WO2008040799A2

WO2008040799A2 - Process for preparing instant forms of aqueous mixed micellar solutions as physiological buffer systems for use in the analysis of in vitro release

Info

Publication number: WO2008040799A2
Application number: PCT/EP2007/060592
Authority: WO
Inventors: Julia Boni; Jennifer Dressman; Martin Wunderlich; Rolf-Stefan Brickl
Original assignee: Boehringer Ingelheim International Gmbh; Boehringer Ingelheim Pharma Gmbh & Co. Kg
Priority date: 2006-10-06
Filing date: 2007-10-05
Publication date: 2008-04-10
Also published as: WO2008040799A3

Abstract

The invention relates to a process for preparing instant forms of aqueous mixed micellar solutions containing phospholipid and non-phospholipid components, particularly FaSSIF Instant, FeSSIF Instant and FeSSIFplus Instant, which are sufficiently stable for storage, comprising preparing a concentrated aqueous mixed micellar solution and subsequently eliminating the solvent under reduced pressure, by lyophilisation or spray drying, using no organic solvent in any of the process steps. The redispersed aqueous mixed micellar solutions are needed particularly for use in the analysis of in vitro release as physiological buffer systems for predicting the in vivo resorption of pharmaceutical active substances.

Description

Process for preparing instant forms of aqueous mixed micellar solutions as physiological buffer systems for use in the analysis of in vitro release

The invention relates to a process for preparing instant forms of aqueous mixed micellar solutions which are stable enough for storage, wherein the mixed micelles comprise

(A) at least one phospholipid,

(B) one or more non-phospholipid substances selected from among bile acids, bile acid salts, bile acid derivatives and salts of bile acid derivatives, as well as

(C) optionally one or more non-phospholipid substances selected from among triacylglycerides and the breakdown products thereof,

and the molar ratio of the components (A) : (B) is from 0.1 :1 to 1 :1 and optionally the molar ratio of the components (C) : (B) is from 0.1 :1 to 6:1 ,

wherein the process comprises the preparation of a concentrated aqueous mixed micellar solution and subsequent elimination of the solvent under reduced pressure, by lyophilisation or spray drying, and the use of organic solvents is ruled out.

Even when the present invention relates to mixed micellar solutions these are generally physically dispersions or emulsions.

Moreover the invention relates to the instant forms of aqueous mixed micellar solutions that are stable enough for storage and to a process for preparing ready-to- use aqueous mixed micellar solutions, characterised by the addition of water or aqueous buffer to the instant form according to the invention. The process according to the invention is thus suitable for preparing a storable mixed micellar formulation for the rapid and reproducible preparation of aqueous mixed micellar solutions for use as release media. Aqueous mixed micellar solutions of this kind are required particularly for use in the analysis of in vitro release as physiological buffer systems for predicting the in vivo resorption of pharmaceutical active substances. In another aspect the present invention therefore relates to the use of the ready-to-use mixed micellar solution redispersed from an instant form, as a biorelevant medium or as a physiological buffer system in the analysis of the in vitro release of pharmaceutical active substances.

Background to the invention

Liposomal systems which constitute pure lipid dispersions, and dispersed mixed micelles which are formed by the addition of bile salts to liposomal systems and their use in pharmaceutical formulations have long been known. ^{(1 )}' ⁽²⁾' ⁽³⁾' ⁽⁴⁾. In the pharmaceutical, cosmetic and food supplement industries, in particular, phospholipid mixed micellar systems are used in all kinds of applications, e.g. for the manufacture and analysis of drugs. The systems are characterised in that they form mixed micelles (Ref: Shankland W 1970. The equilibrium and structure of lecithin-cholate mixed micelles. Chemistry and Physics of Lipids 4:109-130) which may contain an active substance in dissolved, dispersed, suspended or emulsified form depending on the end use envisaged. Examples of such systems are pharmaceutical formulations such as Konakion^® MM and Valium^® MM. Further examples are the mixed micellar solutions FaSSIF and FeSSIF⁽¹⁾' ⁽²⁾' ⁽³⁾ known from the literature and used in pharmaceutical analysis.

Processes for preparing aqueous mixed micellar solutions are described in the patent literature, for example in DE 27 30 570 A1 , WO 90/08534, DE 39 38 030 A1 , EP 0 730 860 and EP 0 252 004

According to the known methods, either all the constituents are dissolved in an organic solvent, then by elimination of the solvent under reduced pressure in the rotary evaporator a lipid film is produced and by the addition of aqueous buffer solution the mixed micellar dispersion is produced or a mixture of water and a water- miscible organic solvent is used in which at least some of the components are initially present in dissolved or emulsified form, and then the mixed micellar dispersion is obtained by elimination of the organic solvent. Another alternative method of preparation described in the prior art consists of spray drying the constituents dissolved in an organic solvent and producing the mixed micellar dispersion by the addition of aqueous buffer solution.

