WO1994012154A1

WO1994012154A1 - Lipid containing formulation and method for its preparation

Info

Publication number: WO1994012154A1
Application number: PCT/GB1993/002393
Authority: WO
Inventors: Roger Randal Charles New
Original assignee: Cortecs Limited
Priority date: 1992-11-23
Filing date: 1993-11-22
Publication date: 1994-06-09
Also published as: GB9224502D0; AU5531494A

Abstract

A method of preparing a pharmaceutical formulation of an active substance which is insoluble in conventional solvents, comprising co-dissolving the active substance and an amphipathic lipid in an organic acid or acid anhydride and then removing or neutralising the acid or acid anhydride. The resulting formulations may be liposomal or alternatively may take the form of lipid suspensions in aqueous solution or lipid-water gels. Active substances such as albendazole, which are sparingly soluble in conventional solvents, are particularly suited to formulation by the method of the invention.

Description

LIPID CONTAINING FORMULATION AND METHOD FOR ITS PREPARATION

The present invention relates to lipid-containing formulations and to the preparation of these formulations. In particular, the invention relates to formulations of active substances which are poorly soluble both in water and in organic solvents and, in particular, liposome formulations of these active substances.

Liposomes provide a convenient means for the in vivo delivery of active agents, such as pharmaceuticals or nutritional substances. The various processes conventionally used for their preparation involve, as a first step, the dispersion or dissolution of lipid material in a solvent optionally together with the active agent (s). For example, one method described by Batzri and Korn in Biochim. Biophys . Acta 298 1015 (1973) comprises the solubilisation of lipids in ethanol, followed by the addition of the resulting solution to a large volume of water. A second method (Hauser and Gains, Pro . Natl . Acad. Sci 79 1683 (1982)) involves so- called "pH-induced vesiculation", whereby an aqueous dispersion of negatively-charged lipids is formed by mechanical means followed by the addition thereto of sodium hydroxide, to restore the solution to neutrality. A third example of a reported method (WO 85/00751) comprises the co-dissolution of lipid and the active agent to be encapsulated in a monophase mixture of an organic solvent and up to 20% water, followed by solvent removal.

A major problem encountered in these and other known processes for liposo e preparation is that the active agent which it is desired to encapsulate in the liposomes is frequently found to be insoluble in conventional solvents (organic or aqueous) such as are used to disperse or dissolve the constituent lipids. This limits the range of active agents which can be administered by means of liposomes. Conventional solvents, such as chloroform, also have the disadvantage of being toxic and yet difficult to remove completely from the liposomes which are produced.

The formulation of active agents which are insoluble in conventional solvents is a serious problem since not only is it hard to make liposomes using such substances but, indeed, it is extremely difficult to provide any formulation for insoluble active substances. The formulation for the administration of such insoluble active substances would therefore be an advantage in a wide variety of fields.

Moreover, it has surprisingly been found that certain organic acids can act as a convenient solvent for active agents which are insoluble in conventionally used organic and aqueous solvents and that lipid-containing formulations such as liposomes may be produced from solutions from these active agents in organic acids.

Therefore, in a first aspect of the present invention, there is provided a process for the preparation of a lipid-containing formulation, the method comprising co- dissolving an amphipathic lipid and an active agent in a liquid organic acid or a liquid organic acid anhydride; and subsequently neutralising or removing the organic acid or anhydride. The choice of amphipathic lipid will depend on the type of product formulation which is required. The formulation may be in the form of liposomes, in which case, at least one amphipathic liposome-forming liquid must be present.

Examples of amphipathic, liposome-forming lipids include phospholipids, including phosphatidic acid, phosphatidyl cholines (PC's), sphingomyelin, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl serine, cardiolipin, and natural lecithins such as those derived from egg yolk or soya bean extract. Other liposome-forming lipids are well known to those skilled in the art.

Alternatively, the formulation may be a lipid suspension in aqueous solution, for example an emulsion or a micellar dispersion. A third possibility is that the formulation is a lipid-water gel. The liposome-forming amphipathic lipids listed above may be included in such formulations but, alternatively, amphipathic non-liposome forming lipids can be used.

Examples of amphipathic, non-liposome-forming lipids include cholesterol and esters and derivatives thereof including other steroids such as ergosterol, fatty acids

(both saturated and unsaturated) , hydroxy fatty acids, lyso-phospholipids and tocopherols and derivatives thereof and also polyoxyethylated glycol monoethers, polyoxyethylated fatty acids and polyoxyethylated sorbitan fatty esters or polysorbates, such as those sold under the trade mark TWEEN. The polyoxyethylated compounds, when combined with phospholipids, are particularly useful in the formation of gels. Other non- liposome-forming lipids include those sold under the trade marks BRIJ and TRITON X100.

