BACKGROUND OF THE INVENTION For many types of drug substances there is a problem to create depot formulations for use in vivo, for example in the case of neuroleptic, antidepressive, anti-psychotic, antibiotic, antimicrobial, antiviral, antidiabetic and anti-Parkinson drugs.

There are also many therapies using hormones and peptides, for example growth hormones and insulin, as well as cytostatic drugs, which suffer from the lack of suitable depot formulations.

For drugs administered intermittently via intravenous injection or by simple oral administration, peaks and troughs in drug concentration are often observed. High drug concentrations may be toxic, whereas low drug concentrations may be subthera- peutic. Controlled release systems aim at maintaining nearly constant in vivo therapeutic drug concentrations for an extended period of time. There are today on the market several delivery systems for controlled and in particular sustained release of drug substances, well-known to those skilled in the art.

Examples are depot systems based on polymer systems from which the active compound is released through diffusion from a non- biodegradable matrix, or through biodegradation of the matrix, or, in the case of water-soluble polymers, through dissolution of the polymer in the biological fluids. The non-biodegradable polymers do not undergo any significant change in the body. They are frequently used in implants, which often need to be eliminated by surgery. Also the biodegradable polymer systems are a potential risk of causing irritation to the site of implantation, which is also the case for water-soluble polymers

during their dissolution and degradation in the body. Another disadvantage with polymeric systems is related to their capacity of incorporation of active substances, which in many cases is low and they are therefore often restricted to highly potent drug substances. A practical problem is that different drugs require different polymer systems to meet their respective specific requirements in terms of incorporation level and release criteria.

Lipid oil systems, such as solutions or suspensions in triglyceride oils, so called fixed oils (USP XXIII), are also used for sustained release. Disadvantages with said systems are that only a limited number of compounds can be incorporated, including drugs which have been esterified with fatty acyl groups to pro-drugs, and that the release rate of such compounds cannot be influenced. This implies that said systems are of limited value as parenteral depot systems. The use of other non- dispersed lipid carriers, i. e. oily vehicles, in pharmaceutical products is quite limited. The use of such systems for oral delivery is based on the self-emulsifying properties of the lipid system and an immediate release of the active compound in the gastrointestinal tract.

Other lipid systems than the oils and oily vehicles are dispersions, such as lipid emulsions and liposomes, which after intravenous administration offer only limited sustained release of incorporated drug substances. However, there are reports in the literature of intramuscularly or subcutaneously injected liposomes, which do work as sustained release delivery systems, but the recognised difficulties are low encapsulation capacity and poor storage stability.

In order to avoid the disadvantages with dispersions a number of thermodynamically stable lipid systems have been developed. They are, however, based on the interaction of water with amphiphilic lipids to form stable liquid crystalline phases. Such systems have hitherto found very limited use in pharmaceutical applications.

PRIOR ART WO 01/66086, in the name of Lipocore Holding AB, refers to a lipid carrier for controlled release of a bioactive substance, the. structure of which is retained in an aqueous environment. The carrier composition comprises a triglyceride oil, a polar lipid selected from the group of phosphatidyl- ethanolamine and monohexosylceramide, and ethanol. The carrier consists entirely of a lipid, or oil, phase, and is therefore well suited for incorporating lipophilic substances; the carrier is, however, incapable of dissolving lipophobic, hydrophilic substances.

US 4,610, 868, in the name of The Liposome Company, Inc, refers to lipid matrix carriers, LMCs, which provide for sustained release of bioactive agents in vivo or in vitro. The LMCs are described as globular structures with a diameter ranging from about 500 to about 100,000 nm composed of a hydro- phobic compound and an amphiphatic compound. Said globular structures are prepared in a cumbersome process involving dissolution of the lipid mixture in an organic solvent, agita- tion of the organic solution in an aqueous phase and evaporation of the organic solvent. The polar lipid used in the preparation of w/o-emulsions is in all examples EPC, that is egg phosphati- dylcholine. Although a sustained release is achieved, effect durations for more than a week cannot be obtained.

US 5,912, 271, in the name of Astra AB, refers to a composition comprising one or more local anaesthetic agents, one or more polar lipids, a triacylglycerol and optionally water, with the objective to obtain faster onset of anaesthesia. When sphingolipid materials are used the preferred sphingolipid is sphingomyelin or products derived from sphingomyelin. In all examples containing sphingolipid materials the sphingomyelin content of the materials lies between 60 and 98 %.

JP2000-119178A, in the name of Shionogi & Co. , Ltd., refers to a composition comprising a ceramide, monohydric lower alcohol, oil and water, which composition is capable of forming a transparent solution in an equilibrium state, i. e. not an

emulsion. The disclosed ceramides can all be dissolved in the composition without heating. Preferred ceramides are ceramide 1, ceramide 3 and ceramide 6 obtained from Cosmoferm B. V. , Delft, The Netherlands. Said ceramides do not have a glycosyl moiety attached to the sphingoid group, that is they are not glycol- ceramides. The disclosed composition is intended for topical use.

There is still need for a pharmaceutical carrier system, which is stable, tissue friendly and easy to manufacture, and which enables a significantly sustained release of a variety of drug substances, in combination with a sufficient capacity for incorporation thereof.

DESCRIPTION OF THE DRAWINGS Figure 1 shows the dissolution profiles obtained with Safranine O as a marker substance, from carrier compositions of the invention.

Figure 2 shows the dissolution profiles obtained with pyridoxine hydrochloride as a marker substance, from carrier compositions of the invention.

DESCRIPTION OF THE INVENTION It has now surprisingly been found that a w/o-emulsion carrier of the composition stated below has the ability to retain its cohesive structure with incorporated compounds in an aqueous environment, and therefore can be used for controlled release, such as sustained release, of an incorporated bioactive substance. The oil phase of the w/o-emulsion carrier of the invention is based on lipid components, which are either normal components of the human cells and membranes, or present in significant amounts in the human diet. This means that said lipids are biocompatible with human tissues and are metabolised in the same way as the corresponding endogenous lipids.

The invention refers to a w/o-emulsion carrier composition for controlled release of a bioactive substance, comprising an oil phase and an aqueous phase dispersed in the

continuous oil phase, which is characterised, in that the oil phase contains at least one non-polar oil and monoglycosyl- ceramide, whereby the carrier composition is able to form a cohesive structure which is retained in an aqueous environment.

The invention also refers to a w/o-emulsion carrier composition for controlled release of a bioactive substance, comprising an oil phase and an aqueous phase dispersed in the continuous oil phase, which is characterised in that the oil phase contains at least one non-polar oil, monoglycosylceramide, and ethanol, whereby the carrier composition is able to form a cohesive structure which is retained in an aqueous environment.

By cohesive structure which is retained in an aqueous environment is meant that the carrier composition forms a macroscopically continuous, typically single, body in the aqueous medium. This cohesive structure in aqueous media differs significantly from the discrete lipid particles, which are normally obtained when a lipid emulsion is dispersed into an aqueous environment. Further, the cohesive composition of the present invention is designed to be administered as such, and not to be dispersed or suspended in any media prior to administration.