These processes are laborious and highly time-consuming, on the one hand, or have the disadvantage that the mixed micellar dispersion is not obtained free from organic solvents, which is a particular disadvantage for pharmaceutical use or for use as a biorelevant medium.

On the one hand aqueous mixed micellar solutions are used to improve the formulation qualities and resorption of substances, and on the other hand they may be used as media in the analysis of the release of different formulations in vitro, e.g. for the investigation of food effects. In vitro diagnosis is a tool for improving formulations under development and should be the best possible predictor of in vivo releases. Therefore it makes sense to simulate physiological in vivo conditions using a simplified media model as effectively as possible. Known models, such as the above-mentioned biorelevant media FaSSIF and FeSSIF^{(1) (2) (3)}, simulate the physiological state with regard to the pH, the osmolality, the buffer capacity and the concentrations of lecithin and bile salts. Lecithin and bile salts form mixed micelles and may thus act as solubilisers to improve the release of substances (Ref: Alkan- Onyuksel H, Ramakrishnan S, Chai HB, Pezzuto JM 1994. A mixed micellar formulation suitable for the parenteral administration of taxol. Pharmaceutical Research 11 :206-212).

FaSSIF is a model for the pre-prandial small intestine or for the conditions prevailing therein. Because of the major fluctuations between individuals, the composition is given in ranges (Ref.: Kalantzi L, Goumas K, Kalioras V, Abrahamsson B, Dressman JB, Reppas C 2006. Characterization of the human upper gastrointestinal contents under conditions simulating bioavailability/bioequivalence studies. Pharmaceutical Research 23: 165-176):

FaSSIF bile salt 1 -5 mM (preferably 2 - 3.5 mM) lecithin 0.2 - 1.3 mM (preferably 0.5 - 1 mM) buffer substance phosphate or maleate buffer capacity 3 - 15 mmol/l/ΔpH (preferably 7.5 - 12.5 mmol/l/ΔpH)

PH 5 - 7 (preferably 6 - 7) osmolality 100 - 300 mosmol/kg (preferably 200 - 300 mosmol/kg)

NaCI 100 - 300 mM (preferably 100 - 150 mM)

The contents of bile salts and lecithin are small, the buffer capacity relatively poor. The osmolality is adjusted using NaCI, KCI or CaCI₂.

For example FaSSIF has the following preferred composition:

FaSSIF sodium taurocholate 3 mM lecithin 0.75 mM

NaCI 106 mM

NaH₂PO₄ 29 mM

PH 6.5 osmolality 270 mosmol/kg

The preferred composition of FaSSIF contains a phosphate buffer (pH 6.5, buffer capacity 10 mEq per litre and pH unit) instead of the natural bicarbonate buffer, to avoid pH instabilities. The content of bile salts and lecithin is typical of the pre- prandial state. The medium is made isoosmolar with NaCI; instead of NaCI it is also possible to use equimolar amounts of KCI or CaCI₂.

FeSSIF constitutes a model of the postprandial small intestine or the conditions prevailing therein. Because of the major fluctuations between individuals the composition is given in ranges (time of measurements: approx. 120 min after taking food; Ref.: Kalantzi L, Goumas K, Kalioras V, Abrahamsson B, Dressman JB, Reppas C 2006. Characterization of the human upper gastrointestinal contents under conditions simulating bioavailability/bioequivalence studies. Pharmaceutical Research 23:165-176):

FeSSIF bile salt 3.5 - 20 mM (preferably 7.5 - 15 mM) lecithin 0.5 - 4 mM (preferably 2 - 4 mM) buffer substance acetate or citrate buffer capacity 10 - 35 mmol/l/ΔpH (preferably 25 - 35 mmol/l/ΔpH) pH 4 - 7 (preferably 5 - 6.5) osmolality 270 - 720 mosmol/kg (preferably 600 - 700 mosmol/kg)

NaCI 150 - 600 mM (preferably 180 - 220 mM)

The contents of bile salts and lecithin are increased by the emptying of the gall bladder, while the buffer capacity is greatly increased. The osmolality is adjusted using NaCI, KCI or CaCI₂.

