AU2002301328B2

AU2002301328B2 - Drug delivery system using membrane mimetics

Info

Publication number: AU2002301328B2
Application number: AU2002301328A
Authority: AU
Inventors: Pankaj Modi
Original assignee: GENEREX PHARM Inc; Generex Pharmaceuticals Inc
Current assignee: Generex Pharmaceuticals Inc
Priority date: 1998-09-27
Filing date: 2002-09-30
Publication date: 2003-07-31
Anticipated expiration: 2019-09-23

Description

DRUG DELIVERY SYSTEM USING MEMBRANE MIMETICS Technical Field The present invention relates to an improved delivery system for the administration of large-molecule pharmaceuticals, e.g. drugs, vaccines and hormones. In particular it relates to pharmaceuticals which may be administered through the oral and nasal membranes, or by pulmonary access. One method of administration is by means of an aerosol into the mouth, for buccal or pulmonary application.

Background Art New methods of delivering large macromolecules (proteins and peptides) continue to be sought. One of the avenues investigated concerns the use of membrane-mimetic amphiphiles. A study of membrane-mimetic amphiphiles extends back to the first decade of the 20th century. Experiments using physical and chemical methods have shown that such molecules assume preferred arrays in the presence of water. Formation of these arrays, which includes micelles, monolayers and bimolecular layers is driven by the need of the polar head groups, which may be ionogenic or not, to associate with water, and the need of the polar hydrophobic tails to be excluded from water, (Small, D; Handbook of Lipid Research, vol. 4,1986; Tanford, J: The Hydrophobic Effect, John Wiley Sons, 1980; Fendler, J. Membrane Chemistry, 1982). Exactly which type of structure is assumed depends upon the nature of the amphiphile, its concentration, the presence of other amphiphiles, temperature and the presence of salts and other solutes in the aqueous phase.

Membrane-mimetic amphiphiles include molecules that are insoluble in water but can take up water, and molecules that have appreciable solubility in water under limiting conditions. The former amphiphites do not form molecularly disperse solutions in water but may swell considerably with water to form lamellar phases. The latter amphiphiles can, at some temperatures, form solutions of dispersed monomers in water and often undergo the following sequence as the concentration in water is increased: monomeric solution to micellar solution. The manufacture of non-phospholipid liposomes, depends on the manipulation of environmental variables g. temperature, hydration and composition) in an -2appropriate temporal sequence so as to cause non-phospholipid amphiphiles to form liposomal structures.

Gebicki et al. (Nature, 243,232,1973: Chem. Phys. Lipids, 16,142,1976; Biochem. Biophys. Res. Commun. 80,704,1978; Biochemistry, 17,3759,1978) demonstrated the formation of water containing vesicles enclosed by oleic acid.

Others, as disclosed for example in U. S. Patents 4 772 471 and 4 830 857, and in J. Microencapsul. 4,321,1987, have made lipid vesicles from single tailed ether or esters derivatives of polyglycerol. These liposomes were found suitable for cosmetic products. Murakami et al Am. Chem. Soc, 101,4030,1979; J. Am Oil Chem Soc. 66,599,1989) formed single compartment vesicles with one or more bilayer walls composed of cationic amphiphiles involving amino acid residues.

Kaler et al (Science, 245,1371, 1989) demonstrated that appropriate aqueous mixtures of single-tailed cationic and anionic surfactants spontaneously form single-walled vesicles, presumably via salt formation. Others have developed methods for manufacture of paucilamellar, nonphospholipid liposomes that can be formed from a variety of amphiphiles as well as from certain phospholipids. The liposomes have two or more membranes surrounding an amorphous core, each membrane being composed of amphiphile molecules in bilayer array. The core accounts for most of the vesicle volume and encapsulating substances.

The above-mentioned non-phospholipid based liposomes are mainly used for the delivery of moisturizers and cosmetic ingredients used topically or externally as creams or moisturizers. In some cases such liposomes may be used as an ointment for delivery of some pharmaceutical products. Many ingredients utilized in the above products have been found to be inadmissible in the human body and are not approved by the regulatory agencies around the world for the purpose of oral administration and as a vehicle for delivery of macromolecules (proteins and peptides) as life saving therapeutics. Furthermore, other nonphospholipid based liposomes have been developed for non-pharmaceutical applications, e.g. water-borne oil paints, surface cleansers, heavy duty industrial cleansers and skin-cleansing detergents.

Certain aspects of the present invention aims at the development of oral formulations consisting of mixture of certain non-phospholipid based membranemimetic amphiphiles (suitable and approved by the regulating agencies for oral formulation of human pharmaceutical products) in combination with specific phospholipids to form multilamellar liposomes which are very stable and are smaller than the pores of the gastrointestinal (GI) tract.

Relatively very little progress has been made in reaching the target of safe and effective oral formulations for peptides and proteins. The major barriers to developing oral formulations for proteins and peptides include poor intrinsic permeability, lumenal and cellular enzymatic degradation, rapid clearance, and chemical stability in the GI tract. Pharmaceutical approaches to address these barriers, which have been successful with traditional small, organic drug molecules, have not readily translated into effective peptide and protein formulations. Although the challenges are significant, the potential therapeutic benefits remain high especially in the field of diabetes treatment using insulin.

Researchers have explored various administration routes other than injection for proteins and peptides. These routes include administration through oral, intranasal, rectal, vaginal cavities for the effective delivery of large molecules.

Out of the above four mentioned routes oral and nasal cavities have been of greatest interest. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid a first pass effect of hepatic metabolism, and avoid exposure of the drug to a hostile GI environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.

Further, there is a good potential for prolonged delivery of large molecules through these membranes.

The oral routes have received far more attention than have the other routes.

The sublingual mucosa includes the membrane of ventral surface of the tongue and the floor of the mouth whereas the buccal mucosa constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible. This route has been investigated clinically for the delivery of a substantial number of drugs.

Various mechanisms of action of penetration of large molecules using enhancers have been proposed. These mechanisms of action, at least for drugs include reducing viscosity and/or elasticity of mucous layer, facilitating transcellular transport by increasing the fluidity of the lipid bilayer of membranes, -4facilitating paracellular transport by altering tight junction across the epithelial cell layer, overcoming enzymatic barriers, and increasing the thermodynamic activity of drugs (Critical Rev. 117-125,1992).

Many penetration enhancers have been tested so far and some have been found effective in facilitating mucosal administration of large molecular drugs.

However, hardly any penetration enhancing products have reached the market place. Reasons for this include lack of a satisfactory safety profile respecting irritation, lowering of the barrier function, and impairment of the mucocilliary clearance protective mechanism. It has been found that some of the popular penetration enhancers, especially those related to bile salts, and some protein solubilizing agents, impart an extremely bitter and unpleasant taste. This makes their use impossible for human consumption on a day to day basis. Several approaches were utilized to improve the taste of the bile salts based delivery systems, but none of them are commercially acceptable for human consumption to date. Approaches utilized include patches for buccal mucosa, bilayer tablets, controlled release tablets, liposome formulations, use of protease inhibitors, bucally administered film patch devices, and various polymer matrices. Further the problem is compounded because of the localized side effect of a patch which often results in severe tissue damage in the mouth.

The absorption of proteins and peptides is believed to be enhanced by the diffusion of large molecules entrapped in liposomal form through the aqueous pores and the cell structure perturbation of the tight paracellular junctions.