The aqueous phase is dispersed as microscopic aqueous droplets in the continuous oil phase. Aqueous phase refers to any aqueous solution or dispersion with water as a solvent or dispersing medium.

The non-polar oil can be a triglyceride oil, a mineral oil or a mixture thereof. A suitable oil, as exemplified below, in the w/o-emulsion carrier composition of the invention is a triglyceride oil, or in other words a triacylglycerol oil, wherein the acyl groups are derived from unsaturated or saturated fatty acids or hydroxy fatty acids having 8-22 carbon atoms. The triglyceride oil can be selected from the group of natural vegetable oils consisting of, but not limited to, almond oil, coconut oil, maize oil, wheat germ oil, soybean oil, sesame oil, palm oil, safflower oil, evening primrose oil, sunflower oil, rape seed oil, linseed oil, corn oil, cottonseed oil,

peanut oil, olive oil, or from the group of fractionated oils consisting of, but not limited to, refined vegetable oils, and medium chain triglyceride oil (also called fractionated coconut oil), or from the group of semi-synthetic oils consisting of, but not limited to, acetylated monoglyceride oils, or from the group of animal oils, consisting of, but not limited to, butter oil, marine oils, such as fish oil. The triglyceride oil is preferably selected from the group consisting of medium chain triglyceride oil, sesame oil, evening primrose oil, sunflower oil, coconut oil, soybean oil, corn oil, fish oil, or a mixture thereof. Examples of mineral oils are hydrocarbon oil, liquid paraffin. Any mixture of oils derived from any of the mentioned groups can also be used in the carrier composition of the invention. From a regulatory point of view the non-polar oil is preferably selected from the oils accepted for parental or oral use according to national and international regulatory authorities.

Monoglycosylceramides are monosaccharide-containing derivatives of ceramides. Ceramides are N-acylated sphingoids. A sphingoid is a long-chain aliphatic amino alcohol. A mono- glycosylceramide can be represented by the general formula I OH R glycosyl R"X NH n 0 I wherein R'and R"represent optionally substituted hydrocarbon chains.

The basic chemical structure of a sphingoid is repre- sented by the compound originally called dihydrosphingosine, now referred to as sphinganine, or more specifically 2-amino-1, 3- octadecanediol. There are many sphinganine homologues, which can differ in chain-length, degree of unsaturation and presence of substituents, such as hydroxyl, oxo, methyl, etc. Examples of sphinganine homologues, or sphingoids, are: 4-sphingenine

(sphingosine), icosasphinganine (C20-dihydrosphingosine), 4- hydroxysphinganine (phytosphingosine), 4-hydroxyicosasphinganine (C20-phytosphingosine), 4-hydroxy-8-sphingenine (dehydrophyto- sphingosine), 4,8-sphingadienine (sphingadienine), 4-hexadeca- sphingenine (C16-sphingosine), hexadecasphinganine (C16-dihydro- sphingosine) and heptadecasphingenine (C17-sphingosine).

The acyl chains linked to the amide nitrogen of naturally occurring ceramides can have a chain length from about 10 to 28 carbon atoms, more often from 16 to 26, and may contain one or more double bonds, and may contain one or more substitu- ents, such as hydroxyl, oxo and lower alkyl, such as methyl.

Obviously, synthetically produced ceramides can have much more diverse acyl chains linked to the amide nitrogen, in terms of chain length, double bonds, substituents, etc.

The invention especially refers to a carrier composition wherein the acyl chains linked to the amide nitrogen of the monoglycosylceramides are derived from unsaturated or saturated fatty acids having 10-28 carbon atoms (that is R"having 9-27 carbon atoms).

According to another aspect of the invention the composition contains monoglycosylceramides wherein the sphingoid base has 6-28 carbon atoms (that is R'has 3-25 carbon atoms).

Preferred monoglycosylceramides according to the invention are monohexosylceramides, CMH, and in particular monoglucosylceramide or monogalactosylceramide. Monohexosyl- ceramide can be described by the following formula II OH R !--o hexosyl R"X ÑH o 0 II wherein R'and R"are defined as in formula I.

Monohexosylceramides, CMH, also sometimes called cerebrosides, can be of synthetic or semi-synthetic origin, or obtained from milk or other dairy products, or from animal

organs or materials, such as brain, spleen, liver, kidney, erythrocytes, or from plant sources.

In monohexosylceramide from bovine milk the most commonly occurring sphingoid is sphingenine. Formula III below refers to a monoglucosylceramide based on sphingenine wherein R1 and R2 represent optionally substituted hydrocarbon chains.

Examples of other sphingoids present are hexadeca- sphingenine, hexadecasphinganine, heptadecasphingenine and sphinganine. In monohexosylceramide from bovine milk the acyl chains linked to the amide nitrogen of the ceramides range in chain length from about 12-28 carbon atoms, where the four most common acyl chains, C16: 0, C22: 0, C23: 0 and C24: 0, account for about 80 % by weight, as determined by gas chromatography. The average acyl chain length is about 22 carbon atoms and the fraction of unsaturated acyl chains is about 5 % by weight, as determined by gas chromatography.

According to a preferred aspect of the present invention the composition contains monohexosylceramides obtainable from bovine milk.

An advantage of the monoglycosylceramides over phospho- lipids and many other polar lipids is the relatively high chemical stability due to a less tendency of oxidation and hydrolysis.

The release properties of the w/o-emulsion carrier system of the invention is depending on the composition of the oil phase, the composition of the aqueous phase and the ratio aqueous phase/oil phase, and can be controlled by selecting the proportions of the oil phase components and the aqueous phase components, and selecting the ratio aqueous phase/oil phase (the

w/o ratio). Said proportions can also be selected to optimise the incorporation of specific bioactive substances, or to control the viscosity of the carrier composition.

A w/o-emulsion carrier composition, which is suitable for parenteral administration, e. g. subcutaneous, intraperi- tonal, intramuscular or intradermal injection, or for oral administration, can preferably consist of an oil phase of 30- 99.9 % by weight of non-polar oil, such as a triglyceride oil or a suitable mineral oil, in combination with 0.1-40 % by weight of monoglycosylceramide, and up to 30 % by weight of ethanol, and an aqueous phase, in a w/o ratio of up to 80/20 by weight.

If ethanol is not needed for the incorporation of the active substance, the carrier composition can consist of an oil phase of 60-99.9% by weight of non-polar oil, such as a tri- glyceride oil or a suitable mineral oil, in combination with 0.1-40 % by weight of monoglycosylceramide, and an aqueous phase, in a w/o ratio of up to 80/20 by weight. A preferred w/o ratio is 10/90-70/30, by weight, of the respective phases. In order to get an injectable preparation the non-polar oil should preferably be liquid at ambient temperature.