For example FeSSIF has the following preferred composition:

FeSSIF sodium taurocholate 15 mM lecithin 3.75 mM

NaCI 203 mM sodium acetate 144 mM

PH 5.0 osmolality 670 mosmol/kg

The preferred composition of FeSSIF contains an acetate buffer (pH 5.0, buffer capacity 75 mEq per litre and pH unit). The content of bile salts and lecithin as well as the buffer capacity are significantly increased compared with FaSSIF and reflect the contributions of the food eaten and the secretions. The osmolarity may be adjusted using NaCI or an equimolar amount of KCI or CaC^; this is somewhat higher, compared with FaSSIF.

To obtain a better simulation of the food eaten, dietary fats (triacylglycerides) and the breakdown products thereof may also be added to FeSSIF, which is hereinafter referred to as FeSSIFplus:

FeSSIFplus bile salt 3.5 - 14.5 mM (preferably 5 - 10 mM) lecithin 0.5 - 4 mM (preferably 1.5 - 3 mM) glycerol monooleate 1 - 6.5 mM (preferably 4 - 6 mM) sodium oleate 10 - 40 mM (preferably 25 - 35 mM) buffer substance phosphate or maleate buffer capacity 5 - 40 mmol/l/ΔpH (preferably 10 - 30 mmol/l/ΔpH) pH 5 - 7 (preferably 5.5 - 6) osmolality 270 - 520 mosmol/kg (preferably 350 - 430 mosmol/kg)

NaCI 100 - 600 mM (preferably 125 - 200 mM)

For example FeSSIFplus has the following preferred composition:

FeSSIFplus sodium taurocholate 7.5 mM lecithin 2 mM glycerol monooleate 5 mM sodium oleate 3O mM

NaCI 15O mM sodium citrate 35 mM

PH 5.8 osmolality 390 mosmol/kg

The preferred composition of FeSSIFplus contains a citrate buffer (pH 5.8, buffer capacity 25 mEq per litre and pH unit). The content of bile salts and lecithin as well as the buffer capacity are lower compared with FeSSIF, as the medium simulates the postprandial state approx. 2 - 3 hours after eating and at this stage some of the food eaten and electrolytes have already been resorbed. The osmolarity may be adjusted using NaCI or an equimolar amount of KCI or CaC^; this is somewhat higher, compared with FaSSIF, and somewhat lower compared with FeSSIF.

The preparation of the biorelevant media, e.g. FaSSIF, FeSSIF and FeSSIFplus, using the methods described in the prior art is time-consuming and not always reproducible. To prepare the biorelevant media FaSSIF, FeSSIF and FeSSIFplus, first of all the non-phospholipid substance (B), here usually the sodium salt of taurocholic acid, is dissolved in aqueous buffer. Then the phospholipid substance, here usually egg lecithin, dissolved in dichloromethane is added, the organic solvent is eliminated in the rotary evaporator, optionally the non-phospholipid substances (C), here usually glycerolmonooleate and the sodium salt of oleic acid, which have also been dissolved in dichloromethane, are added and the organic solvent is again eliminated in the rotary evaporator. The mixed micellar solution is then diluted to the desired final concentration using aqueous buffer.

The disadvantages of this method of preparing aqueous mixed micellar solutions are the large amount of time taken, the poor reproducibility with regard to the micelle size and particularly the poor storage stability of the media or aqueous mixed micellar solutions or biorelevant media of not more than one day, which demands that the preparation be made freshly as required, possibly every day.

The aim of the present invention was therefore to prepare a storage-stable precursor of aqueous mixed micellar solutions as an "instant form", particularly FaSSIF Instant, FeSSIF Instant and FeSSIFplus Instant, from which the ready-to-use aqueous mixed micellar solutions, such as the biorelevant release media, e.g. FaSSIF, FeSSIF and FeSSIFplus, can be produced as necessary within a few minutes by the addition of water or aqueous buffer solution. Brief summary of the invention

In a first aspect the invention relates to a process for preparing a storable instant form of an aqueous mixed micellar solution or mixed micellar dispersion comprising the steps of

(i) preparing a concentrated aqueous mixed micellar solution comprising

(A) at least one phospholipid,

(B) one or more non-phospholipid substances selected from among bile acid, bile acid salts, bile acid derivatives and salts of a bile acid derivative as well as

(C) optionally one or more non-phospholipid substances selected from among triacylglycerides and the breakdown products thereof, wherein

the molar ratio of the components (A) : (B) is from 0.1 :1 to 1 :1 and the molar ratio of the components (C) : (B) is from 0.1 :1 to 6:1 ,

and

(ii) subsequently eliminating the solvent under reduced pressure, by lyophilisation or spray drying, using no organic solvents in any of the process steps.