It has now been found that improvements in penetration and absorption of certain formulations can be achieved by mixing the formulation with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants, especially when delivered applied to the buccal mucosa) through aerosol devices, e.g. metered dose inhalers (MDIs). Metered dose inhalers are a proven technology and a popular drug delivery form for many kinds of drug. The use of the present novel formulations and excipients can improve the quality (in terms of absorption), stability and performance of MDI formulations.

The formulation ingredients are selected specifically to give enhancement in the penetration through the pores and facilitate the absorption of the drugs to reach therapeutic levels in the plasma. With the proper formulation changes and changes in administration technique, the formulation can be delivered to the deep lungs, through the nasal cavity and the buccal cavity.

Pressurized inhalers also offer a wide dosing range, consistent dosing efficiency. In this local delivery greater than 95% of the dose is reached to the target area. The smaller particle size (4-15 microns) of pressurized inhalers also enhances dosing due to broader coverage within the buccal cavity. In this situation, increased coverage can help more absorption of drug like insulin.

Furthermore, because these devices are self-contained, the potential for contamination is avoided.

Disclosure of the Invention In one aspect, the present invention provides a mixed liposome pharmaceutical formulation with multilamellar vesicles, comprising: i) a pharmaceutical agent; ii) water; iii) an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to 10 wt./wt. of the total formulation; iv) at least one membrane-mimetic amphiphile; and v) at least one phospholipid, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma-linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PGdiammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxocholanylglycine and alkali metal salts thereof, octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanol X-oleyl ether, wherein X is from 9 to and combinations thereof, and wherein the phospholipid is selected from the group consisting of, phospholipid GLA (glycolic, lactic acid), phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, -6dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, unsaturated lecithin, saturated lecithin and tysolecithin, and combinations thereof, and wherein the amount of each membrane-mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. of the total formulation, and the total concentration of membrane-mimetic amphiphiles and phospholipids is less than 50 wt./wt. of the formulation.

Preferably the mixed liposome pharmaceutical formulation has a pH of between 6.0 and The preferred number of membrane mimetic amphiphiles are from 2 to The preferred number of phospholipids are from 1 to 4.

In one embodiment, the alkali metal C8 to C22 alkyl sulphate is sodium lauryl sulphate.

In embodiments where the pharmaceutical agent is a protein at least one protease inhibitor may be added to the formulation to inhibit degradation of the pharmaceutical agent by the action of proteolytic enzymes. Of the known protease inhibitors, most are effective at concentrations of from 1 to 3 wt./wt. of the formulation.

Non-limiting examples of effective protease inhibitors are bacitracin, soyabean trypsin, aprotinin and bacitracin derivatives, e. g. bacitracin methylene disalicylate. Bacitracin is the most effective of those named when used in concentrations of from 1.5 to 2 wt./wt. Soyabean trypsin and aprotinin may be used in concentrations of about 1 to 2 wt./wt. of the formulation.

In one embodiment, the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid and mixtures thereof, the concentration such absorption enhancing compound being from about 1 to about 5 wt./wt. In another embodiment, suitable for delivery through oral mucosal membranes, the formulation contains sodium lauryl sulphate, and combinations selected from the group consisting of: i) sodium salt of trihydroxy-oxo-cholanyl glycine, sphingomyelin and stearamide DEA; ii) sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipid GLA; -7iii) ceramide and stearamidopropyl phosphatidyl PG-diammonium chloride; iv) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin; v) octylphenoxypolyethoxyethanol and saturated lecithin; vi) sodium hyaluronate, polydecanol 9-lauryl ether, lecithin and evening of primrose oil, and vii) monoolein, saturated lecithin, sodium hyaluronate and evening of primrose oil.

In yet another embodiment, suitable for topical delivery transdermally, the formulation contains sodium lauryl sulphate and combinations selected from the group consisting of: i) lecithin, sodium hyaluronate, glycolic acid and propylene glycol, and ii) sodium hyaluronate, sphingomyelin, glycolic acid and propylene glycol.

Preferably the lecithin is saturated lecithin.

It will be recognized by those skilled in the art that for many pharmaceutical formulations it is usual to add at least one antioxidant to prevent degradation and oxidation of the pharmaceutically active ingredients. It will also be understood by those skilled in the art that colorants, flavouring agents and non-therapeutic amounts of other compounds may be included in the formulation.

In one embodiment the antioxidant is selected from the group consisting of tocopherol, deteroxime mesylate, methyl paraben, ethyl paraben and ascorbic acid and mixtures thereof. A preferred antioxidant is tocopherol.

The formulation suitable for delivery through oral mucosal membranes may be in chewable form, in which case it will be necessary to add ingredients suitable for such form. Such ingredients include guar gum, powdered acacia, carrageenin, beeswax and xanthan gum.

In a preferred embodiment, the mixed liposome pharmaceutical formulation may take the form of an aerosol. In this embodiment the formulation also includes a propellant, which is liquid under pressure, and a phenolic compound.

Another aspect of the invention provides an aerosol pharmaceutical formulation with multilamellar vesicles comprising: -8a) an intermediate formulation which comprises: i) a pharmaceutical agent; ii) water; iii) an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to wt./wt. of the total formulation; iv) at least one membrane-mimetic amphiphile; v) at least one phospholipid, and vi) a phenolic compound in a concentration of from 1 to 10 wt./wt. of the total formulation; and b) a propellant which is liquid under pressure, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma-linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl

PG-

diammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxocholanylglycine and alkali metal salts thereof, octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanol X-oleyl ether, wherein X is from 9 to and combinations thereof, and wherein the phospholipid is selected from the group consisting of, phospholipid GLA (glycolic, lactic acid), phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, polysiloxy pyrrolidone linoleyl phospholipid, sphingomyelin, ceramides, cephalin, triolein, unsaturated lecithin, saturated lecithin and lysolecithin, and combinations thereof, and wherein the amount of each membrane-mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. of the total formulation, and the total concentration of membrane-mimetic amphiphiles and phospholipids is less than 50 wt./wt. of the formulation, and wherein the phenolic compound is selected from the group consisting of phenol and methyl phenol.

-9- Preferably the aerosol pharmaceutical formulation with multilamellar vesicles has a pH of between 6.0 and The preferred number of membrane mimetic amphiphiles are from 2 to The preferred number of phospholipids are from 1 to 4.

In one embodiment, the alkali metal C8 to C22 alkyl sulphate is sodium C8 to C22 alkyl sulphate, and preferably is sodium lauryl sulphate.

Preferably, the phenol is selected from the group consisting of phenol and methyl phenol.

Preferably, the ratio of pharmaceutical agent, e.g. insulin, to propellant is from 5:95 to 25:75.

In a preferred embodiment, the propellant is selected from the group consisting of C1-C2 dialkyl ether, butanes, fluorocarbon propellant, hydrogencontaining fluorocarbon propellant, chlorofluorocarbon propellant, hydrogencontaining chlorofluorocarbon propellant, and mixtures thereof.

In another embodiment, the intermediate formulation also contains a compound selected from glycerin, polyglycerin and mixtures thereof in an amount of from 1-40 wt./wt. of the intermediate formulation.

In a further embodiment, the weight ratio of intermediate formulation to propellant is from 5:95 to 25:75.

In one embodiment, the alkali metal C8 to C22 alkyl sulphate is in a concentration of from 2 to 8 wt./wt. of the intermediate formulation.