The upper limit of the content of monoglycosylceramide is restrained by the possibility of achieving a homogeneous oil phase during the preparation of the carrier and by the high viscosity obtained in the oil phase and the carrier at too high contents thereof. If the oil phase has a high viscosity, it is difficult to disperse the aqueous phase in the oil phase, and for parenteral formulations a carrier of high viscosity is not practical, but a carrier of high viscosity can be used in oral formulations. To facilitate the preparation of a carrier with a high content of monoglycosylceramides, that is to achieve a homogeneous oil phase and to reduce the viscosity of the oil phase, the content of ethanol can be varied. A high content of ethanol will facilitate the achievement of a homogeneous oil phase and lower the viscosity of both the oil phase and the w/o- emulsion carrier. The upper limit of the w/o ratio is restrained by the possibility of achieving a homogeneous w/o-emulsion. At

too high w/o ratios the oil phase cannot disperse the entire aqueous phase. The maximum w/o ratio differs with the composi- tion of the oil phase and the aqueous phase.

Depending on the special features wanted of the w/o- emulsion carrier composition, the content of monoglycosyl- ceramide, as well as the w/o ratio, may be adjusted. The per- formance of the w/o-emulsion carrier composition in aqueous environments is also depending on the choice of non-polar oil, the content of ethanol and the presence of possible excipients.

In a w/o-emulsion carrier composition having a high w/o ratio, a high content of monoglycosylceramide may be necessary for the carrier to stay cohesive in an aqueous environment.

According to a preferred aspect the present invention refers to a w/o-emulsion carrier composition wherein the content of monoglycosylceramide is 0.1-20 % by weight of the oil phase, preferably 0.2-10 %.

The composition of the invention can also contain one or more excipients in an amount not negatively affecting the bio- active substance or the release thereof.

Excipient can be defined as any component, other than the bioactive substance, included in the carrier composition.

Excipients can be incorporated for the purpose of modifying physical or chemical properties of the composition, or as inert bulk, or volume, materials. The excipient can contribute to such properties of the carrier composition as stability, solubility, polarity, viscosity, release properties, appearance, patient acceptability, and ease of production. Excipients are for instance antimicrobial preservatives, antioxidants, stabilisers, emulsifiers, complexing agents, thickeners and penetration enhancers.

As examples of excipients can be mentioned glycerol, ethylene glycol, polyethylene glycols, propylene glycol, poly- propylene glycols, fatty alcohols, sterols, such as cholesterol, monoglycerides, diglycerides, tetraglycol, propylene carbonate and copolymers of polyethylene oxide and polypropylene oxide, or a mixture thereof.

The w/o-emulsion carrier of the present invention is prepared in a relatively easy manner with few constituents, compared to other common w/o-emulsions and other depot systems.

A mixture of monoglycosylceramide, non-polar oil, and optionally ethanol is stirred in a sealed vial at an elevated temperature, typically 80 °C, until a homogeneous oil phase has been obtained, normally for 10 minutes. After the heating the oil phase is allowed to cool whereupon a macroscopically homogeneous, turbid oil phase of semi-solid consistency is formed. The aqueous phase is then added to the oil phase, and dispersed into the oil phase simply by supplying mechanical energy at a temperature from 0 °C to about 60 °C. A macroscopically homogeneous w/o-emulsion, often of a cream-like consistency, is then obtained. Energy can be provided by vigorous mechanical or magnetic stirring, vortex- ing, shaking or by other means of agitation. Noteworthy is that the aqueous phase does not have to be heated during the prepara- tion, which is a substantial advantage when thermally labile substances are to be incorporated into the aqueous phase.

Another advantage in the preparation of the carrier composition of the invention is that no organic solvents, other than optionally ethanol, is used during the preparation. Organic solvents may be detrimental to the native antigenic properties of vaccines, leading to undesirable immune responses upon immunisation.

The common feature of the different compositions of the present invention is the coherent appearance of the carrier composition when brought into contact with different aqueous media. This has been observed in many different aqueous media such as distilled water, 0.1 M HC1 (pH 1), 0.1 M NaOH (pH 13), buffer solution that mimics the salt concentration and pH of human blood and interstitial fluids (20 mM Hepes, 150 mM NaCl, 0.01 W w/w NaN3, pH 7.4), solutions that mimic the salt concen- tration, pH and pepsin concentration of human gastric juice (2.0 g NaCl, 3.2 g pepsin, 80 ml 1 M HC1, distilled water up to 1000

ml) and an acidic saline (70 mM NaCl, pH 1.0). The fact that the carrier composition of the present invention retains its cohesive structure when added to such diverse aqueous media as described above, makes it possible to use the carrier composi- tion for controlled release in a number of different applications.

The invention refers to the use of a w/o-emulsion carrier as described for the preparation of a depot formulation for injection for controlled release of a bioactive substance in vivo. Preferred ways of administration are by subcutaneous, intraperitonal, intramuscular or intradermal injection. The results of the sustained release examples indicate that the w/o- emulsion carrier composition can act as a parenteral depot system for several months in vivo.

The use of the invention for parenteral depot applica- tions is obvious, but other uses are also obvious to the man skilled in the art. The w/o-emulsion carrier composition can for example be used for oral controlled release delivery of a drug substance. A specific aspect of the invention is therefore the use of a w/o-emulsion carrier according to the invention for the preparation of an oral formulation for controlled release of a bioactive substance in vivo. Because of the coherent appearance in aqueous solutions mimicing the human gastric juice it is furthermore appealing to think of applications where the carrier protects the drug substances in the gastric environment.

Another possible use for the w/o-emulsion carrier of the present invention is for taste masking of drugs in pharmaceuti- cal products for oral administration.

Depot formulations are of general interest to the pharmaceutical industry. The invention also refers to a pharma- ceutical formulation for controlled release of a bioactive substance, which formulation consists of a w/o-emulsion carrier composition as previously described, and a bioactive substance dissolved or dispersed in said carrier.

According to a preferred aspect the invention refers to a pharmaceutical formulation wherein the w/o-emulsion carrier

composition consists of an oil phase of 30-99.9 t by weight ot non-polar oil, such as a triglyceride oil or a suitable mineral oil, in combination with 0.1-40 % by weight of monoglycosyl- ceramide, and up to 30 % by weight of ethanol, and an aqueous phase, in a w/o ratio of up to 80/20 by weight, in addition to the bioactive substance.

According to another preferred aspect the invention refers to a pharmaceutical formulation wherein the w/o-emulsion carrier composition consists of an oil phase of 60-99. 9% by weight of non-polar oil, such as a triglyceride oil or a suitable mineral oil, in combination with 0.1-40 % by weight of monoglycosylceramide, and an aqueous phase, in a w/o ratio of up to 80/20 by weight, in addition to the bioactive substance.

A pharmaceutical formulation of the invention can in addition contain one or more excipients in combination with the carrier of the invention.

The most obvious way of incorporating a bioactive substance in the w/o-emulsion of the present invention is by dissolving the substance in the aqueous phase. A range of water- soluble substances has also been incorporated into the carrier composition by such means to demonstrate the possibilities to incorporate water-soluble substances, see Examples 17-25. The ability of the w/o-emulsion of the present invention to form macroscopically homogeneous w/o-emulsions of cream-like consist- ency, even when the aqueous phase has a considerably high ionic strength or has a very acidic or basic pH value, see Examples 28-31, implies that the carrier is suitable for incorporating salts and other highly ionic substances in high amounts, and also for incorporating substances requiring extreme pH values to dissolve or to retain stable, see Test of stability of formula- tion of Example 17. Further, in the dispersed aqueous phase, which can be degassed, the carrier can provide an aqueous environment with low oxygen content. This makes the carrier composition suitable for substances that are easily oxidised or by other means degraded by the presence of oxygen in aqueous environments.