The active agent must be soluble in the organic acid or anhydride and may be a pharmaceutical, a nutritional substance, an immunological agent, a cosmetic substance, an agrochemical, a biocide or a substance for inclusion in a paint composition.

Although the method is suitable for all active agents, including those which can be conventionally formulated, in liposomes or otherwise,^' it is particularly suitable for active agents which are insoluble or sparingly soluble in conventional organic or inorganic solvents.

In many cases, therefore, the active substance will insoluble or sparingly soluble in solvents such as water, halogenated or non-halogenated alkanes, alcohols, cyclic ethers such as tetrahydrofuran (THF) or dioxane, carbonyl or thiocarbonyl containing solvents such as dimethyl- sulphoxide (DMSO) and dimethylformamide (DMF) and aromatic solvents such as pyridine.

Examples of active agents which are particularly suitable for use in the process- of the present invention include pharmaceutical substances such as benzimidazoles, for examplealbendazole (methyl-5-propylthio-lH-benzimidazol- 2-yl-carbamate) ; polyene antibiotics such as amphotericin B; carotenoids; proteins and peptides for example peptide hormones such as insulin; antigens and polyamino acid conjugates.

Albendazole is a benzimidazole pharmaceutical used in the treatment of systemic and intestinal infection by helminths, for example in the treatment of hydatid disease. It is difficult to achieve effective blood concentrations of albendazole either by direct intravenous injection, because of the drugs limited solubility, or by oral administration, because of its poor penetration of the gut. In addition, it has, so far, been difficult to administer albendazole in a liposome formulation because of the limited solubility of the drug in conventional aqueous and organic solvents. However, the method of the present invention now makes a liposome formulation of albendazole possible.

As used herein, the term "organic acid" refers to a compound having a melting point not greater than 100°C and containing at least one carbon atom and at least one acid moiety. Acid moieties are well known to those skilled in the art and examples include carboxylic (C0₂H) , phosphoric (PO(OH))₂, phosphinic (P(OH)₂), sulphonic (S0₃H) and sulphinic (SO,H) acids.

The term "organic acid anhydride" refers to the anhydride of an organic acid as defined above.

It is preferred that the organic acid is substantially anhydrous to avoid hydrolysis of the lipids and this is particularly important when the amphipathic lipid is cholesterol or a derivative thereof. If water is present in the acid it is preferably present at no more than 10% by weight, more preferably no more than 5% by weight and even more preferably no more than 1% by weight. Most preferably the acid should be as dry as possible and contain no more than 0.1% by weight of water. The acid may suitably be dried over zeolitic molecular sieves. The acid is used in an amount sufficient to dissolve the active ingredient. Preferred acids for use in the invention are mono- or di- carboxylic acids. The organic acid may contain from 1 to 20 carbon atoms, although acids having from 1 to 10 carbon atoms are preferred and most preferably, the acid has from 1 to 4 carbon atoms.

The carbon chain may be either substituted or unsubstituted and suitable substituents include halogen, particularly chlorine or fluorine, and hydroxyl groups. As mentioned above, carboxylic acids are particularly suitable for use in the present invention and the carboxylic acid may be one whose alkali metal salts are soluble in water, particularly if a liposome suspension is to be formed. On the other hand, if a gel is required, it is preferable to use an acid having poorly water-soluble salts, for example with alkaline earth metals. Examples of suitable organic acids include monocarboxylic acids, such as glacial acetic acid and formic acid, although formic acid is less preferred because it cannot be obtained very dry and because it tends to act as a reducing agent towards the lipid material. Derivatives and longer chain homologues of these monocarboxylic acids are also suitable; examples include longer chain acids, homologues such as propionic acid, butyric acid and isobutyric acid, derivatives of monocarboxylic acids, such as trichloroacetic acid, trifluoroacetic acid and phenyl acetic acid, hydroxylated derivatives of monocarboxylic acids such as glycolic acid and D,L-lactic acid and 4-hydroxybutyric acid. Dicarboxylic acids and derivatives thereof, such as glutaric acid and malic acid, are also suitable for use in the process of the present invention.

When an acid anhydride is used, this should be a liquid and be readily hydrolysable. Suitable examples include maleic anhydride and citraconic anhydride.