In a further embodiment, the methyl phenol is m-cresol.

In another embodiment, the alkali metal C8 to C22 alkyl sulphate is sodium lauryl sulphate.

In yet another embodiment, the total concentration of membrane mimetic amphiphiles is from about 1 to about 25 wt./wt. In yet another embodiment, the propellant is selected from the group consisting of tetrafluoroethane, tetrafluoropropane, dimethylfluoropropane, heptafluoropropane, dimethyl ether, n-butane and isobutane.

In yet another embodiment, the mixed liposome pharmaceutical formulation is contained in an aerosol dispenser. As such, another aspect of this invention provides a pressurized container or an aerosol dispenser, containing an aerosol pharmaceutical formulation in an aerosol form as described above.

The present invention also provides a metered dose aerosol dispenser with the propellant and intermediate formulation therein.

The present invention also provides a method for administering aerosol pharmaceutical formulations of the present invention, by spraying a predetermined amount of the formulation into the mouth with a metered dose spray device.

The present invention also provides a method for administration of a pharmaceutical agent in a buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a predetermined amount of an aerosol formulation with multilamellar vesicles comprising: a) an intermediate formulation comprising: i) a pharmaceutical agent; ii) water; iii) an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to wt./wt.% of the total formulation; iv) at least one membrane-mimetic amphiphile; v) at least one phospholipid; and vi) a phenolic compound in a concentration of from 1 to 10 wt./wt. of the total formulation; and b) a propellant, which is a liquid under pressure, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PGdiammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, octylphenoxypolythoxyethanol, and combinations thereof, and wherein the phosphotipid is selected from the group consisting of, phospholipid GLA (glycolic, lactic acid), phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, polysiloxy pyrrolidone linoleyl phospholipid, -11 sphingomyelin, ceramides, cephalin, triolein, unsaturated lecithin, saturated lecithin and lysolecithin, and combinations thereof, and wherein the phenolic compound is selected from the group consisting of phenol and methyl phenol, and wherein the amount of each membrane-mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. of the total formulation, and the total concentration of membrane-mimetic amphiphiles and phospholipids is less than 50 wt./wt. of the formulation.

Preferably the propellant is selected from the group consisting of C1 to C2 dialkyl ether, butanes, fluorocarbon propellant, hydrogen-containing fluorocarbon propellant, chlorofluorocarbon propellant, hydrogen-containing chlorofluorocarbon propellant, and mixtures thereof, In one embodiment, the metered dose spray dispenser is vigorously shaken immediately prior to administration of the pharmaceutical agent.

The pharmaceutical agent may be selected from a wide variety of macromolecular agents, depending on the disorder being treated, generally with molecular weights greater than about 1000 and especially between about 1000 and 2 000 000. Pharmaceutical agents useful in the present invention include insulin, heparin, low molecular weight heparin, hirugen, hirulos, hirudine, interferons, interleukins, cytokines, mono and polyclonal antibodies, chemotherapeutic agents, vaccines, glycoproteins, bacterial toxoids, growth hormones, parathyroid hormone (PTH), luteinizing hormones, oestrogens, androgens, calcitonins, insulin like growth factors (IGF), glucagon like peptides (GLP-1 and GLP-2), steroids and retinoids, injectable large molecule antibiotics, protein based thrombolytic compounds, platelet inhibitors, DNA, gene therapeutics, RNA and antisense oligonucleotides, and small molecule drugs.

Modes For Carrying Out The Invention When developing new pharmaceutical formulations, it is desirable to provide dosage forms suitable for administering drugs to humans and animals through oral, nasal, pulmonary and transdermal mucosal routes and to allow easy accessibility to the sites of administration. Local absorption of macromolecular drugs is desirable over a prolonged period to maximize drug absorption.

Furthermore, it is desirable to minimize tissue damage and provide acceptable -12tissue compatibility of the dosage form. It is preferable to provide systems which are pain free and easy to be administered with great flexibility, in order to gain high acceptance and compliance of any therapy by patients.

It has been found that macromolecular drugs may be administered in mixed liposomal formulations in which particle sizes (1 to 4 nm) are smaller than any pores of mucosal surfaces.

The present invention provides an improved method for delivery of macromolecular (high molecular weight) pharmaceutical agents, particularly through the skin or membranes in the nose, mouth, lungs, vagina or rectum. The preferred delivery is through oral or nasal cavities or through the lungs. Even more preferred is delivery into the buccal cavity using a metered dose dispenser.

The pharmaceutical agents cover a wide spectrum of agents, including proteins, peptides, hormones, vaccines and drugs. The molecular weights of the macromolecular pharmaceutical agents are preferably above 1000, especially between 1000 and 2 000 000.

For example, hormones which may be administered with the present invention include human growth hormones, parathyroid hormones, follicular stimulating hormones, luteinizing hormones, androgens, oestrogens, prostaglandins, somatropins, gonadotropins, erythropoietin, interferons, interleukins, steroids and cytokines.

Vaccines which may be administered with the present invention include bacterial and viral vaccines such as vaccines for hepatitis A, hepatitis B, hepatitis C, influenza, tuberculosis, canary pox, chicken pox, measles, mumps, rubella, pneumonia, BCG, HIV, helicobacter pylori and AIDS.

Bacterial toxoids which may be administered using the present invention include diphtheria, tetanus, pseudomonas and mycobacterium tuberculosis.

Examples of specific cardiovascular or thrombolytic agents include heparin, low molecular weight heparin, hirugen, hirulos and hirudine.

Small molecules may also be administered using the present invention. For example, opioids, narcotics, analgesics, NSAIDS, steroids, anaesthetics, hypnotics and pain killers, may be administered with the aerosol formulation of the present invention.

For insulin-containing and some other formulations, the formulation may also contains at least one inorganic salt which opens channels in the -13gastrointestinal tract and may provide additional stimulation to release insulin.

Non-limiting examples of inorganic salts are sodium, potassium, calcium and zinc salts, especially sodium chloride, potassium chloride, calcium chloride, zinc chloride and sodium bicarbonate.

It will be recognized by those skilled in the art that for many pharmaceutical formulations it is usual to add at least one antioxidant to prevent degradation and oxidation of the pharmaceutically active ingredients. It will also be understood by those skilled in the art that colorants, flavouring agents and non-therapeutic amounts of other compounds may be included in the formulation. Typically flavouring agents are menthol and other fruit flavors.

The antioxidant is selected from the group consisting of tocopherol, deteroxime mesylate, methyl paraben, ethyl paraben and ascorbic acid and mixtures thereof. A preferred antioxidant is tocopherol.

Non-limiting examples of effective protease inhibitors are bacitracin, soyabean trypsin, aprotinin and bacitracin derivatives, e.g. bacitracin methylene disalicylate. Bacitracin is the most effective of those named when used in concentrations of from 1.5 to 2 wt./wt. Soyabean trypsin and aprotinin two may be used in concentrations of about 1 to 2 wt./wt. of the formulation.

It is believed that the phenolic compounds act mainly as preservatives and complexing agents to stabilize drugs, e.g. insulin. Besides their function as a stabilizer and preservative, they may also act as antiseptic agents and furthermore may help in absorption. The methyl phenol may be o-cresol, m-cresol or p-cresol, but m-cresol is preferred.