The use of the carrier composition of the present invention is by no means limited to the ability of the carrier composition to dissolve the bioactive substance in the aqueous phase. Other bioactive substances which are not soluble in the aqueous phase may be incorporated into the oil phase, by dis- solving and/or dispersing the substance in the oil phase, see Example 26. The purpose of an aqueous phase in such cases is merely to make the carrier cream-like or to alter the appearance and performance of the carrier.

The semi-solid consistency, which can be obtained, of the carrier composition, makes it possible to disperse and suspend solid crystalline or amorphous structures homogeneously into the carrier composition and prevent sedimentation upon storage, see Example 27. This is a possible way of incorporating bioactive substances which are neither soluble in the aqueous phase nor the oil phase.

The carrier composition is also appealing to use for incorporating a multitude of bioactive substances in the same formulation due to the ability of the carrier to dissolve both water, oil and ethanol soluble drugs.

The bioactive substance can be defined as a biologically active substance, or material, which can be used within human or veterinary medicine for diagnosis, treatment or prevention of disease, or to affect the structure or function of the human or animal body.

The invention especially refers to a pharmaceutical formulation wherein the bioactive substance is selected from the group consisting of neuroleptic, antidepressive, antipsychotic, antibiotic, antimicrobial, antiviral, anti-inflammatory, antitumour, nootropic, psychotomimetic and anti-Parkinson drugs, drugs used in bone disorders, contraceptives, psychostimulants, lipids, steroids, hormones, proteins, peptides, amino acids, minerals and vitamins.

A pharmaceutical formulation can comprise more than one bioactive substance.

EXAMPLES OF COMPOSITIONS Examples of carrier compositions with different oils Several different oils were tested in the system. The relative proportions of the oil phase components (RP oil phase) and the ratio aqueous phase/oil phase (w/o) were tried to be kept to monoglycosylceramide/oil/ethanol : 5/85/10 % w/w, and aqueous phase/oil phase : 40/60 % w/w in all examples. The procedure for preparing the different compositions was the same in all examples. Monoglycosylceramide, oil and ethanol were mixed in a sealed 10 ml glass vial and stirred at 80 °C for 10 minutes to form a homogeneous oil phase. When brought back to room temperature a macroscopically homogeneous, turbid oil phase of semi-solid consistency was formed. After the oil phase had attained room temperature, the aqueous phase was weighed into the-glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial. The resulting emulsion was in all examples, except Example 7 Castor oil, a macroscopically homogeneous w/o-emulsion of cream-like consistency.

Materials As monoglycosylceramide was used monohexosylceramide, CMH, prepared from whey concentrate by means of chromatographic fractionation to a purity of >99 % from Lipid Technologies Provider AB, Sweden.

The non-polar oils were: MCT oil (medium chain triglyceride oil) from Croda Oleochemicals, England; Evening primrose oil (chromatographic fractionated) from Scotia LipidTeknik AB, Sweden; Sunflower oil (chromatographic fractionated) from Scotia LipidTeknik AB, Sweden; Coconut oil (chromatographic fractionated) from Scotia LipidTeknik AB, Sweden;

Soybean oil (chromatographic fractionated) from Scotia LipidTeknik AB, Sweden; Corn oil (chromatographic fractionated) from Scotia LipidTeknik AB, Sweden; Castor oil from Apoteksbolaget AB, Sweden; Fish oil from Lipid Technologies Provider AB, Sweden; Mineral oil (USP) from Sigma-Aldrich.

Ethanol (99.5 %) from Kemetyl AB, Sweden.

Distilled water.

Example 1. MCT oil 0. 0597 g CMH was mixed with 1.0100 g MCT oil and 0.1201 g ethanol to form the oil phase. 0.7900 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5.0/84. 9/10.1 w/w, w/o : 39.9/60. 1 % w/w.

Example 2. Evening primrose oil 0.0591 g CMH was mixed with 0. 9992 g evening primrose oil and 0.1209 g ethanol to form the oil phase. 0.7885 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5.0/84. 7/10.3 % w/w, w/o : 40.1/59. 9 % w/w.

Example 3. Sunflower oil 0.0604 g CMH was mixed with 1.0281 g sunflower oil and 0.1218 g ethanol to form the oil phase. 0.8285 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5. 0/84.9/10. 1 % w/w, w/o : 40.6/59. 4 % w/w.

Example 4. Coconut oil 0.0610 g CMH was mixed with 1.0403 g coconut oil and 0.1217 g ethanol to form the oil phase. 0.8022 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5. 0/85.1/10. 0 % w/w, w/o : 39.6/60. 4 % w/w.

Example 5. Soybean oil 0.0604 g CMH was mixed with 1. 0375. g soybean oil and 0.1213 g ethanol to form the oil phase. 0.8174 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5.0/85. 1/9.9 % w/w, w/o: 40.1/59. 9 % w/w.

Example 6. Corn oil 0.0604 g CMH was mixed with 1.0253 g corn oil and 0.1210 g ethanol to form the oil phase. 0.8022 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5.0/85. 0/10.0 % w/w, w/o: 39.9/60. 1 % w/w.

Example 7. Castor oil (comparative) 0.0595 g CMH was mixed with 1. 0077 g castor oil and 0.1190 g ethanol to form the oil phase. 0. 7981 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5.0/85. 0/10.0 % w/w, w/o : 40.2/59. 8 % w/w.

Example 8. Fish oil 0.0581 g CMH was mixed with 0.9992 g fish oil and 0.1160 g ethanol to form the oil phase. 0.7887 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 5.0/85. 2/9.9 % w/w, w/o: 40.2/59. 8 % w/w.

Example 9. Mineral oil 0.0600 g CMH was mixed with 1.1606 g mineral oil and 0.1244 g ethanol to form the oil phase. 0.9649 g of distilled water was added to the oil phase. RP oil phase; CMH/oil/ethanol : 4.5/86. 3/9.2 % w/w, w/o: 41. 8/58. 2 % w/w.

Comparative examples with different sphingolipid materials In order to evaluate the properties of different sphingolipid materials the following compositions, Example 12- 16, were prepared and compared to the compositions according to the invention, Example 10-11.

The relative proportions, RP, of the carrier components sphingolipid/MCT oil/ethanol are given for each composition in % w/w.

Materials The following sphingolipid materials were used in the examples: CMH (monohexosylceramide), prepared from whey concentrate by means of chromatographic fractionation to a purity of >98 % (Scotia LipidTeknik AB); CDH (dihexosylceramide), prepared from whey concentrate by means of chromatographic fractionation to a purity of >98 % (Scotia LipidTeknik AB); m-SL, milk sphingolipids containing approximately 70 % sphingomyelin, 10 % CMH and 10 % CDH, prepared from whey concentrate by means of chromatographic fractionation (Scotia LipidTeknik AB); Sphingomyelin, prepared from whey concentrate by means of chromatographic fractionation to a purity of >99 % (Scotia LipidTeknik AB); Glucosylceramide C8: 0 (glucosylceramide with a C8: 0 acyl chain linked to the amide nitrogen) from Avanti Polar Lipids, Inc., USA; Ceramide C24: 0 (ceramide with a C24: 0 acyl chain linked to the amide nitrogen) from Avanti Polar Lipids, Inc. , USA.