In the first step of the process of the present invention, the organic solvent or anhydride is in liquid form so that it can act as a solvent for the amphipathic lipid and the active agent. Therefore, the process will be conducted at a temperature of the melting point of the organic acid or anhydride if it is not liquid at room temperature (20 to 25°). However, elevated temperatures which would be deleterious to the active agent and/or the lipid over the course of the dissolution step should be avoided and, therefore, the dissolution step will preferably be conducted at not more than 100°C. More preferably, the dissolution temperature will be up to 90°C and, most preferably, up to 60°C.

The active agent and any lipids may be added to the organic acid or anhydride simultaneously or sequentially, and, if necessary, heated to form the requisite solution. The solution formed by co-dissolution step may be a clear solution or it may, instead, include dispersed lipid material present in its own saturated solution.

When cholesterol and esters and derivatives thereof are used it is, as already briefly mentioned, usually necessary for the organic acid used to be substantially free of traces of water. Preferably the cholesterol and phospholipid are co-dried from solution in an organic solvent, for example onto the surface of a vessel, and the organic acid then added. In addition, dissolution of cholesterol and esters and derivatives thereof is also helped by warming the acid to about 60°C. After co-dissolving the amphipathic lipid and the active agent in the liquid organic acid or anhydride, it is then necessary either to neutralise or remove the acid or anhydride.

The acid or anhydride may either be partially or completely neutralised or, alternatively, may be removed, for example by lyophilisation.

Neutralisation is typically achieved by the addition of a basic substance so that the final pH ranges from acidic to slightly alkaline, typically in the range from pH 5 to 8, preferably pH from 5 to 7. The particular pH chosen depends upon the stability of the active agent and of the lipid formulation. A final pH of greater than about 9 prejudices the integrity of liposomes and a slightly acidic pH is preferred for liposomal formulations.

The basic substance may be an aqueous alkaline solution of, for example, a hydroxide or a carbonate of an alkali metal or an alkaline earth metal or ammonium. Aqueous sodium hydroxide and ammonium hydroxide are examples of suitable alkaline solutions. Solid hydroxides or carbonates of alkali metals or alkaline earth metals may also be used, as may ammonium carbonate. A preferred example of a solid carbonate is finely divided sodium carbonate decahydrate (Na₂C0₃.10H₂O) . Addition of the basic substance may, if required, be followed by dialysis to remove the salt which forms uoon neutralisation.

The choice of base may determine the physical form of the lipid formulation obtained. When the acid is one which forms soluble alkali metal salts, use of an alkali metal hydroxide or ammonium hydroxide will lead to the formation of liposomes. When an alkaline earth metal base is used to neutralise an acid which has poorly soluble alkaline earth metal salts, a gel will be formed.

The pH is controlled either by using a standard pH electrode or, particularly when the process is conducted on a small scale, by adding a suitable indicator to the alkaline solution and monitoring the colour change. Xylenol blue is one example of a suitable indicator. Especially in the case of forming liposomes, the neutralisation step will be monitored to avoid accidental addition of excess base resulting in undesirably high final pH.

When lyophilisation is employed the organic acid is removed without there usually being the need for subsequent treatment to neutralise the acid. Once the solution of lipid and active agent in organic acid has been lyophilised, it may suitably be hydrated in aqueous solution.

The formulation may be exposed to other conventional procedures either before or after dialysis, or after lyophilisation, to alter the size characteristics of liposomes or to break up non-liposomal particles for instance. Such procedures include sonication, microfluidisation and membrane extrusion.

Formulations prepared according to the process of the invention and containing active substances which are insoluble in conventional solvents are also new and, therefore, in a second aspect of the invention there is provided a formulation comprising an active substance which is insoluble or sparingly soluble in organic or aqueous solvents and which is obtainable by a process according to the first aspect.

The formulation of the invention may also include other ingredients, for example additional lipids may be present to modify the form or size or physical properties of the lipid-containing domains of the formulations. Examples of further types of lipid include non-polar lipids and lipids containing polar or hydrophilic head groups which include functional groups such as phosphate, phosphonate, sulphate, carboxyl, amino pyridinium, hydroxyl or sulphydryl groups.

Preferred amphipathic lipids and active agents are as mentioned above for the first aspect. However, a particularly preferred type of active agent is a benzimidazol such as albendazole. Such formulations are particularly useful in the treatment of systemic and intestinal infection by helminths, for example in the treatment of hydatid disease.

