.

The hydrophobic preparations of the present invention can be prepared using a method comprising:

(i) associating the hydrophilic species with the amphiphile in a liquid medium such that, in the liquid medium, there is no chemical interaction between the amphiphile and the hydrophilic species;

(ii) removing the liquid medium to leave an array of amphiphile molecules with their hydrophilic bead groups orientated towards the hydrophilic species; and

(iii) providing a mixture of medium chain mono- and diglycerides around the hydrophilic species/amphiphile array.

Such methods are disclosed in WO-A-9513795. In particular, the hydrophobic preparations of the present invention can be prepared by methods disclosed in PCT patent application No. PCT/GB97/00749 which comprise:

(i) associating the hydrophilic species with the amphiphile in the presence of a hydrophobic phase; and

(ii) removing any hydrophilic solvent which is present;

wherein the hydrophilic solvent removal step is carried out under conditions which maintain the hydrophobic phase in a solid state.

Preferably, when using this method, the hydrophilic species and the amphiphile are first dissolved in a hydrophilic solvent, eg an aqueous solvent, often water alone, and this solution is then brought into association with the glyceride mixture. The hydrophilic solvent removal step is conveniently achieved by lyophilisation, such that it is carried out at temperatures which will ensure that the glyceride mixture is maintained in the solid state until all the water has been removed. Under certain circumstances, the oil may become liquid during lyophilisation, as a result of local rises in temperature in parts of the solid block (usually at the surface and edges) where all the hydrophilic solvent has already been removed. Here the cooling effect deriving from sublimation of hydrophilic solvent no longer exists, and in those areas the oil will melt. This situation will lead to the production of a satisfactory end-product providing that the oil is allowed to drain away from the remainder of the solid block as soon as it appears (if not, then accumulating oil will form a layer which prevents further removal of hydrophilic solvent).

Alternatively, the temperature during lyophilisation can be maintained such that the oil remains solid even after the hydrophilic solvent has been driven off. In this case following lyophilisation, the temperature of the preparation is elevated to produce the single phase preparation. This can often simply be achieved by bringing the lyophilised preparation up to room temperature which in turn will cause the glyceride mixture to return to the liquid state. Other methods for removal of hydrophilic solvent may also be employed, eg spray drying.

The hydrophobic preparation of the present invention are extremely versatile and have many applications. They may either be used alone or they may be combined with an aqueous phase to form an emulsion or similar two phase composition which forms a second aspect of the invention.

In this aspect of the invention there is provided a two phase composition comprising a hydrophilic phase and a hydrophobic phase, the hydrophobic phase comprising a hydrophobic preparation of the invention which is obtainable by the methods described above.

Generally, in this type of composition, the hydrophobic phase will be dispersed in the hydrophilic phase.

As mentioned herein the hydrophobic preparations of the invention have particular advantages in that the hydrophilic species is readily taken up, eg into the bloodstream following oral administration. They are therefore particularly suitable for the oral delivery of protein for example. However, the skilled man will appreciate that the preparations of the invention will also provide advantages for other routes of administration, eg topical or vaginal. This, in a third aspect the present invention provides a pharmaceutical formulation comprising a hydrophobic preparation of the invention. Pharmaceutical formulations within the scope of the invention include capsules, tablets and other presentations. In addition, in a fourth aspect the present invention provides the use of a hydrophobic preparation of the invention in the preparation of a medicament for oral delivery of a hydrophilic species, for instance a hydrophilic macromolecule such as a protein. The invention can also be used to modify the immune response, whereby stimulation or suppression.

Preferred features of each aspect of the invention are equally applicable to each other aspect mutatis mutandis.

Claim 1 of 9 Claims

What is claimed is:

1. A hydrophobic preparation comprising:

(i) an oil phase comprising one or more medium chain monoglycerides and one or more medium chain diglycerides, said mono- and di-glycerides having chain lengths of 8 to 10 carbon atoms, said oil phase optionally comprising an additional compound selected from oleic acid, glycerol mono-oleate or a gelucire, wherein the monoglyceride(s) make(s) up 40-90% of the total amount of oil present;

(ii) at least one amphiphile selected from the group consisting of phosphatidyl choline, phosphatidic acid, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl serine or lyso derivatives thereof, octyl glucoside, tocopherol succinate, cholesterol hemisuccinate, sodium docusate, hydroxypropyl cellulose, and a bile salt, said bile salt being selected from the group of bile salts consisting of cholate, deoxycholate, chenodeoxycholate, ursodeoxycholate, taurocholate, taurodeoxycholate, tauroursodeoxycholate, taurochenodeoxycholate, glycholate, glycodeoxycholate, glycoursodeoxycholate, glycochenodeoxycholate, lithocholate, taurolithocholate and glycolithocholate; and

(iii) a hydrophilic species solubilized or dispersed in the mixture of mono- and di-glycerides; wherein the hydrophilic species is not normally soluble in the mono- or di-glycerides and is selected from the group consisting of proteins, glycoproteins, nucleic acids analogues thereof and polysaccharides; said preparation containing no free water molecules.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.