Example 10. CMH 0.0608 g CMH was mixed with 1.7676 g MCT oil and 0.2026 g ethanol in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form the oil phase. When brought back to room temperature a macroscopically homogeneous, turbid oil phase of semi-solid consistency was formed. After the oil phase had attained room temperature, 1.0048 g of distilled water was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a

magnetic stirring bar included in the glass vial. The resulting emulsion was a macroscopically homogeneous w/o-emulsion of cream-like consistency. RP oil phase; CMH/MCT oil/ethanol : 3.0/87. 0/10.0 % w/w, w/o : 33.1/66. 9 % w/w.

Example 11. CMH without ethanol 0.0666 g CMH was mixed with 2.1558 g MCT oil in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form the oil phase. When brought back to room temperature an initially macroscopically homogeneous, turbid oil phase of semi- solid consistency was formed. After the oil phase had attained room temperature, 1.0891 g of distilled water was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial. The resulting emulsion was a macro- scopically homogeneous w/o-emulsion of cream-like consistency.

RP oil phase; CMH/MCT oil : 3.0/97. 0 % w/w, w/o : 32. 9/67. 1 % w/w.

Example 12. CDH (comparative) 0.0736 g CDH was mixed with 2.1449 g MCT oil and 0. 2567 g ethanol in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form a homogeneous oil phase. When brought back to room temperature an initially macroscopically homogeneous, turbid oil phase of semi-solid consistency was formed. After the oil phase had attained room temperature, 1.2175 g of distilled water was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial. The result was an inhomogeneous emulsion with unemulsified aqueous phase. RP oil phase; CDH/MCT oil/ethanol : 3.0/86. 7/10.4 % w/w, w/o : 33.0/67. 0 % w/w.

Example 13. m-SL (comparative) 0.0875 g milk sphingolipids was mixed with 2.5452 g MCT oil and 0.2945 g ethanol in a sealed 10 ml glass vial. The

mixture was stirred at 80 °C for 10 minutes to form a homogeneous clear oil phase. When brought back to room temperature an in- homogeneous oil phase of milk sphingolipid sediment in MCT oil was formed. After the oil phase had attained room temperature, 1.4509 g of distilled water was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial. The result was a two phase system consisting of one oil phase and one aqueous phase. RP oil phase; milk sphingo- lipids/MCT oil/ethanol : 3.0/86.9/10.1 %w/w, w/o : 33.1/66.9 %w/w.

Example 14. Sphingomyelin (comparative) 0.0840 g sphingomyelin was mixed with 2.4467 g MCT oil and 0. 2796 g ethanol in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form a homogeneous clear oil phase. When brought back to room temperature an inhomo- geneous milky oil phase of sphingomyelin sediment in MCT oil was formed : After the oil phase had attained room temperature, 1.3840 g of distilled water was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial. The result was a two phase system consisting of one oil phase and one aqueous phase. RP oil phase; sphingomyelin/MCT oil/ethanol : 3.0/87. 1/9.9 % w/w, w/o : 33. 0/67. 0 % w/w.

Example 15. Glucosylceramide C8: 0 (comparative) 0.0279 g glucosylceramide C8: 0 was mixed with 0.8140 g MCT oil and 0.0917 g ethanol in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form the oil phase. When brought back to room temperature an inhomogeneous grainy oil phase was formed. After the oil phase had attained room temperature, 0.4933 g of distilled water was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar

included in the glass vial. The result was an inhomogeneous milky, grainy mixture of oil and aqueous phases. RP oil phase; glucosylceramide C8: 0/MCT oil/ethanol : 3.0/87. 2/9.8 % w/w, w/o : 34.6/65. 4 % w/w.

Example 16. Ceramide C24: 0 (comparative) 0.0315 g ceramide C24: 0 was mixed with 0.9210 g MCT oil and 0.1044 g ethanol in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form the oil phase. When brought back to room temperature an inhomogeneous grainy oil phase was formed. After the oil phase had attained room tempera- ture, 0.5275 g of distilled water was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial. The result was an inhomogeneous mixture of oil and aqueous phases. RP oil phase; ceramide C24: 0/MCT oil/ethanol : 3.0/87. 1/9.9 % w/w, w/o : 33.3/66. 7 % w/w.

Examples of pharmaceutical formulations The following examples demonstrate the ability of the carrier to incorporate bioactive substances. The examples also demonstrate the versatile ways of incorporating a bioactive substance; by dissolving the bioactive substance in the aqueous phase, by dissolving the bioactive substance in the oil phase or by disperse, suspend, the bioactive substance in the carrier.

The procedure for preparing the different formulations was the same in Example 17-25, where the bioactive substance was dissolved in the aqueous phase. Monoglycosylceramide, oil and ethanol were mixed in a sealed 10 ml glass vial and stirred at 80 °C for 10 minutes to form a homogeneous oil phase. When brought back to room temperature a macroscopically homogeneous, turbid oil phase of semi-solid consistency was formed. After the oil phase had attained room temperature the aqueous phase was weighed into the glass vial. The mixture was then shaken vigo- rously at room temperature on a vortex apparatus, with a

magnetic stirring bar included in the glass vial. The resulting emulsion was in all examples a macroscopically homogeneous w/o- emulsion of cream-like consistency. The relative proportions of the oil phase components (RP oil phase) and the ratio aqueous phase/oil phase (w/o) as well as the total concentration of bioactive substance are given in all examples.

Materials As monoglycosylceramide was used monohexosylceramide, CMH, prepared from whey concentrate by means of chromatographic fractionation to a purity of >96 W from Scotia LipidTeknik AB, Sweden.

MCT oil (medium chain triglyceride oil) from Croda Oleochemicals, England.

Ethanol (99. 5 %) from Kemetyl AB, Sweden.

Glycerol (99. 8) from Apoteksbolaget AB, Sweden.

Distilled water. pH 3 buffer (50 ml 0.10 M potassium hydrogen phthalate, 20 ml 0.10 M HC1, add distilled water up to 100 ml.) The bioactive substances were: Aminolevulinic acid hydrochloride [5451-09-2] from Sigma- Aldrich; Ascorbic acid (vitamin C) [50-81-7] from Sigma-Aldrich; Aspartame [22839-47-0] from AB R. Lundberg, Sweden. (Model for a small peptide); Pyridoxine hydrochloride (Vitamin B6 hydrochloride) [58-56-0] from Sigma-Aldrich; Tinzaparin sodium [9041-08-1] from LEO Pharmaceutical Products, Denmark; Fluphenazine hydrochloride [146-56-5] from Sigma-Aldrich; Vancomycin hydrochloride [1404-93-9] from Sigma-Aldrich; Calcium lactate [5743-47-5] from Sigma-Aldrich; Buspirone hydrochloride [33386-08-2] from Sigma-Aldrich; Cyclosporin [59865-13-3] from Sigma-Aldrich; Metronidazole benzoate [13182-89-3] from Jucker Pharma, Sweden.