In a second aspect the invention relates to a process for preparing ready-to-use aqueous mixed micellar solutions, characterised in that in another step

(iii) water or aqueous buffer is added to the instant form obtained after carrying out steps (i) and (ii) and in this way the ready-to-use aqueous mixed micellar solution is reconstituted. In a third aspect the invention relates to an instant form of an aqueous mixed micellar solution free from organic solvents and substantially anhydrous, which is sufficiently stable for storage, comprising

(A) at least one phospholipid,

obtainable according to the process described as the first aspect of the invention.

In a fourth aspect the present invention relates to the use of the ready-to-use mixed micellar solution redispersed from an instant form according to the invention, as a biorelevant medium or as a physiological buffer system in the analysis of the in vitro release of pharmaceutical active substances.

Detailed description of the invention

In a preferred embodiment of the process corresponding to the first aspect of the invention,

in step (i) the non-phospholipid component (B) selected from among bile acid, bile acid salts, bile acid derivatives and salts of a bile acid derivative is dissolved or suspended in water or an optionally buffered aqueous solution, at a temperature between 0 and 50⁰C, preferably between 20 and 40⁰C, the phospholipid component (A) and optionally the non-phospholipid component (C) is either added directly to the solution or suspension containing the component (B) and dispersed therein, at a temperature between 0 and 50⁰C, preferably between 20 and 40⁰C, or however dispersed separately in water or an optionally buffered aqueous solution and the aqueous systems thus obtained containing component (A), (B) and optionally (C) are combined,

then the resulting suspension is homogenised, for example by ultrasound and/or high pressure filtration, while if both methods of homogenisation are used the high pressure filtration is preferably carried out after the ultrasound treatment. For the high pressure filtration a pore size of between 0.2 and 0.5 μm is expediently chosen. A particle size distribution in the nm range is obtained with a high degree of reproducibility.

Step (i) is preferably carried out at a temperature of for example 10 to 40°C, e.g. at 15 to 25°C, but particularly without any external introduction of heat. Preferably the non-phospholipid component (B) is first of all dissolved or suspended in water or aqueous buffer solution in a concentration of 1 - 95 % (m/V) and particularly in a concentration of 10 - 60 % (m/V).

The phospholipid component (A) and optionally the non-phospholipid component (C) is then preferably added to the solution or suspension containing component (B) and emulsified with stirring, for example in a concentration of 0.5 - 35 % (m/V) and particularly in a concentration of 5 - 20 %.

The molar ratio of components (A) : (B) with regard to all aspects of the invention is 0.1 :1 to 1 :1 , preferably 0.2:1 to 0.4:1 , but particularly preferably about 0.25:1. The molar ratio of the optionally added component (C) : (B) is 0.1 :1 to 6:1 , preferably 1 :1 to 5:1 , but particularly preferably about 4:1 , with regard to all aspects of the invention.

The solvents used may be aqueous buffer solutions with added salt, but pure water is preferably used. The buffer substances used are for example the following acids and their associated salts as well as mixtures thereof: phosphoric acid, acetic acid, citric acid, malic acid, oxalic acid, succinic acid, fumaric acid, tartaric acid, phthalic acid, boric acid, malonic acid, glutaric acid, glutamic acid and aspartic acid. The salts used to adjust the osmolality are NaCI, KCI or CaC^. The pH values of the solvents are between 4-8, preferably between 5-7. If aqueous buffer and salt solutions are used, both buffer substance and also salt are dissolved before the addition of the non-phospholipid substance.

Surprisingly it has been found that after components (A) and (B) and optionally (C) have been combined in step (i), a clear solution or dispersion is formed within a few hours with stirring at ambient temperature. By treating with ultrasound the time taken for clarification can be shortened to about 30 min and in addition a high reproducibility can be achieved in terms of the particle size. Further improvement to the reproducibility is achieved by subsequent filtration, e.g. high pressure filtration through a 0.45 μm or 0.2 μm filter.

Preferably the elimination of the solvent in step (ii) is carried out by lyophilisation or spray drying, particularly preferably by lyophilisation, thus rendering the mixed micelles suitable for storage.

The dispersion obtained in step (i) of the process according to the invention may then be lyophilised gently in the freeze-dryer without the addition of further excipients. The freeze-drying may for example be carried out by freezing the dispersion to -40⁰C (1 K/min), subsequent main drying at 0 - 20⁰C and 0.28 mbar and after-drying at 30°C and 0.01 mbar. For this purpose the dispersion is placed in metered amounts for example in dishes as a bulk material or in vials or blisters for later use, frozen and the water is eliminated in vacuo. The advantage of this process is the low stress to which the mixed micelles are subjected during drying. After drying is complete, the lyophilisate may be sealed directly under a nitrogen gas current and this results in an instant form which is stable (particularly with regard to the hydrolysis of the phospholipid components) and storable as the precursor of an aqueous mixed micellar solution. Another possible method of eliminating the solvent is spray drying, in which the solvent water is removed by spray drying from the dispersion obtained according to step (i). Both the pure aqueous mixed micellar solution and also the solution after the addition of buffer can be dried by the spray-drying method. The spray drying may for example be carried out at an entry temperature of 60 to 130⁰C, preferably 70 to 110⁰C, particularly preferably about 80°C, and an exit temperature of 40 to 90⁰C, preferably 50 to 80°C, particularly preferably about 70°C.