As will be understood, the concentration of the pharmaceutical agent is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in an animal or human. The concentration or amount of pharmaceutical agent administered will depend on the parameters determined for the agent and the method of administration, e.g. oral, nasal, transdermal, pulmonary. For example, nasal formulations tend to require much lower -14concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 100 times in order to provide a suitable nasal formulation.

Preferred methods of forming mixed non-phospholipid membrane mimetic amphiphiles and phospholipid are based on the phase behaviour of lipid amphiphiles and phospholipids. Such methods use high turbulence or high shear methods of mixing, e.g. turbines or high velocity nozzles. For example, the membrane-mimetic amphiphiles may be injected at high velocity, e.g. through nozzles, into an aqueous phase of the phospholipid. Alternatively, the membrane mimetic amphiphiles and the phospholipids may be mixed in a mixing chamber into which the phospholipids are injected at high velocity through one or more nozzles and the membrane-mimetic amphiphiles are also injected at high velocity through one or more nozzles. Other ingredients, such as sodium C8 to C22 alkyl sulphate, phenol and/or m-cresol, protease inhibitors may be premixed with either the membrane-mimetic amphiphile or the phospholipid. The velocity and mixing of the two liquids depends in part on the viscosities of the materials and nozzle diameters, e.g. 10 to 15 m/s through 0.5 to 1.0 mm diameter nozzle apertures.

Typically the ratio of the membrane-mimetic amphiphile aqueous solution to the phospholipid solution is about 5:1 to about 20:1 and the temperature of mixing is typically from about 10°C to 200C.

It may sometimes be necessary to heat the membrane-mimetic amphiphiles and other ingredients in order to yield a homogeneous aqueous solution prior to mixing with the phospholipids. The nature of the pharmaceutical may also dictate the temperature range at which mixing may take place. The temperature of mixing is typically room temperature or below, but may be higher than room temperature for certain formulations. The resulting formulation contains multi-lamellar liposomal vesicles. If the formulation has been heated during mixing, it is sometimes desirable to cool the mixture while still being mixed, in order to assist in the formation of the multilamellar vesicles.

Mixed multilamellar vesicles formed by the present process are very small in size, e.g. less than 10 nm, and are stable under most storage conditions.

Preferably, the membrane-mimetic amphiphile solution is injected into the phospholipid solution through tangentially placed nozzles in a small cylindrical mixing chamber.

Preferably, one or two nozzles are used for the membrane-mimetic amphiphile solution and one or two alternating nozzles for the phospholipid solution. The two liquids are preferably delivered to the nozzles by flow-controlled positive displacement pumps.

The phenol and/or m-cresol may be added to stabilize the formulation and protect against bacterial growth. An isotonic agent such as glycerin may also be added. The phenol and/or m-cresol and glycerin may be added after the membrane-mimetic amphiphile and phospholipids have been mixed, if desired, rather than with the other ingredients.

After formation of the pharmaceutical formulation, the formulation is charged to a pressurizable container. Preferably the container is a vial suitable for use with a metered dose dispenser, e.g. a metered dose inhaler or applicator.

Then the vial is charged with propellant. As the propellant is introduced into the vial, there is great turbulence in the vial and the propellant and pharmaceutical formulation become mixed. Some of the formulations with glycerin or polyglycerin in them tend not to separate on standing. Others may separate. For those aerosol formulations which are substantially homogeneous, it may not be necessary to shake the vial before use, although, through habit with other formulations, many users may shake the vial. Shaking the vial is recommended, however, in order to assure good accuracy of pharmaceutical dispensing from "shot" to "shot" and from the first shot to the last from the container. As is known, in order to deliver the pharmaceutical agent to the lung, it is necessary for the user to breathe deeply when the aerosol spray from the pressurized container is released. Without breathing in, the pharmaceutical agent is delivered to the buccal cavity. The method chosen will depend on a number of factors, including the type of pharmaceutical agent, the concentration in the aerosol, the desired rate of absorption required and the like.

The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFC 134 a (1,1,1,2 tetrafluoroethane).

Although the present invention has such wide applicability, the invention is described hereinafter with particular reference to insulin and its analogues, which are used for the treatment of diabetes.

-16- In the case of insulin, which is intended for administration through nasal or oral cavities or the lungs, an aqueous buffer solution may be made first by adding aqueous alkali metal C8 to C22 alkyl sulphate, e.g. sodium lauryl sulphate, to powdered insulin, and then stirring until the powder is dissolved and a clear solution is obtained. The buffer solution may also contain sodium salicylate.

Typical concentrations of sodium salicylate and sodium lauryl sulphate in the aqueous solution are about 3 to 20 wt./wt. of each compound in the solution.

Typically, insulin is present in the solution in an amount which will give a concentration of about 2 to 4 wt./wt. of the final formulation.

The buffer solution is then added to liquid which comprises a membranemimetic amphiphile or a phospholipid while mixing vigorously, to form multilamellar liposomal vesicles.

The membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma-linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PGdiammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxocholanylglycine and alkali metal salts thereof, octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanol X-oleyl ether, wherein X is from 9 to and combinations thereof. Preferably X is 9,10 or The phospholipid is selected from the group consisting of phospholipid GLA, phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, unsaturated lecithin, saturated lecithin and lysolecithin.

Each of the membrane-mimetic amphiphiles and phospholipids are present in a concentration of from 1 to 10 wt./wt. of the total formulation.

-17- Preferred salts of hyaluronic acid are alkali metal hyaluronates, alkaline earth hyaluronates and aluminium hyaluronate. The preferred salt is sodium hyaluronate. The preferred concentration of hyaluronic acid or pharmaceutically acceptable salts of hyaluronic acid is from 1 to 5 wt./wt. of the total formulation.

An even more preferred range is from 1.5 to 3.5 wt./wt. of the total formulation.

The phenol and/or m-cresol may be added with the membrane mimetic amphiphile, the phospholipid or at any other time during mixing.

It will be recognized by those skilled in the art that for many pharmaceutical formulations it is usual to add at least one antioxidant to prevent degradation and oxidation of the pharmaceutically active ingredients. It will also be understood by those skilled in the art that colorants, flavouring agents, salts, protease inhibitors, other pharmaceutically acceptable compounds and non-therapeutic amounts of other compounds may be included in the formulation. Typical flavouring agents are menthol, sorbitol and fruit flavours.

The antioxidant may be selected from the group consisting of tocopherol, deteroxime mesylate, methyl paraben, ethyl paraben and ascorbic acid and mixtures thereof. A preferred antioxidant is tocopherol.

In general the size of the multi-lamellar liposomal vesicles particles is about from 1 to 10 nm, and preferably from 1 to 5 nm. Such a size distribution ensures effective absorption of the formulation, and therefore the pharmaceutical agent, through the membranes, for example the membranes in the oral and nasal cavities.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the nasal and oral cavities, it is often desirable to increase, e.g. double or triple, the dosage which is normally required through injection of administration through the gastrointestinal tract.

As will be understood, the amount of each component of the formulation will vary depending on the pharmaceutical agent and the site of application.

For oral application, sodium C8 to C22 alkyl sulphate and sodium edetate are insufficient on their own and must be combined with at least one membranemimetic amphiphile and at least one phospholipid to promote the oral absorption of macromolecules to achieve therapeutic effects. The effect is enhanced by delivery of the macromolecules by aerosol, with the additions of phenol and/or m-cresol to -18the formulation and using a propellant, particularly a hydrogen-containing fluorocarbon or a hydrogen-containing chlorofluorocarbon.