Example 17. Aminolevulinic acid dissolved in the aqueous phase 0.0849 g CMH was mixed with 1.8284 g MCT oil and 0.2146 g ethanol to form the oil phase. 1.8370 g of a 19.8 % w/w aminolevulinic acid hydrochloride pH 3 buffered aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 4.0/85. 9/10.1 % w/w, w/o: 46.3/53. 7 % w/w. Total concentration of aminolevulinic acid hydrochloride 9.2 % w/w.

Example 18. Ascorbic acid dissolved in the aqueous phase 0.0735 g CMH was mixed with 1.2551 g MCT oil and 0.1495 g ethanol to form the oil phase. 0.9903 g of a 10.0 % w/w ascorbic acid aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 5.0/84. 9/10.1 % w/w, w/o: 40.1/59. 9 % w/w. Total concentration of ascorbic acid 4.0 % w/w.

Example 19. Aspartame dissolved in the aqueous phase 0.0575 g CMH was mixed with 0.9833 g MCT oil and 0.1154 g ethanol to form the oil phase. 0.7745 g of a 3.0 % w/w aspartame aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 5.0/85. 0/10.0 % w/w, w/o: 40. 1/59. 9 % w/w. Total concentration of aspartame 1.2 % w/w.

Example 20. Pyridoxine hydrochloride dissolved in the aqueous phase 0.0457 g CMH was mixed with 0.0117 g cholesterol, 0.9805 g MCT oil and 0.1127 g ethanol to form the oil phase. 0.8001 g of a 10.0 % w/w pyridoxine hydrochloride aqueous solution was added to the oil phase. RP oil phase; CMH/cholesterol/MCT oil/ethanol : 4.0/1. 0/85.2/9. 8 % w/w, w/o: 41.0/59. 0 % w/w. Total concentration of pyridoxine hydrochloride 4.1 % w/w.

Example 21. Tinzaparin sodium dissolved in the aqueous phase 0.2532 g CMH was mixed with 4.5540 g MCT oil and 0.2517 g ethanol to form the oil phase. 2.1651 g of a 6.7 % w/w tinzaparin sodium aqueous solution was added to the oil phase.

RP oil phase; CMH/MCT oil/ethanol : 5.0/90. 0/5.0 % w/w, w/o:

30.0/70.0 %w/w. Total concentration of tinzaparin sodium 2.

%w/w.

Example 22. Fluphenazine hydrochloride dissolved in the aqueous phase 0. 0597 g CMH was mixed with 1.0228 g MCT oil and 0.1190 g ethanol to form the oil phase. 1.2080 g of a 10.0 % w/w fluphenazine hydrochloride aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 5. 0/85. 1/9. 9 % w/w, w/o : 50. 1/49. 9 % w/w. Total concentration of fluphenazine hydrochloride 5. 0 % w/w.

Example 23. Vancomycin hydrochloride dissolved in the aqueous phase 0. 0600 g CMH was mixed with 1.0181 g MCT oil and 0.1208 g ethanol to form the oil phase. 1.2129 g of a 10.0 % w/w vancomycin hydrochloride aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 5. 0/84. 9/10. 1 % w/w, w/o : 50. 3/49. 7 % w/w. Total concentration of vancomycin hydro- chloride 5. 0 % w/w.

Example 24. Calcium lactate dissolved in the aqueous phase 0. 0618 g CMH was mixed with 1.0627 g MCT oil and 0.1227 g ethanol to form the oil phase. 1.2133 g of a 10.0 % w/w calcium lactate aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 5.0/85. 2/9.8 % w/w, w/o: 49. 3/50. 7 % w/w. Total concentration of calcium lactate 4. 9 % w/w.

Example 25. Buspirone hydrochloride dissolved in the aqueous phase 0. 1004 g CMH was mixed with 1. 7209 g MCT oil and 0. 2079 g ethanol to form the oil phase. 2. 0290 g of a 10.0 % w/w buspirone hydrochloride aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 4. 9/84. 8/10. 2 % w/w, w/o : 50. 0/50. 0 % w/w. Total concentration of buspirone hydrochloride 5. 0 % w/w.

Example 26. Cyclosporin dissolved in the oil phase 0.0960 g CMH was mixed with 1.5350 g MCT oil, 0.1938 g ethanol and 0.0960 g cyclosporin in a sealed 10 ml glass vial.

The mixture was stirred at 80 °C for 10 minutes to form a homo- geneous oil phase. When brought back to room temperature a macroscopically homogeneous, turbid oil phase of semi-solid consistency was formed. After the oil phase had attained room temperature, 1.2747 g of a 5.0 % w/w NaCl aqueous solution, the aqueous phase, was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial, to form a macroscopically homogeneous w/o-emulsion of cream-like consistency. Relative proportions; CMH/MCT oil/ethanol : 5.3/84. 1/10.6 % w/w, aqueous phase/oil phase : 39.9/60. 1 % w/w. Total concentration of cyclosporin 3.0 % w/w.

Example 27. Metronidazole benzoate suspended in the carrier 0.0368 g CMH was mixed with 0.9401 g MCT oil, 0. 1250 g ethanol, 0.1250 g glycerol and 0.8037 g metronidazole benzoate in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form a homogeneous oil phase. When brought back to room temperature a macroscopically homogeneous, turbid oil phase of semi-solid consistency was formed. As soon as the oil phase had attained room temperature, 0.5022 g of distilled water, the aqueous phase, was weighed into the glass vial. The mixture was then shaken vigorously at room. temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial, to form a macroscopically homogeneous w/o-emulsion of cream-like consistency. Relative proportions; CMH/MCT oil/ethanol/glycerol : 3.0/76. 6/10.2/10. 2 % w/w, aqueous phase/oil phase : 19.8/80. 2 % w/w. Total concentration of metronidazole benzoate 31.7 % w/w.

Examples demonstrating the incorporation of salt and variation of the pH of the aqueous phase The ability of the carrier of the present invention to form macroscopically homogeneous w/o-emulsions of cream-like consistency even when the aqueous phase has a considerable high ionic strength or has a pH value far from neutral pH, is demon- strated in the following examples. The procedure for preparing the different compositions was the same in all examples. Mono- glycosylceramide, oil and ethanol, the same as in the previous examples, were mixed in a sealed 10 ml glass vial and stirred at 80 °C for 10 minutes to form a homogeneous oil phase. When brought back to room temperature a macroscopically homogeneous, turbid oil phase of semi-solid consistency was formed. After the oil phase had attained room temperature the aqueous phase was weighed into the glass vial. The mixture was then shaken vigo- rously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial. The resulting emulsion was in all examples a macroscopically homogeneous w/o- emulsion of cream-like consistency.