Suitable phospholipid components (A) for the purposes of the invention are

pure phosphatidylcholine or

a mixture of phospholipids of natural origin, for example egg lecithin or soya lecithin with a high concentration of phosphatidylcholine, preferably at least 80%, but particularly preferably at least 96% based on the dry mass, or

phosphatidylcholines of general formula

wherein R¹ and R², which may be identical or different, represent saturated and mono- or polyunsaturated straight-chain fatty acid groups with 14 to 18 C atoms, preferably with 16 to 18 C atoms. Examples of R¹ and R² are the myristoyl, palmitoyl, stearoyl, oleoyl and elaidoyl group. Suitable non-phospholipid components (B) for the purposes of the invention are derivatives of 5β-cholanic-24-acid of general formula

wherein R³ and R⁴ each independently of one another denote a hydroxy group or a hydrogen atom and R⁵ denotes a hydroxy group, the taurine or glycine group, including the salts thereof, particularly the sodium and potassium salts thereof.

Examples of non-phospholipid components (B) are preferably cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, hyocholic acid, hyodesoxycholic acid, sodium taurocholate, sodium taurodesoxycholate, sodium glycocholate, sodium taurochenodesoxycholate, potassium taurocholate, potassium taurodesoxycholate, potassium glycocholate and potassium taurochenodesoxycholate, but particularly sodium taurocholate, sodium taurodesoxycholate, sodium glycocholate, sodium taurochenodesoxycholate, potassium taurocholate.

Suitable non-phospholipid components (C) for the purposes of the invention are selected from among saturated, mono- or polyunsaturated straight-chain fatty acids with 8 to 18 C atoms, the salts and compounds thereof of general formula

wherein R⁶ to R⁸ each independently of one another denote a hydrogen atom or a saturated, mono- or polyunsaturated straight-chain fatty acid group (acyl group) with 8 to 18 C atoms.

The breakdown products that occur in the gastrointestinal tract are predominantly free fatty acids R⁶-OH and monoacylglycerides of formula (III), wherein R⁶ and R⁸ denote hydrogen atoms and R⁷ denotes one of the above-mentioned fatty acid groups, wherein the free fatty acids are usually saturated or monounsaturated and the esterified fatty acid group is usually mono- or polyunsaturated.

Preferred non-phospholipid components (C) are 2-glycerolmonooleate, lauric acid, myristic acid, myhstoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid and behenic acid, the salts, particularly the sodium salts thereof, of which the sodium salt of oleic acid is particularly preferred, including the mixtures thereof.

The process for preparing ready-to-use aqueous mixed micellar solutions according to the second aspect of the invention consists in the fact that after steps (i) and (ii) have been carried out the instant form obtained, which is present as a powdered mixed micellar system, is redispersed in another step (iii) in water or an aqueous buffer directly before use. The redispersing may be carried out in a few seconds and leads to mixed micellar solutions that can be used directly, which have reproducible micelle sizes and a high batch homogeneity in terms of their particle size, viscosity, surface tension, osmolality and density.

The third aspect of the invention comprises an instant form free from organic solvents and sufficiently stable for storage, as the precursor of an aqueous mixed micellar solution comprising

(A) at least one phospholipid,

(B) one or more non-phospholipid substances selected from among bile acid, bile acid salts, bile acid derivatives and salts of a bile acid derivative as well as (C) optionally one or more non-phospholipid substances selected from among triacylglycerides and the breakdown products thereof, wherein

the molar ratio of the components (A) : (B) is from 0.1 :1 to 1 :1 , preferably between 2:10 to 4:10, but particularly preferably about 1 :4 and the molar ratio of the optionally added component (C) : (B) with regard to all aspects of the invention is from 0.1 :1 to 6:1 , preferably between 1 :1 to 5:1 , but particularly preferably about 4:1 ,

which may be obtained by the process described as the first aspect of the invention.