The oral aerosol formulations may be mixed with a suitable propellant and delivered with a suitable applicator.

Preferred formulations for oral or nasal application have the following combinations, in addition to sodium lauryl sulphate: i) sodium salt of trihydroxy-oxo-cholanyl glycine, sphingomyelin and stearamide DEA; ii) sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipid GLA; iii) phospholipid GLA, polydecanol 9-lauryl ether and octylphenoxyethoxyethanol; iv) ceramide and stearamidopropyl phosphatidyl PG-diammonium chloride; v) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin; vi) octylphenoxypolyethoxyethanol and saturated lecithin; vii) lecithin, evening of primrose oil and trihydroxy-oxocholanylglycine; viii) sodium hyaluronate, trihydroxy oxo-cholanylglycine, lecithin and evening of primrose oil; ix) sodium hyaluronate, saturated lecithin, and evening of primrose oil; x) sodium hyaluronate and saturated lecithin; xi) sodium hyaluronate and sphingomyelin; xii) stearamidopropyl phosphatidyl PG-diammonium chloride and ceramide; xiii) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin; xiv) sodium hyaluronate, polydecanol 9-lauryl ether, lecithin and evening of primrose oil, and xv) monoolein, saturated lecithin, sodium hyaluronate and evening of primrose oil.

Some preferred formulations for transdermal application have the following absorption enhancing compound combinations, in addition to sodium lauryl sulphate and sodium edetate: i) sodium hyaluronate, saturated lecithin, glycolic -19acid and propylene glycol; ii) sodium hyaluronate, sphingomyelin, glycolic acid and propylene glycol.

For topical applications, enhanced skin penetration can be obtained with a combination of glycolic lactic acid propylene glycol with the liposomes.

The therapeutic formulations of the present invention can be stored at room temperature or at cold temperature. Storage of drugs is preferable at a cold temperature, e.g. 4 0 C, to prevent degradation of the drugs and to extend their shelf life.

As indicated hereinbefore, generally, oral, pulmonary, transdermal and nasal are the favoured sites of the administration but the formulation can be applied to the rectal and vaginal mucosa. According to the physiologically active peptide or protein used, the dosage form and the site of administration a specific administration method can be selected.

The formulation of this invention is generally prepared as microfine multilamellar liposomal vesicle particles (1 to 10 nm or less) by the virtue of its preparation methods used and combinations suitable characteristics of the membrane mimetic amphiphiles and phospholipids.

Administration of the formulation is by methods generally known in the art.

For oral and nasal application, sprays are preferable. Other methods include the use of drops, chewable tablets, chewable gum, suppositories, lotions and ointments.

Utilization of atomizer or aerosol spray devices (metered dose inhalers or nebulizers) can be used to further reduce the particle size for effective inhalation from the nasal or oral cavity so the drug may successfully reach to the specific site, especially the lungs, and be absorbed.

It is also possible to utilize a drug delivery system such that an enteric coating is applied to the gelatin capsule to cause the micelles to be released only in the duodenum or in the proximity of the large intestine and not in the stomach.

As indicated hereinbefore, oral or pulmonary administration may be desirable. Preferably, the pharmaceutical is administered using a metered dose dispenser, in which the pharmaceutical formulation is delivered with a propellant.

To prepare an aerosol formulation, phenol and/or methyl cresol, e.g. mcresol, may be added to stabilize the formulation and protect against bacterial 20 growth. An isotonic agent such as glycerin may also be added. The formulation is then put into an aerosol dispenser and the dispenser charged with the propellant.

The propellant, which is under pressure, is in liquid form in the dispenser. In the present invention, when the formulation of the present invention is in a dispenser, the aqueous phase may be separated from the propellant phase.

Preferably, however, the ratios of the ingredients are adjusted by simple experimentation so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is hydrofluoroalkane (HFA) 134a (1,1,1,2 tetrafluoroethane).

A particular advantage with the use of metered dose dispensers is that the formulation can be delivered in a relatively precise dose, e.g. titratable to injection within 1 unit of insulin dose. The droplet size of the formulation preferably falls between 1-5 pm in order for droplets to penetrate buccal mucosa or to reach to the deep lung surface. Thus, the present invention is suitable for delivery of drugs such as insulin for the treatment of diabetes.

The pressurized dispenser also offers a wide dosing range and consistent dosing efficiency. With such a delivery, greater than about 95% of the dose may reach the target area. The smaller particle size (1-5 pm) obtained using pressurized dispensers also enhances dosing due to broader coverage within the lung cavity. In this situation, increased coverage can help more absorption of a drug like insulin. Furthermore, because these devices are self-contained, potential contamination is avoided.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. It will be understood that the amounts of certain ingredients may need to be limited in order to avoid formulations which produce foam when sprayed rather than forming a fine spray.

For absorption through the oral cavities, it is often desirable to increase, e.g.

double or triple, the dosage which is normally required through injection or administration through the gastrointestinal tract.

-21 As will be understood, the amount of each component of the formulation will vary depending on the pharmaceutical agent and the site of application.

Administration of the formulation into the buccal cavity is by spraying the formulation into the mouth, without inhalation, so that the droplets stay in the mouth rather than be drawn into the lungs.

The invention is illustrated by reference to the following examples.

Example 1 26 000 units (1000 mg) of insulin crystals were suspended in 150 mL 0.3M hydrochloric acid and the solution was stirred to dissolve the crystals completely.

The pH was adjusted to 7.0 by neutralizing with 0.3M sodium hydroxide. The final volume was adjusted to 260 mL to give 100 units/mL insulin concentration.

To 10 mL of insulin solution, 50 mg of sodium lauryl sulphate was added and dissolved completely. In 50 mL of water, 50 mg trihydroxy-oxocholanylglycine and 50 mg polydecanol 20-oleyl ether were added and dissolved and then mixed with the insulin solution. This mixture was then sprayed under pressure into a 1 wt. solution of phospholipid GLA. This procedure gave a mixed amphiphile insulin solution with 50 units/mL.

The structure of the mixed amphiphile insulin was examined under a light microscope and the particle size was analysed by laser light scattering. The average particle size was estimated to be about 2 to 10 nm.

In one set of tests, ten diabetic human volunteers who normally took insulin by injection three times a day, were studied. The volunteers were tested with insulin, taken orally. The volunteers fasted from midnight prior to the test, with no food being taken during the 4 hour study.

Each of the volunteers received 10 units insulin. In one test, the oral insulin was administered with a metered dose spray. In another test, the insulin was administered by injection. Blood glucose levels, in mmol/L, were monitored every 15-30 minutes by Bayer's Glucometer Elite.

The average results for the ten volunteers, of the trial were as follows: -22- Table I Time(minutes) Oral Insulin Injection units) (10 units) 0 11.0 10.5 10.6 10.5 10.2 10.4 9.3 10.2 8.6 7.0 8.2 120 6.5 6.8 150 5.9 180 5.1 4.7 The results show that the oral insulin formulation, within the scope of the present invention, at an equivalent dosage, is comparable with the injected insulin.

Example II To 10 mL of the insulin solution prepared in Example I, 50 mg of sodium lauryl sulphate was added and dissolved completely. In 50 mL of water, 50 mg lauramidopropyl betain and 50 mg polydecanol 9-lauryl ether were added and dissolved and then mixed with the insulin solution. This mixture was then sprayed under pressure into a 1 wt. solution of Phospholipon-H (trade mark) saturated lecithin. This procedure gave a multilamellar, mixed amphiphile insulin solution with 50 units/mL.