Example 28. NaCl 0.0767 g CMH was mixed with 1.2996 g MCT oil and 0.1531 g ethanol to form the oil phase. 1.5214 g of a 25.4 % w/w NaCl aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 5. 0/85.0/10. 0 % w/w, w/o: 49.9/50. 1 % w/w.

Total concentration of NaCl 12.7 % w/w.

Example 29. CaCl2 0.0782 g CMH was mixed with 1.3245 g MCT oil and 0.1568 g ethanol to form the oil phase. 1.5555 g of a 7.0 % w/w CaCl2 aqueous solution was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 5.0/84. 9/10.1 % w/w, w/o : 49.9/50. 1 % w/w.

Total concentration of CaCl2 3.5 % w/w.

Example 30.0. 1 M HCl 0.1045 g CMH was mixed with 1.9804 g MCT oil and 0.1336 g ethanol to form the oil phase. 0.3754 g of 0.1 M HC1 (pH 1) was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 4.7/89. 3/6.0 % w/w, w/o : 14.5/85. 5 % w/w.

Example 31.0. 1 M NaOH 0.1041 g CMH was mixed with 1.9723 g MCT oil and 0.1218 g ethanol to form the oil phase. 0.4014 g of 0.1 M NaOH (pH 13) was added to the oil phase. RP oil phase; CMH/MCT oil/ethanol : 4.7/89. 7/5.5 % w/w, w/o : 15.4/84. 6 % w/w.

TEST OF STABILITY OF THE FORMULATION OF EXAMPLE 17 To demonstrate the ability of the w/o-emulsion carrier of the present invention to incorporate substances, which demand extreme pH values to retain stable, the stability of aminolevu- linic acid was examined by means of high performance liquid chromatography. The pharmaceutical formulation of Example 17 was stored at room temperature for approximately 3 months, protected from light, before the analysis was made.

Materials HPLC Column Ultrasphere C8,150*4. 6 mm, 5p, Beckman, USA.

Guard column Zorbax SB-C18, 12.5*4. 6 mm, 5u, Agilent Technologies, USA.

Phosphoric acid from Merck, Germany.

1-octanesulphonic acid from Sigma-Aldrich.

Acetonitrile from Merck Eurolab AB, Sweden.

Hexane from Merck Eurolab AB, Sweden.

Isopropanol from Merck Eurolab AB, Sweden.

Liquid chromatographic system; Dionex P580 pump 1 ml/min, Auto sampler Dionex Gina 50 100 pi and Lamda UV detector SPD-6A at 263 nm. The signal from the detector was recorded by Gynkotek Chromeleon Chromatography Data System version 4.32.

Mobile phase; 90 % 50 mM phosphoric acid containing 5 mM 1-octanesulphonic acid and 10 W acetonitrile.

Analysis Approximately 200 mg of the pharmaceutical formulation of Example 17 was accurately weighed and dissolved in 4 ml hexane/isopropanol: 4/1 by volume in a test tube, 2 ml distilled water was added and the mixture was vigorously shaken and slightly heated for 10 to 20 seconds in a water bath at 50 °C.

After centrifugation at 3500 rpm for 1 min the upper organic layer was discarded and the water phase was washed twice more with 4 ml of the hexane/isopropanol mixture. The resulting water phase was diluted up to 10 ml with the mobile phase and injected on the chromatographic system described above. The aminolevu- linic acid concentration in the samples was evaluated by means of a three point standard curve (1-3 mg/ml). The sample was prepared in duplicate. The analysis showed a 92 and 94 % recovery of aminolevulinic acid, respectively.

TEST OF THE RELEASE PROPERTIES OF THE CARRIER COMPOSITION In the following examples sustained release properties of w/o-emulsion systems of the present invention are illustrated by the incorporation and release of three different marker substances, Safranine O, pyridoxine hydrochloride and ascorbic acid. To show how it is possible to control the behaviour of the system by altering the amount of monoglycosylceramide, the non- polar oil and the presence of any excipients (as cholesterol) in the oil phase and by altering the aqueous phase to oil phase ratio, but also to examine the influence on the system of the incorporated marker substance, a factorial design was made for the release studies of Safranine O (Saf O) and pyridoxine hydro- chloride (B6-HCl). For the release study of ascorbic acid (vita- min C) only one experiment was made where the amount of ascorbic acid released was compared to the total amount incorporated.

Materials As monoglycosylceramide was used monohexosylceramide, CMH, prepared from whey concentrate by means of chromatographic fractionation to a purity of >96 % from Scotia LipidTeknik AB, Sweden.

Cholesterol from Apoteksbolaget AB, Sweden.

MCT oil (medium chain triglyceride oil) from Croda Oleochemicals, England.

Sesame oil from Croda Oleochemicals, England.

Ethanol (99.5 %) from Kemetyl AB, Sweden.

Distilled water.

Safranine O (Saf O) [477-73-6] from Labora Chemicals, Sweden.

Pyridoxine hydrochloride (B6-HCl) [58-56-0] from Sigma- Aldrich.

Ascorbic acid (vitamin C) [50-81-7] from Sigma-Aldrich.

Spectra/Por@ Membrane MWCO 6000-8000 with weighted closures, from Kebo Lab AB, Sweden.

The dissolution medium, a buffer solution consisting of 20 mM Hepes, 150 mM NaCl, 0.01 % w/w NaN3, pH 7.4.

Dissolution equipment A conventional USP dissolution bath, PTWS has been modified so that it can be used with smaller volumes. The lids to the original vessels have been modified so a 50 ml round bottomed flask can be placed in them. The original paddles were made smaller to fit into these new vessels, which hang inside the original vessels, which were filled with water. The tempera- ture in the water bath was set to 38.1 °C, which corresponds to a temperature of 37.2-37. 3 °C inside the 50 ml vessel.

Preparation of the formulations For each formulation the oil phase was prepared by mixing monoglycosylceramide, cholesterol (when included), non-

polar oil and ethanol in a sealed 10 ml glass vial. The mixture was stirred at 80 °C for 10 minutes to form a homogeneous oil phase. After the oil phase had attained room temperature and become turbid, macroscopically homogeneous and of semi-solid consistency, the aqueous phase with the dissolved marker sub- stance was weighed into the glass vial. The mixture was then shaken vigorously at room temperature on a vortex apparatus, with a magnetic stirring bar included in the glass vial, to form a macroscopically homogeneous w/o-emulsion of cream-like consistency. The w/o-emulsion was finally transferred to 2 ml syringes to ease the filling of the Spectra/Pors Membrane. The composition of the formulations examined is given in Table 1 and Table 2.