For example the phospholipid component (A) consists of a pure phosphatidylcholine, such as dipalmitoylphosphatidylcholine or palmitoyloleoylphosphatidylcholine, or of a mixture of phospholipids of natural origin such as egg lecithin or soya lecithin with a high concentration of phosphatidylcholine, preferably at least 80%, particularly preferably at least 96% based on the dry mass, wherein the phosphatidylcholine has the following fatty acid composition, for example: in each case about 33% palmitic acid, 27% oleic acid, 17% linoleic acid, 14% stearic acid and 9% other fatty acids,

the non-phospholipid component (B) consists of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, hyocholic acid, hyodesoxycholic acid, sodium taurocholate, sodium taurodesoxycholate, sodium glycocholate or sodium taurochenodesoxycholate, but particularly of sodium taurocholate, sodium taurodesoxycholate, sodium glycocholate or sodium taurochenodesoxycholate, of which sodium taurocholate is particularly preferred,

moreover the molar ratio of the components (A) : (B) is for example 1 :3 to 1 :5, but particularly preferably about 1 :4 and the molar ratio of the optional component (C) : (B) is 0.1 :1 to 6:1 , preferably 1 :1 to 5:1 , but particularly preferably about 4:1. For example, mention may be made of FaSSIF Instant, FeSSIF Instant and FeSSIFplus Instant as instant forms of the biorelevant media FaSSIF, FeSSIF and FeSSIFplus which are free from organic solvents, substantially anhydrous, and sufficiently stable for storage, comprising

(A) egg-phosphatidylcholine,

(B) sodium taurocholate and

(C) optionally glycerol monooleate and/or sodium oleate

in a molar ratio of components (A) : (B) of about 1 :4 and, if component C is present, in a molar ratio of component (C) : (B) of about 4:1 , for example

sodium taurocholate 3 mM egg-phosphatidylcholine 0.75 mM or

sodium taurocholate 15 mM egg-phosphatidylcholine 3.75 mM or

sodium taurocholate 7.5 mM egg-phosphatidylcholine 2 mM glycerol monooleate 5 mM sodium oleate 30 mM.

Preferably FaSSIF Instant, FeSSIF Instant and FeSSIFplus Instant are prepared according to the invention without the addition of buffer and salts.

The fourth aspect of the present invention comprises using the ready-to-use mixed micellar solution obtained from the instant form produced by the process according to the invention and redispersed after the addition of water, as a biorelevant medium or as a physiological buffer system in release tests, for example on pharmaceutical forms (analysis of in vitro release of pharmaceutical active substances). In this context special mention should be made of the instant forms referred to hereinbefore as being preferred, but especially FaSSIF Instant, FeSSIF Instant and FeSSIFplus Instant. The high reproducibility of the media produced also results in a high reproducibility of the releases. As releases are generally carried out in defined volumes (usually 200 to 1000 ml) it is possible to prepare or decant the freeze-dhed mixed micelles in batches for the preparation of the aqueous mixed micellar solution. Suitable methods include for example freeze-drying in vials or blisters or the decanting of the bulk-dried mixed micelles into special containers with the addition of desiccants, for example calcium chloride, calcium oxide, 3λ molecular sieve, 4λ molecular sieve or silica gel. For drying in vials or blisters the concentrated mixed micellar solution is decanted to suit the final concentration and lyophilised in the freeze-dryer. The subsequent sealing should take place in the drying chamber or under a nitrogen gas current. The portion packs thus obtained may be redispersed with water or with aqueous buffer directly in the release vessel. No further treatment is required. The reproducibility of the aqueous mixed micellar solutions prepared is evaluated according to the parameters of particle size, particle size distribution, surface tension, density, osmolality and viscosity. With regard to all the parameters higher reproducibility can be achieved with the method of preparation according to the invention than with the processes described in the prior art. For example, using the process according to the invention, after redispersion of the instant forms aqueous mixed micellar solutions are readily obtained which reproducibly conform to the following parameters:

particle size: D90 value between 30-60 nm (FaSSIF), 5-15 nm (FeSSIF) or 10-20 nm

(FeSSIFplus); particle size distribution: PdI value between 0.2-0.3 (FaSSIF), 0.12-0.17 (FeSSIF) and 0.1-0.15 (FeSSIFplus) surface tension: 53-58 mN/m (FaSSIF), 46-49 mN/m (FeSSIF) and 48-50 mN/m (FeSSIFplus) density: 1.0055-1.0065 g/cm³(FaSSIF), 1.0125-1.0135 g/cm³(FeSSIF) and 1.0115-

1.0125 g/cm³ (FeSSIFplus); osmolality: 260-280 mosmol/kg (FaSSIF), 660-690 mosmol/kg (FeSSIF) and 385-405 mosmol/kg (FeSSIFplus); viscosity: 0.95-1.05 cP (FaSSIF), 1.05-1.15 cP (FeSSIF) and 1.15-1.25 cP (FeSSIFplus). The values depend on the buffer used and are given here by way of example for the standardised media.