In formulations containing albendazole, the weight ratio of lipid to albendazole may range from 99:1 to 1:99, for example from 75:25 to 25:75 or from 60:40 to 40:60. A ratio from 5:1 to 1:1, especially 3:1, is preferred. Liposomes are formed with a lipid:albendazole weight ratio of from 2:1 to 99:1. Lipid complexes are formed with a lipid:albendazole weight ratio of from 2:1 to 1:99. Ratios for formulations containing other active agents will generally be similar to the above.

There is also provided a method of medical treatment, which comprises administering to a host suffering from or exposed to the risk of, systemic or intestinal infection by helminths, for example hydatid disease, an effective non-toxic dose of a lipid-containing formulation comprising a lipid material and albendazole. The formulation is typically administered orally, in an amount which provides a conventional pharmaceutically effective dosage, typically about lOmg per kg body weight, of albendazole per day.

The formulation may be a lipid suspension in an aqueous solution or a lipid-water gel, but it is preferred the formulation takes the form of liposomes.

Formulations produced in accordance with the invention may be formulated for administration by any route, for example in suspension or as a gel suitable for oral, intravenous or topical administration. Depending on the intended application, they are typically formulated for administration together with one or more pharmaceutically or cosmetically acceptable carriers or diluents.

Preferred carriers and diluents will be selected according to the route of administration. For oral administration, the carriers and diluents may be liquids or solids and the lipid-containing formulations may be formulated as a solution, suspension or mixture. For parenteral administration the carrier or diluent will generally be a sterile, pyrogen-free liquid, preferably water for injection. The carrier or diluent may optionally include accessory ingredients such as preservatives, anti-oxidants, flavouring agents, colouring agents, suspending agents, buffers and isotonic agents. For topical administration the carrier or diluent will be a suitable solvent, cream or lotion base and the formulation may further comprise optional accessory ingredients such as preservatives, antioxidants, suspending agents, and buffers.

When the invention is applied to an active agent which has limited solubility in conventional aqueous or organic solvents, administration to a host of the lipid- containing formulation thus produced may provide a means of increasing the bioavailability of that particular active agent as compared with conventional means of administration.

The invention will be further illustrated by the following Examples.

EXAMPLE 1

Preparation of albendazole liposomes using glacial acetic acid

Egg phosphatidyl choline (Lucas Meyer Ovothin 200, 20mg) was dissolved in glacial acetic acid (200μl) at room temperature, with shaking. Albendazole (up to lOmg) was then dissolved in the resulting solution.

Once the solid had all dissolved, sodium hydroxide (2M, 1.6ml) was introduced, with mixing, to yield a fluid milky suspension. The pH of the resulting suspension was monitored using xylenol blue as indicator, and found to be close to neutrality.

The suspension was dialysed overnight at 4°C against physiological saline (2 litres) to remove sodium acetate, it being ensured that both internal and external compartments of the dialysis chamber were well mixed. EXAMPLE 2

Preparation of cholesterol-containing liposomes using glacial acetic acid

Egg phosphatidyl choline (Lucas Meyer Ovothin 200, 20mg) was dissolved in anhydrous glacial acetic acid (20μl) in a glass round-bottomed test tube. Cholesterol, which is insoluble in glacial acetic acid alone, (lOmg) was added to the solution and mixed well until it dissolved.

Sodium hydroxide (10M, 320μl) was then added to the solution, with mixing, to yield a firm white gel. Distilled water (2ml) was then added to the gel with mixing, which gave a milky suspension.

the suspension was dialysed overnight as described in Example 1.

EXAMPLE 3

Preparation of albendazole liposomes using formic acid Alb'endazole (lOmg) was dissolved in 200ml formic acid

(90%, approx, 23.5M) in a round-bottomed 10ml glass test tube. Solid egg yolk phosphatidyl choline (Lucas Meyer

Ovothin 200, 25mg) was dissolved in the resulting solution.

Sodium hydroxide (2M, 2.5ml) was added rapidly to the solution, to yield a homogeneous suspension of which the pH was 5.5. The pH was brought to 7.3 by the addition of a further two drops of sodium hydroxide (2N) . The suspension was dialysed overnight as described in Example 1. EXAMPLE 4

Preparation of saturated phosphatidyl choline liposomes Anhydrous glacial acetic acid (250μl) was added to hydrogenated soya phosphatidyl choline (Lucas Meyer Epicuron 200H, 25mg) . The two components were mixed well to form a homogeneous paste.

Sodium hydroxide (10M, 0.4ml) was added to the paste with rapid mixing, followed by distilled water (1ml) to yield a homogeneous milky suspension.