The structure of the multilamellar, mixed amphiphile insulin was examined under a light microscope and the particle size was analysed by laser light scattering. The average particle size was estimated to be about 2 to 10 nm.

In one set of tests, ten healthy human volunteers were studied. The volunteers were tested with insulin, taken orally and taken by injection. The volunteers fasted from midnight prior to the test, with no food being taken during the 4 hour study.

Each of the volunteers received 10 units insulin. In one test, the oral insulin was administered with a metered dose spray. In another test, the insulin was -23administered by injection. Blood glucose levels, in mmol/L, were monitored every minutes by Bayer's Glucometer Elite.

The average results for the ten volunteers, of the trial were as follows: Table II Time (minutes) Oral Insulin Injection units) (10 units) 0 5.5 5.3 5.0 5.2 4.6 4.2 4.2 3.8 120 4.0 3.6 150 3.6 3.3 180 3.1 The results show that the oral insulin formulation, within the scope of the present invention, at an equivalent dosage, is comparable with the injected insulin.

Example Ill To 10 mL of the insulin solution prepared in Example I, 50 mg of sodium lauryl sulphate was added and dissolved completely. This mixture was then sprayed under pressure into a 1 wt. solution of Phospholipon-H (trade mark) saturated lecithin. This procedure gave a multilamellar, mixed amphiphile insulin solution with 50 units/mL.

This formulation, which is outside the scope of the present invention, was tested on 10 healthy volunteers and compared to injected insulin, as in Example II.

The average results for the ten volunteers, of the trial were as follows: -24- Table III Time (minutes) 0 120 150 180 Oral Insulin units) 5.7 5.8 5.5 5.4 5.3 5.4 5.3 Injection (10 units) 5.9 5.7 4.8 The results show that the oral insulin formulation, outside the scope of the present invention, at an equivalent dosage, had little effect. This is probably because the insulin was not absorbed, and degraded faster.

Example IV To 10 mL of the insulin solution prepared in Example 1, 100 mg of sodium lauryl sulphate was added and dissolved completely.

The average results for the ten volunteers, of the trial were as follows: Table IV Time (minutes) 0 120 150 180 Oral Insulin units) 6.1 6.0 5.8 5.7 5.6 5.5 5.6 Injection (10 units) 5.9 5.7 5.2 4.7 4.3 3.7 3.3 The results show that the oral insulin formulation, outside the scope of the present invention, at an equivalent dosage, had little effect.

Example V mL of the insulin solution prepared in Example I was added to a 1 wt. solution of Phospholipon-H saturated lecithin.

The average results for the ten volunteers, of the trial were as follows: Table V Time (minutes) Oral Insulin Injection units) (10 units) 0 6.2 5.9 6.3 5.6 6.2 6.4 4.6 120 6.5 4.1 150 6.4 3.8 180 6.5 3.2 The results show that the oral insulin formulation, outside the scope of the present invention, at an equivalent dosage, had no effect.

ExampleVI To 10 mL of the insulin solution prepared in Example I, 50 mg of sodium lauryl sulphate was added and dissolved completely. In 50 mL of water, 50 mg trihydroxy-oxo-cholanylglycine and 50 mg stearamide DEA were added and dissolved and then mixed with the insulin solution. This mixture was then sprayed under pressure into a 1 wt. solution of sphingomyelin. This procedure gave a mixed amphiphile insulin solution with 50 units/mL.

The structure of the mixed amphiphile insulin was examined under a light microscope and the particle size was analysed by laser light scattering.

This formulation, which is within the scope of the present invention, was tested on 10 diabetic volunteers and compared to injected insulin, as in Example I.

The average results for the ten volunteers, of the trial were as follows: -26- Table VI Time (minutes) Oral Insulin Injection units) (10 units) 0 7.8 6.5 5.3 5.1 120 4.8 4.6 150 4.1 4.2 180 3.6 The results show that the oral insulin formulation, within the scope of the present invention, at an equivalent dosage, is comparable with the injected insulin.

Example VII To 10 mL of the insulin solution prepared in Example 1, 100 mg of sodium lauryl sulphate was added and dissolved completely. In 50 mL of water, 100 mg sodium hyaluronate, 0.5 mL glycolic acid and 0.5 mL propylene glycol were added and dissolved and then mixed with the insulin solution. This mixture was then sprayed under pressure into a 1 wt. solution of Phospholipon-H (trade mark) saturated lecithin.

In one set of tests, ten healthy human volunteers were studied. The volunteers were tested with insulin, applied topically and taken by injection. The volunteers fasted from midnight prior to the test, with no food being taken during the 4 hour study.

Each of the volunteers received 10 units insulin. In one test, the insulin was administered topically to a 2 cm 2 area of the back of the hand. In another test, the insulin was administered by injection. Blood glucose levels, in mmol/L, were monitored every 30 minutes by Bayer's Glucometer Elite.

The average results for the ten volunteers, of the trial were as follows: -27- Table VII Time (minutes) Topical Insulin Injection units) (10 units) 0 5.5 5.3 5.3 5.3 5.0 4.9 4.6 120 4.8 4.3 150 4.7 180 4.5 3.8 The results show that the topical insulin formulation, within the scope of the present invention, at an equivalent dosage, is comparable with the injected insulin.

In the specification the terms "comprising" and "containing" shall be understood to have a broad meaning similar to the term "including" and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the terms "comprising" and "containing" such as "comprise", "comprises", "contain" and "contains".

It will of course be realised that while the foregoing has been given by way of illustrative example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

Claims

1. A mixed liposome pharmaceutical formulation with multilamellar vesicles, comprising: i) a pharmaceutical agent, ii) water; iii) an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to 10 wt./wt. of the total formulation; iv) at least one membrane-mimetic amphiphile, and v) at least one phospholipid, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma- linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxo- cholanylglycine and alkali metal salts thereof, and octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanol X-oleyl ether, wherein X is from 9 to 20, and combinations of any two or more thereof, and wherein the phospholipid is selected from the group consisting of phospholipid GLA (glycolic, lactic acid), phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, lecithin, saturated lecithin and lysolecithin, and combinations of any two or more thereof, and wherein the amount of each membrane mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. of the total formulation, and the total concentration of membrane mimetic amphiphiles and phospholipids is less than wt./wt.% of the formulation.

2. An aerosol mixed liposome pharmaceutical formulation with multilamellar vesicles, comprising: a) an intermediate formulation comprising: i) a pharmaceutical agent; ii) water; iii) an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to 10 wt./wt% of the total formulation; iv) at least one membrane-mimetic amphiphile; v) at least one phospholipid, and vi) a phenolic compound in a concentration of from 1 to wt./wt.% of the total formulation, and b) a propellant which is selected from the group consisting of C1 to C2 dialkyl ether, butanes, fluorocarbon propellant, hydrogen-containing fluorocarbon propellant, chlorofluorocarbon propellant, hydrogen-containing chlorofluorocarbon propellant, and mixtures of any two or more thereof, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma- linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxo- cholanylglycine and alkali metal salts thereof, and octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanol X-oleyl ether, wherein X is from 9 to 20, and combinations of any two or more thereof, and wherein the phospholipid is selected from the group consisting of phospholipid GLA (glycolic, lactic acid), phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, lecithin, saturated lecithin and lysolecithin, and combinations of any two or more thereof, wherein the amount of each membrane mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. of the total formulation, and the total concentration of membrane mimetic amphiphiles and phospholipids is less than wt./wt.% of the formulation, and wherein the phenol is selected from the group consisting of phenol and methyl phenol.