Abbreviations:

D90 value: 90% of the particles have a particle size less than or equal to the value specified;

PdI value: polydispersity index; cP: centi-Poise.

Literature: (¹⁾ Pharmaceutical Research, Vol. 15, No. 5, 1998, 698-705;

⁽²⁾ Journal of Pharmacy and Pharmacology, 2004, 56: 453-462

⁽³⁾ European Journal of Pharmaceutical Sciences 11 Suppl. 2 (2000), S73-S80

⁽⁴⁾ European Journal of Pharmaceutics and Biopharmaceutics 40, 1994, 147-156.

The following Examples are intended to illustrate the invention:

Example 1

For the process for preparing the aqueous mixed micellar solutions for lyophilisation, which is described hereinbefore as step (i), the compositions listed below and designated Examples 1.1 -1.8 in Table 1 were used. All the phospholipid substances contained lecithins with at least 80% phosphatidylcholine and all the non- phospholipid substances had a purity of at least 98%.

The freeze-drying parameters in the preparation of the Examples were as follows: Freezing the aqueous mixed micellar solutions from ambient temperature to -40⁰C at a rate of 1 K/min and maintaining this temperature for 3 hours. Lowering the pressure during the main drying to 0.28 mbar and heating to 0⁰C over 10 hours and to 20⁰C over 5 hours. Increasing the vacuum during the after-drying to 0.001 mbar and increasing the temperature to 30°C for 3 hours. Then the drying chamber was ventilated to a pressure of 800 mbar with nitrogen and the vials were sealed. The mixed micelles dried in dishes were individually metered into containers with desiccant (calcium chloride) under a drying air current or decanted in bulk. The blisters were sealed under a drying air current.

Example 2

The dried storage form consists of a phospholipid and a non-phospholipid substance in the proportions described in Table 1 under 1.1 -1.6, optionally with the addition of the amounts of buffer and salt specified in Table 1 under 1.7-1.8, while after dissolving and dispersing in water the water content was reduced to less than 1 % by lyophilisation and the powders obtained were sealed in airtight manner so as to be impervious to water vapour. In this way, storable powders are obtained for preparing mixed micellar solutions or dispersions and above all biorelevant media.

Example 3

The powders may be produced for preparing ready-to-use mixed micellar solutions and dispersions in the solvents described in Table 1 under 1.1 -1.8. In order to prepare aqueous mixed micellar solutions from lyophilisate, amounts of lyophilisate corresponding to the final volume were weighed out or the pre-metered amounts were redispersed in the appropriate dispersing agent (water or buffer) and used directly. For example, for the preparation of a) FaSSIF, 2.2 g of the powder prepared according to Table 1 under 1.1 were redispersed in 1 litre of 29 mM phosphate buffer, pH 6.5, with the addition of

106 mM NaCI and for the preparation of b) FeSSIF, 11.2 g of the powder prepared according to Table 1 under 1.3 were redispersed in 1 litre of 144 mM acetate buffer, pH 5.0, with the addition of 203 mM NaCI and for the preparation of c) FeSSIFplus, 16.61 g of the powder prepared according to Table 1 under 1.4 were redispersed in 1 litre of 35 mM citrate buffer, pH 6.0, with the addition of 15O mM NaCI.

Example 4

In order to prepare biorelevant media for release analysis the lyophilisates according to Table 1 1.1-1.8 may be redispersed with solvent and used as the release medium. For a release test in a) 900 ml FaSSIF with n=6, 12.12 g of the powder prepared according to Table 1 under 1.1 is redispersed in 5.4 litres of 29 mM phosphate buffer, pH 6.5, and for a release test in b) 900 ml FeSSIF with n=6, 60.6 g of the powder prepared according to Table 1 under 1.3 is redispersed in 5.4 litres of 144 mM acetate buffer, pH 5.0, and for a release test in c) 900 ml FeSSIFplus with n=6, 89.7 g of the powder prepared according to

Table 1 under 1.3 is redispersed in 5.4 litres of 35 mM acetate buffer, pH 6.0, and used directly as media.