Table 1. Composition of the formulations without cholesterol Formulation Oil phase Aqueous Total % w/w phase % w/w % w/w CMH Oil EtOH Markerw o Marker substance phase phase R1 2.0 MCT, 9.9 Saf O, 15.2 84.8 0.12 88.1 0.80 R2 8.0 MCT, 9.9 Saf O, 15.0 85.0 0.12 82.1 0.80 R3 8. 0 MCT, 10. 1 Saf o, 60.1 39.9 0.12 81. 9 0.20 R4 2. 0 MCT, 10. 4 Saf O, 60.1 39.9 0.12 87. 6 0.20 R5 2. 0 Sesame, 9. 9 Saf O, 15.0 85.0 0. 12 88.1 0.80 R6 7. 9 Sesame, 10. 1 Saf O, 15.4 84.6 0.12 82. 0 0.80 R7 8. 0 Sesame, 9. 8 Saf O, 60. 1 39.9 0.12 82.2 0.20 R8 2. 0 Sesame, 10. 1 Saf O, 60.0 40.0 0.12 87.9 0.20 R9 2. 0 MCT, 10. 1 B6-HCl, 15. 2 84.8 1.52 87. 9 10.00 R10 8. 0 MCT, 10. 1 B6-HCl, 15.0 85.0 1.50 81.9 10.00 R11 8. 0 MCT, 9. 9 B6-HCl, 60. 1 39. 9 1.50 82.1 2.50 R12 2. 0 MCT, 10. 0 B6-HCl, 60.0 40.0 1.50 88.0 2.50 R13 5. 0 MCT, 10. 4 B6-HCl, 39. 9 60.1 1.50 84.6 3.75 R14 5. 0 MCT, 10. 1 Vitamin C, 40. 1 59.9 4.01 Example 18 84.9 10.00 Table 2. Composition of the formulations with cholesterol Formulation Oil phase Aqueous Total % w/w phase % w/w % w/w CMH Chol MCT EtOH Marker w o Marker substance phase phase R15 4. 0 1.0 85. 2 9. 8 B6-HCl, 41.0 59.0 4.10 Example 20 10.00 R16 3. 0 2.0 85. 1 9. 9 B6-HCl, 40.0 60.0 4.00 10. 00 KIT2. 0 3.1 84.9 10. 0 B6-HC1, 40.0 60.0 4.00 10.00

Release studies 25 ml dissolution medium, was added to the 50 ml inner vessel and allowed to reach the right temperature, approximately 37.3 °C, before the experiments started. The stirring rate was set to 80 rpm. The Spectra/Por@ Membrane was soaked in distilled water for at least 30 minutes before use. Approximately 0.4 g of the formulation was weighed into a piece of the Spectra/Por@ Membrane and the membrane was locked at both ends with weighted closures. The formulation in its membrane was put in the medium and samples were taken after specific times.

The dissolution medium was used as a blank on the W spectrophotometer. To take a sample, the peristaltic pump which is adherent to the flow cuvette system of the UV-spectrophoto- meter, was used. The flow cuvette was filled with sample and the absorbance was measured at 521 nm (Saf O), 324 nm (pyridoxine hydrochloride) and 265-290 nm (ascorbic acid). Afterwards the pump was allowed to work in the reverse direction and the sample was returned to the inner vessel. The cuvette system was finally rinsed thoroughly with dissolution medium.

Results from the release studies In Table 3 the amount of marker substance released, expressed as a percentage of the amount of marker substance in- corporated, are shown. The dissolution profiles from the experi- ments stated in Table 1 and Table 2 are shown in Figure 1 and Figure 2, except formulation R14. The chosen examples show how the dissolution profiles varies depending on the oil, the amount of monoglycosylceramide, the amount of aqueous phase in the system, and also on the marker substance.

Table 3. Release in % of amount incorporated marker substance after 160 hours Formulation Oil phase Total Marker Amount % w/w substance released (%) after 160 h CMH Chol Oil Aq. phase % w/w R1 2. 0 0 MCT 15. 2 Saf o 2. 17 R2 8. 0 0 MCT 15. 0 Saf O 7. 91 R3 8.0 0 MCT 60.1 Saf O 10.87 R4 2.0 0 MCT 60.1 Saf O 8.55 R5 2. 0 0 Sesame 15. 0 Saf 0 2. 16 R6 7. 9 0 Sesame 15. 4 Saf O 4. 90 R7 8. 0 0 Sesame 60. 1 Saf O 8. 81 R8 2. 0 0 Sesame 60. 0 Saf O 6. 40 R9 2. 0 0 MCT 15. 2 B6-HCl 3. 49 R10 8. 0 0 MCT 15. 0 B6-HC1 3. 71 Rl1 8. 0 0 MCT 60. 1 B6-HCl 5. 73 R12 2.0 0 Mct 60.0 B6-HCl 5. 42 R13 5.0 0 MCT 39.9 B6-HCl 4.96 R14 5. 0 0 MCT 40. 1 Vitamin C 0. 22 R15 4. 0 1. 0 MCT 41. 0 B6-HC1 1. 79 R16 3. 0 2. 0 MCT 40. 0 B6-HC1 0. 97 R17 2. 0 3. 1 MCT 40.0 B6-HCl 1.03

CONCLUSIONS FROM THE EXPERIMENTS - The surprisingly easy method of preparation of the w/o-emulsion carrier composition, and the possibility to use several different oils is demonstrated in Examples 1-9.

- The unique properties of monoglycosylceramides compared to diglycosylceramides, unglycosylated ceramides and other sphingolipid materials are demonstrated by the Comparative Examples 10-16.

- The capacity of the w/o-emulsion carrier composition to incorporate drug substances is clearly demonstrated in Examples 17-27, in which about 0. 5-32 % by weight of eleven structurally very different bioactive substances successfully have been incorporated. The ability of the carrier composition to incorporate aqueous phase soluble, as well as oil phase soluble bioactive substances, and also the possibility to in- corporate substances, which are neither aqueous phase soluble nor oil phase soluble, by dispersing or suspending homogeneously solid crystalline and amorphous structures, is further demon- strated. In all cases the resulting formulation is macro- scopically homogeneous, cream-like and injectable.

- The surprising property of the w/o-emulsion carrier composition to form macroscopically homogeneous w/o-emulsions of cream-like consistency even when the aqueous phase has a consid- erably high ionic strength or has a pH value far from neutral, is demonstrated in the Examples 28-31.

- The ability of the w/o-emulsion carrier composition of the present invention to incorporate substances, requiring extreme pH values to remain stable, is demonstrated in Test of stability of the formulation of Example 17.

- It is clear from the release experiments that the different marker substances are released at different rates from the same w/o-emulsion carrier composition, it is also clear that the release rate for each marker substance depends on the composition of the w/o-emulsion carrier. The results from the studies on Saf O and pyridoxine hydrochloride (B6-HCl) show that the composition of the system can be modified to suit. the

incorporated substance and the desired behaviour of the system.

The depot effect of the w/o-emulsion carrier composition of the present invention is strikingly good. In all but one of the formulations less then 10 % of the total amount of the incorpo- rated marker substance was released in 160 hours. Experience shows that the sustained released obtained in these experiments could correspond to a depot effect of at least one month in vivo.

From the experiments, results and conclusions summarised above it is obvious that the characteristics of the w/o-emul. sion carrier composition of the invention make it especially suitable as a pharmaceutical carrier for controlled release of incorpo- rated bioactive substances. The compositions of the oil phase and the aqueous phase, and the ratio aqueous phase/oil phase of the w/o-emulsion carrier composition can be adjusted to facili- tate the incorporation and protection of various bioactive substances, and to control the release rate from the carrier.