3. A formulation according to claim 1 or claim 2, wherein the alkali metal C8 to C22 alkyl sulphate is sodium lauryl sulphate.

4. A formulation according to any one of claims 1 to 3, wherein there are at least two membrane mimetic amphiphiles.

5. A formulation according to any one of claims 1 to 4, wherein the membrane- mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid and mixtures thereof, the concentration such absorption enhancing compound being from about 1 to about wt./wt.

6. A formulation according to claim 1 or claim 2 which contains sodium lauryl sulphate and combinations selected from the group consisting of: i) sodium salt of trihydroxy-oxo-cholanyl glycine, sphingomyelin and stearamide DEA; ii) sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipid GLA; iii) phospholipid GLA, polydecanol 9-lauryl ether and octylphenoxyethoxyethanol; iv) ceramide and stearamidopropyl phosphatidyl PG-diammonium chloride; v) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin; vi) octylphenoxypolyethoxyethanol and saturated lecithin; vii) lecithin, evening of primrose oil and trihydroxy-oxocholanylglycine; viii) sodium hyaluronate, trihydroxy oxo-cholanylglycine, lecithin and evening of primrose oil; ix) sodium hyaluronate, saturated lecithin, and evening of primrose oil; x) sodium hyaluronate and saturated lecithin; xi) sodium hyaluronate and sphingomyelin; xii) stearamidopropyl phosphatidyl PG-diammonium chloride and ceramide; -31- xiii) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin; xiv) sodium hyaluronate, polydecanol 9-lauryl ether, lecithin and evening of primrose oil; xv) monoolein, saturated lecithin, sodium hyaluronate and evening of primrose oil; xvi) lecithin, sodium hyaluronate, glycolic acid and propylene glycol, and xvii) sodium hyaluronate, sphingomyelin, glycolic acid and propylene glycol.

7. A formulation according to any one of claims 1 to 6, wherein the pharmaceutical agent is selected from the group consisting of insulin, heparin, low molecular weight heparin, low molecular weight heparin, hirugen, hirulos, hirudine, interferons, interleukins, cytokines, mono and polyclonat antibodies, chemotherapeutic agents, vaccines, glycoproteins, hormones, bacterial toxoids, growth hormones, parathyroid hormone (PTH), luteinizing hormones, oestrogen, androgens, calcitonins, insulin like growth factors (IGF), glucagon like peptides (GLP- 1 or GLP-2), steroids and retinoids, injectable large molecule antibiotics, protein based thrombolytic compounds, platelet inhibitors, DNA, Gene therapeutics, RNA, antisense oligonucleotides, small molecule drugs, opioids, narcotics, analgesics, NSAIDS, steroids, anaesthetics, hypnotics, pain killers or any two or more thereof.

8. A formulation according to any one of claims 1 to 7, wherein the pharmaceutical agent is insulin.

9. A formulation according to any one of claims 2 to 8, wherein the intermediate formulation also contains a compound selected from glycerin, polyglycerin and mixtures thereof in an amount of from 1-40 wt./wt. of the intermediate formulation. A formulation according to any one of claims 2 to 9, wherein the weight ratio of intermediate formulation to propellant is from 5:95 to 25:75.

11. A formulation according to any one of claims 2 to 10, wherein the propellant is selected from the group consisting of tetrafluoroethane, tetrafluoropropane, dimethylfluoropropane, heptafluoropropane, dimethyl ether, n-butane and isobutane.

12. A process for making a pharmaceutical formulation comprising: mixing in a high shear mixer a pharmaceutical agent, water, an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to 10 wt./wt.% of the total formulation, at least one membrane-mimetic amphiphile and at least one phospholipid, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma- linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxo- cholanylglycine and alkali metal salts thereof, and octylphenoxypolythoxyethanol, polydecanol X-lauryt ether and polydecanol X-oleyl ether, wherein X is from 9 to and wherein the phospholipid is selected from the group consisting of phospholipid GLA, phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, lecithin, saturated lecithin and lysolecithin, and wherein the amount of each membrane mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. of the total formulation, and the total concentration of membrane mimetic amphiphiles and phospholipids is less than wt./wt. of the formulation; said mixing being continued until the formulation is in multilamellar vesicle form.

13. The process for making an aerosol pharmaceutical formulation comprising: mixing in a high shear mixer a pharmaceutical agent, water, an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to 10 of the total formulation, at least one membrane-mimetic amphiphile and at least one phospholipid, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma- linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxo- cholanylglycine and alkali metal salts thereof, and octylphenoxypolythoxyethanol, polydecanol X-lauryl ether and polydecanol X-oleyl ether, wherein X is from 9 to and wherein the phospholipid is selected from the group consisting of phospholipid GLA, phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, lecithin, saturated lecithin and lysolecithin, and wherein the amount of each membrane mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. of the total formulation, and the total concentration of membrane mimetic amphiphiles and phospholipids is less than wt./wt. of the formulation; said mixing being continued until the formulation is in multilamellar vesicle; and adding a phenolic compound selected from the group consisting of phenol, methyl phenol and mixtures of two or more thereof; and dispensing the resulting formulation into an aerosol container and charging the container with a propellant selected from the group consisting of hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, diethyl ether and a mixture of two or more thereof.

14. A process according to claim 12 or claim 13, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid and mixtures of two or more thereof, the concentration such absorption enhancing compound being from about 1 to about wt./wt.

15. A process according to any one of claims 12 to 14, wherein the alkali metal C8 to C22 alkyl sulphate is sodium lauryl sulphate.

16. A process according to claim 12 or claim 13, wherein phospholipids and amphiphiles comprise a combination selected from the group consisting of: i) sodium salt of trihydroxy-oxo-cholanyl glycine, sphingomyelin and stearamide DEA; ii) sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipid GLA; iii) phospholipid GLA, polydecanol 9-lauryl ether and octylphenoxyethoxyethanol; iv) ceramide and stearamidopropyl phosphatidyl PG-diammonium chloride; v) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin; vi) octylphenoxypolyethoxyethanol and saturated lecithin; vii) lecithin, evening of primrose oil and trihydroxy-oxocholanylglycine; viii) sodium hyaluronate, trihydroxy oxo-cholanylglycine, lecithin and evening of primrose oil; ix) sodium hyaluronate, saturated lecithin, and evening of primrose oil; x) sodium hyaluronate and saturated lecithin; xi) sodium hyaluronate and sphingomyelin; xii) stearamidopropyl phosphatidyl PG-diammonium chloride and ceramide; xiii) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin; xiv) sodium hyaluronate, polydecanol 9-lauryl ether, lecithin and evening of primrose oil; xv) monoolein, saturated lecithin, sodium hyaluronate and evening of primrose oil; xvi) lecithin, sodium hyaluronate, glycolic acid and propylene glycol, and xvii) sodium hyaluronate, sphingomyelin, glycolic acid and propylene glycol.