Explanation of abbreviations:

Ph-type description of the type of phospholipid substance (PC: phosphatidylcholine)

Ph-M amount of phospholipid substance used

NPh-type description of the type of non-phospholipid substance (NaTC: sodium taurocholate; NaTDC: sodium taurodesoxycholate; NaGC: sodium glycocholate; NaTCDC: sodium taurochenodesoxycholate; GMO: glycerolmonooleate; NaO: sodium oleate) NPh-M. amount of non-phospholipid substance used solv-type type of solvent for preparing the mixed micellar solution or dispersion for the lyophilisation (buffer a: 29mM NaH₂PO₄ and 106mM NaCI with pH6.5; buffer b: 144mM C₂H₃NaO₂ and 203mM NaCI with pH 5.0; buffer c: 35mM C₆H₅Na₃O₇ and 15OmM NaCI with pH 6.0)

10 solv-V volume of solvent in which the substances are dissolved or dispersed disp-type type of solvent for preparing the mixed micellar solution to be used by redispersion of the lyophilisate (buffer a: 29mM NaH₂PO₄ and

106mM NaCI with pH6.5; buffer b: 144mM C₂H₃NaO₂ and 203mM NaCI with pH 5.0; buffer c: 35mM C₆H₅Na₃O₇ and 15OmM NaCI with pH 6.0) disp-V volume of solvent in which the lyophilisate is dissolved or redispersed

Claims

Patent Claims

1. Process for preparing a storable instant form of an aqueous mixed micellar solution or mixed micellar dispersion, comprising the steps of

(i) preparing a concentrated aqueous mixed micellar solution comprising

(A) at least one phospholipid,

(B) one or more non-phospholipid substances selected from among bile acid, bile acid salts, bile acid derivatives and salts of a bile acid derivative and

the molar ratio of the components (A) : (B) is from 0.1 :1 to 1 :1 and optionally the molar ratio of the components (C) : (B) is from 0.1 :1 to 6:1 ,

and

2. Process according to claim 1 , characterised in that the phospholipid component (A) is

pure phosphatidylcholine or

a mixture of phospholipids of natural origin or

a phosphatidylcholine of general formula

wherein R¹ and R², which may be identical or different, represent saturated as well as mono- or polyunsaturated straight-chain fatty acid groups with 14 to 18 C atoms, or is a mixture of the above-mentioned phospholipid components,

the non-phospholipid component (B) is a derivative of 5β-cholanic-24-acid of general formula

wherein R³ and R⁴ each independently of one another denote a hydroxy group or a hydrogen atom and R⁵ denotes a hydroxy group, the taurine or glycine group, including the salts thereof, and

the non-phospholipid component (C) is selected from among saturated, mono- or polyunsaturated straight-chain fatty acids with 8 to 18 C atoms, the salts and compounds thereof of general formula

3. Process according to claim 2, characterised in that the molar ratio of the components (A) : (B) assumes a value of 2:10 to 4:10 and optionally the molar ratio of the components (C) : (B) assumes a value of 0.1 :1 to 6:1.

4. Process for preparing ready-to-use aqueous mixed micellar solutions, characterised in that in a further step

(iii) water or aqueous buffer is added to the instant form obtained after carrying out steps (i) and (ii) according to one of claims 1 , 2 or 3 and in this way the ready-to-use aqueous mixed micellar solution is reconstituted.

5. An instant form of an aqueous mixed micellar solution which is free from organic solvents, substantially anhydrous and sufficiently stable for storage, comprising

(A) at least one phospholipid,

(C) optionally one or more non-phospholipid substances selected from among triacylglycerides and the breakdown products thereof, wherein the molar ratio of the components (A) : (B) is from 0.1 :1 to 1 :1 and optionally the molar ratio of the components (C) : (B) is from 0.1 :1 to 6:1.

6. Instant form according to claim 5, characterised in that the phospholipid component (A) is

pure phosphatidylcholine or

a mixture of phospholipids of natural origin or

a phosphatidylcholine of general formula

the optional non-phospholipid component (C) is selected from among saturated, mono- or polyunsaturated straight-chain fatty acids with 8 to 18 C atoms, the salts and compounds thereof of general formula

wherein R to R each independently of one another denote a hydrogen atom or a saturated, mono- or polyunsaturated straight-chain fatty acid group (acyl group) with 8 to 18 C atoms.

7. Instant form according to claim 6, characterised in that the molar ratio of the components (A) : (B) assumes a value of 2:10 to 4:10 and optionally the molar ratio of the components (C) : (B) assumes a value of 0.1 :1 to 6:1.

8. Instant form according to claim 6, comprising

(A) egg-phosphatidylcholine, (B) sodium taurocholate and

(C) optionally glycerol monooleate and/or sodium oleate

in a molar ratio of component (A) : (B) of about 1 :4 and, if component C is present, in a molar ratio of component (C) : (B) of about 4:1.

9. Use of the aqueous mixed micellar solution prepared by the process according to claim 4 or a mixed micellar solution redispersed from an instant form according to one of claims 5 to 8 by the addition of water or aqueous buffer as a biorelevant medium or as a physiological buffer system in the analysis of the in vitro release of pharmaceutical active substances.