EXAMPLE 5

In vivo testing of liposomes

Liposomes containing albendazole, prepared as described in Example 1 and sonicated briefly to give a non- sedimenting suspension, were administered orally to a group of rats at a dose of 0.lmg/animal. The same dosage of free albendazole was administered orally to a group of control rats. Blood concentrations were measured at intervals.

The results obtained are shown in Table 1.

TABLE 1

Note: [Als] = blood concentration of Albendazole sulphoxide sd = standard deviation se = standard error

The results show significant differences in the blood concentrations of the chief metabolite albendazole sulphoxide as between the two groups of rats, indicating that the in vivo delivery of albendazole via liposomes increases the bioavailability of the drug.

Claims

1. A process for the preparation of a lipid-containing formulation, the method comprising co-dissolving an amphipathic lipid and an active agent in a liquid organic acid or a liquid organic anhydride; and subsequently neutralising or removing the organic acid or anhydride.

2. A process as claimed in claim 1, wherein the amphipathic lipid is a liposome-forming lipid.

3. A process as claimed in claim 2, wherein the lipid is a phospholipid such as phosphatidic acid, phosphatidyl choline, sphingomyelin, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl serine, cardiolipin or a natural lecithin.

4. A process as claimed in claim 1, wherein the lipid is a non-liposome-forming lipid.

5. A process as claimed in claim 4, wherein the lipid comprises cholesterol or an ester or derivative thereof, ergosterol, a saturated or unsaturated fatty acid, a hydroxy fatty acid, a lysophospholipid, tocopherol or a derivative of one of these.

6. A process as claimed in any one of claims 1 to 5, wherein the active agent is a pharmaceutical, nutritional substance, an immunological agent, a cosmetic substance, an agrochemical, a biocide or a substance for inclusion in a paint composition.

7. A process as claimed in any one of claims 1 to 6, wherein the active agent is insoluble or sparingly soluble in water, halogenated or non-halogenated alkanes, alcohols, cyclic ethers such as tetrahydrofuran (THF) or dioxane, carbonyl or thiocarbonyl containing solvents such as dimethylsulphoxide (DMSO) and dimethylfor amide (DMF) and aromatic solvents such as pyridine.

8. A process as claimed in any one of claims 1 to 7, wherein the active agent is a benzimidazole such as albendazole, a polyene antibiotic such as amphotericin B, a carotenoid, a protein or peptide such as insulin, an antigen or a polyamino acid conjugate.

9. A process as claimed in any one of claims 1 to 8, wherein the organic acid is a carboxylic, phosphoric, phosphinic, sulphonic or sulphinic acid.

10. A process as claimed in any one of claims 1 to 9, wherein the organic acid comprises glacial acetic acid, formic acid, propionic acid, butyric acid, isobutyric acid, trichloroacetic acid, trifluoroacetic acid, phenylacetic acid, glycolic acid, DL-lactic acid, 4- hydroxybutyric acid, glutaric acid or maleic acid.

11. A process as claimed in claim 10, wherein the acid anhydride is maleic anhydride or citraconic anhydride.

12. A process as claimed in any one of claims 1 to 11, wherein the organic acid or anhydride contains no more than 0.1% by weight of water.

13. A process as claimed in any one of claims 1 to 12, wherein the acid or anhydride is removed by lyophilisation.

14. A process as claimed in any one of claims 1 to 12, wherein the acid or anhydride is partially or completely neutralised.

15. A process as claimed in claim 13, wherein the neutralisation is achieved by the addition of a basic substance such as an aqueous solution of a hydroxide or carbonate of an alkali metal, an alkaline earth metal or ammonium.

16. A formulation comprising an active substance which is insoluble in conventional solvents, the formulation being obtainable by a process as claimed in any one of claims 1 to 15.

17. A formulation as claimed in claim 16, which is a liposomal formulation.

18. A formulation as claimed in claim 16 which is a lip'id suspension in aqueous solution of a lipid-water gel.

19. A formulation as claimed in any one of claims 16 to 18, wherein the active substance is albendazole.

20. A composition as claimed in claim 19 for use in the treatment or prophylaxis of systemic or intestinal infection by helminths.

21. The use of a formulation as claimed in claim 19 in the preparation of an agent for the treatment or prophylaxis of systemic or intestinal infection by helminths.

22. A method for the treatment or prophylaxis of systemic or intestinal infection by helminths, the method comprising administering to a patient an effective amount of a formulation as claimed in claim 19.