17. A process according to any one of claims 12 to 16, wherein the pharmaceutical agent is selected from the group consisting of insulin, heparin, so- called low molecular weight heparin, low molecular weight heparin, hirugen, hirulos, hirudine, interferons, interleukins, cytokines, mono and polyclonal antibodies, chemotherapeutic agents, vaccines, glycoproteins, growth hormones, bacterial toxoids, hormones, parathyroid hormone (PTH), luteinizing hormones, oestrogen, androgens, calcitonins, insulin like growth factors (IGF), glucagon like peptides (GLP- 1 or GLP-2), large molecule antibiotics, protein based thrombolytic compounds, platelet inhibitors, DNA, RNA, gene therapeutics, antisense oligonucleotides ,small molecule drugs, opioids, narcotics, analgesics, NSAIDS, steroids, anaesthetics, hypnotics, pain killers or any two or more thereof.

18. The process according to any one of claims 12 to 17, wherein the pharmaceutical agent is insulin.

19. A process according to any one of claims 12 to 18, wherein the method of mixing is a high turbulence or high shear method of mixing. A process according to claim 19 selected from the group consisting of i) injecting the phospholipid, in liquid form, at high velocity through at least one nozzle into an aqueous phase of the membrane-mimetic amphiphile, ii) injecting the membrane-mimetic amphiphile, in liquid form, at high velocity through at least one nozzle into an aqueous phase of the phospholipid, and iii) injecting the phospholipid, in liquid form, at high velocity through at least one nozzle and the membrane mimetic amphiphile, in liquid form, at high velocity through at least one nozzle into a mixing chamber; and wherein the alkali metal C8 to C22 alkyl sulphate is present with either the phospholipid or membrane-mimetic amphiphile. -36-

21. A process according to claim 20 wherein the velocity the phospholipid and amphiphile liquids is from 0 to 15 m/s through 0.5 to 1.0 mm diameter nozzle apertures.

22. A process according to any one of claims 19 to 21 wherein the ratio of the membrane-mimetic amphiphile aqueous solution to the phospholipid solution is about 1 to about 20: 1.

23. A mixed liposome pharmaceutical formulation with multilamellar vesicles prepared by the process according to any one of claims 12 to 22.

24. An aerosol dispenser containing the mixed liposome pharmaceutical formulation with multilamellar vesicles of any one of claims 2 to 11 or 23.

25. A metered dose aerosol dispenser containing the mixed liposome pharmaceutical formulation with multilamellar vesicles of any one of claims 2 to 11 or 23.

26. A method for administering an aerosol pharmaceutical composition by spraying a predetermined amount of a mixture comprising: a) an intermediate formulation comprising: i) a pharmaceutical agent, ii) water, iii) an alkali metal C8 to C22 alkyl sulphate in a concentration of from 1 to 10 wt./wt. of the total formulation, iv) at least one membrane-mimetic amphiphile, v) at least one phospholipid, and vi) a phenolic compound in a concentration of from 1 to wt./wt.% of the total formulation; and b) a propellant wherein the propellant is selected from the group consisting of C1-C2 dialkyl ether, butanes, fluorocarbon propellant, hydrogen-containing fluorocarbon propellant, chlorofluorocarbon propellant, hydrogen-containing chlorofluorocarbon propellant, and mixtures thereof, wherein the membrane-mimetic amphiphile is selected from the group consisting of hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, -37- dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma- linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxo- cholanylglycine and alkali metal salts thereof, octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanol X-oteyl ether, wherein X is from 9 to 20, and cobinations thereof, and wherein the phospholipid is selected from the group consisting of, phospholipid GLA (glycolic, lactic acid), phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, unsaturated lecithin, saturated lecithin and lysolecithin, and combinations thereof, and wherein the amount of each membrane-mimetic amphiphile and phospholipid is present in a concentration of from 1 to 10 wt./wt. o of the total formulation, and the total concentration of membrane-mimetic amphiphiles and phospholipids is less than wt./wt. of the formulation, and wherein the phenolic compound is selected from the group consisting of phenol and methyl phenol in a concentration of from 1 to 10 wt./wt. of the total formulation.

27. A method according to claim 26, wherein the mixture is administered from a metered dose dispenser.

28. A method according to any one of claims 26 or 27, wherein the propellant is selected from the group consisting of tetrafluoroethane, tetrafluoropropane, dimethylfluoropropane, heptafluoropropane, dimethyl ether and n-butane.

29. A method according to any one of claims 26 to 28, wherein the intermediate formulation also includes a compound selected from glycerin, polyglycerin and mixtures thereof in an amount of from 1-40 wt./wt. of the intermediate formulation. -38- A method according to any one of claims 26 to 29, wherein the pharmaceutical agent is selected from the group consisting of insulin, heparin, so-called low molecular weight heparin, low molecular weight heparin, hirugen, hirulos, hirudine, interferons, interleukins, cytokines, mono and polyclonal antibodies, chemotherapeutic agents, vaccines, glycoproteins, bacterial toxoids, hormones, calcitonins, insulin like growth factors (IGF), glucagon like peptides (GLP-1 or GLP- large molecule antibiotics, protein based thrombolytic compounds, platelet inhibitors, DNA, RNA, gene therapeutics and antisense oligonucleotides.

31. A method according to any one of claims 26 to 30, wherein the pharmaceutical agent is insulin.

32. A method for administering a mixed liposome pharmaceutical formulation with multilamellar vesicles according to any one of claims 26 to 31, wherein the formulation is sprayed into the buccal cavity of a human being, without inhalation.

33. A method of administering an aerosol pharmaceutical formulation including spraying a predetermined amount of the aerosol mixed liposome pharmaceutical formulation of any one of claims 2 to 11 or 23, into the mouth with a metered dose spray device.

34. A mixed liposome pharmaceutical formulation with multilamellar vesicles comprising insulin, water, an alkali metal lauryl sulphate in a concentration of from 1 to 10 wt./wt.% of the total formulation, having dispersed therein trihydroxy-oxo- cholanylglycine and phospholipid GLA each present in a concentration of from 1 to wt./wt.% of the total formulation, wherein the total concentration of trihydroxy-oxo- cholanyiglycine and phospholipid GLA is less than 50 wt./wt/% of the formulation. A mixed liposome pharmaceutical formulation with multilamellar vesicles comprising insulin, water, an alkali metal lauryl sulphate in a concentration of from 1 to 10 wt./wt.% of the total formulation, having dispersed therein lauramidopropyl betain and saturated lecithin each present in a concentration of from 1 to 10 wt./wt.% of the total formulation, wherein the total concentration of lauramidopropyl betain and saturated lecithin is less than 50 wt./wt/% of the formulation. -7 -39-

36. A mixed liposome pharmaceutical formulation with multilamellar vesicles comprising insulin, water, an alkali metal lauryl sulphate in a concentration of from 1 to 10 wt./wt.% of the total formulation, having dispersed therein trihydroxy-oxo- cholanylglycine and sphingomyelin each present in a concentration of from 1 to wt./wt.% of the total formulation, wherein the total concentration of trihydroxy-oxo- cholanylglycine and sphingomyelin is less than 50 wt./wt/% of the formulation.

37. A process of forming a pharmaceutical formulation substantially as hereinbefore defined with reference to the accompanying examples 1, 2 and 6.

38. A mixed liposome pharmaceutical formulation substantially as hereinbefore defined with reference to the accompanying examples 1, 2 and 6. DATED THIS THIRTIETH DAY OF SEPTEMBER

2002. GENEREX PHARMACEUTICALS INC BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS