DD290807A5 - METHOD FOR STABILIZING VESICLES - Google Patents

Abstract

The present invention relates to a novel, advantageous method for stabilizing liposomes. The aim of the invention is the drying or freeze-drying of liposomes, in particular SUV and REV, with the addition of stabilizers, which enables the recovery of a long-term storable product, which allows the resuspension in the simplest way by adding water or buffer. Vesicle sizes and inclusion behavior should correspond to the initial practice separation. According to the invention, vesicles are stabilized by the addition of carbohydrates either during formation or after their formation. Particularly suitable carbohydrates for carrying out the process according to the invention are: D-glucose, D-fructose, sucrose, maltose, a, a-trehalose, cellobiose, lactose. The carbohydrates are added in a molar ratio of at least 1 mol of carbohydrate to 2 mol of lipid. A higher mole fraction of carbohydrate is preferred. Areas of application of the present invention are medicine, pharmacy and life sciences.

Description

Field of application of the invention

Water compartments separated from each other. -

A distinction is made between two-year-olds, one-sided, one-layered © (unilamellareni VeSiKeJq (SU \ yPa.r4ai \ a.dj.oppüloff, D. [1978] Ann. NY Acad., 308, 367-368) with a diameter up to 100 nm, large unilamellar VesjkelrnLüV) with a diameter of 100 to 300 nm and grpßjn multi-lamellar vesicles (MLVl with sizes up to about 5 pm

Liposomes can i.a. be used in the following areas:

T. In research, as models of biological membranes or cells, as they are internal volumes of the external environment

delimit

 2. In the application, as a carrier of active ingredients of various kinds, z.b. of pharmaceuticals, proteins, enzymes and hormones, agrochemicals, genes, antibodies and contrast agents for imaging techniques, e.g.

Computed Tomography (CTI; Magnetic Resonance Tomography (MRI)

 Especially the second area is gaining in importance in recent times.

The preparation of the liposomes and the "encapsulation" of the active ingredient (s) can be carried out in different manners adapted to the intended use.

-2- 290 807 Characteristic of the known state of the art

The storage of lipids or lipids and active ingredients can be carried out in the dry state and under inert conditions (oxygen and light exclusion, low temperatures).

Similar conditions are also sought for the storage of liposomes. So far, however, it has only been possible to work under inert conditions and to achieve relatively short storage times. While oxygen exclusion (shielding gas atmosphere) and light exclusion are relatively easy to enable, freezing is e.g. up to temperatures of

liquid nitrogen (-196 ⁰ G) already accompanied by a number of structural changes of the system. P. Machy et al. (in Liposome Technology, 1984), G. Gregoriadis, ed. Vol. 1, Chap. 16, CRC Press, Inc., Boca Raton, Florida) found that MLV and LUV partially release their vesicle contents upon freezing and thawing.

On the other hand, the freezing of liposomes is recommended in the European patent application 0065292 for the production of storage-stable MLV.

On the other hand, due to the properties of the amphiphilic molecules that form them, the drying of the liposomes is accompanied by much stronger structural and phase state changes (see H. Hauser, [1975] in Water-A Comprehensive Treatise [F. Franks, ed.], Vol , Plenary Press, New York and London). Drying of the liposomes while preserving their structure and the entrapped active ingredient was therefore previously not possible.

FP-PS 2399241 describes the reconstruction of liposomes after dehydration (drying) by the addition of hydrophilic components such as e.g. Polyalcohols and the like This patent relates exclusively to MLV. Immediately after resuspension, about 30% of the liposomes are already destroyed. By reintroducing drug after resuspension, this indication must even be considered as the lower limit. Vesicle size before and after drying is completely absent.

Considering the substantial release of active ingredient and the fact that MLV form spontaneously from lipid and water, a preservation of the vesicle structure does not emerge from the abovementioned patent description.

At present, the problem of storage or long-term storage of liposomes and liposomal delivery systems according to previously known methods is not solved.

In particular, the problem of storage or reconstitution of SUV was still considered impossible in 1984 in the standard work "Liposome Technology" (G. Gregoriadis, ed., 1984, in Liposome Technology Vol. 1, Chap. 18, CRC Press, Inc., Boca Raton, Florida).

However, in living nature, there are examples of how organisms protect against the effects of complete dryness by synthesis of appropriate stabilizers (Anhydrobiosis, Crowe, JH, Crowe, LM [1984] in Biological Membranes, Vol.5, [D. Chapman, Vol. Ed.] Chap. 2, Academic Press, New York, London).

Object of the invention

The object of the invention is to provide a method which achieves improved stability and hence longer shelf life of vesicles and vesicular-based delivery systems. On the other hand, it should be ensured that after a storage time in the simplest way an application ready Liposomenpräparation can be produced, which corresponds in essential parameters and also with respect to the incorporated active ingredients of the initial preparation. Thus, the practical use of liposomal encapsulated drugs is significantly expanded and achieved a safe efficacy under practical conditions.

Explanation of the essence of the invention

The invention solves the problem of overcoming the above-described disadvantages of the prior art and of developing a stabilization method which makes it possible to keep vesicles and vesicular administration systems storable for a long time while maintaining their structure, inclusion behavior and vesicle size , By resuspension, a liposome suspension corresponding to the starting preparation must be obtained which also contains the introduced active ingredients in unchanged form or releases them at the time of application.

A vesicle stabilizing and vesicular - based delivery system has now been found which is characterized in that either in the construction or after formation of small unilamellar vesicles (SUV), large unilamellar vesicles (LUV) and reverse phase - evaporation vesicles (REV) consisting of a lipid, a lipid mixture, a mixture of lipid and surfactant, a synthetic amphiphile, a mixture of lipids and synthetic amphiphiles and / or surfactants, which replaces the water bound to the head group thereof by membrane-stabilizing carbohydrates The active substances may include, for example, pesticides, agrochemicals, food additives, fertilizers, dyes, vitamins, enzymes, monoclonal antibodies, hormones, genes, viruses, cell organelles, chelating agents, drugs such asAnti-inflammatory drugs, antibiotics, protease inhibitors, antineoplastic agents, contrast agents for computed tomography, magnetic resonance tomography or NMR spectroscopy or other imaging techniques.

Suitable membrane-stabilizing carbohydrates are: a) all natural or synthetic monosaccharides,

either an aldose, e.g. D-erythrose, D-threose, D-ribose, D- or L-arabinose, D-xylose, D-lyxose, further D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D- or L-galactose, D-talose, D-idose and D-glucoheptose, or their L analogues,

or a ketosis, e.g. D-erythrulose, D-ribulose, D-xylulose, D-psicose, D-fructose, D-sorbose, D-tagatose, D-alloheptulose, D-glucoheptulose, D-altroheptulose, D-mannoheptulose, D-guloheptulose, D- Idoheptulose, D-galactoheptulose, D-taloheptulose or D-altro-2-heptulose and the L analogues of the above-mentioned.

- a deoxy sugar, ζ.B. 2-deoxy-D-ribose, L-rhamnose or a-L-fucose, further

- an amino sugar, *. B. a D-glucosamine ^ .B. N-acetyl-D-glucosamine, a D-galactosamine.z-B. N-acetyl-D-galactosamine, or

A sugar alcohol, e.g. D-mannitol, D-sorbitol, D-erythritol, D-ribitol, D-xylitol, D-dulcite, D- or L-arabitol or inositol, adonite, maltitol, mannoheptitol, myo-inositol

and further

b) all natural and synthetic disaccharides, e.g. Lactose, maltose, D-cellobiose, sucrose,

a trehalose, e.g. α, α-trehalose or their synthetic enantiomers α, β-neotrehalose or β.β-isotrehalose,

c) all natural and synthetic tri- or oligosaccharides, e.g. B. raffinose, further

d) all water-soluble natural or synthetic polysaccharides, e.g. Glycogen, pectin, alginic acid or inulin, heparin, dextran or dextrin, carboxymethyl chitin

or mixtures of the carbohydrates mentioned.

Carbohydrates which are particularly suitable for carrying out the process according to the invention are: D-glucose, D-fructose, sucrose, maltose, α, α-trehalose, cellobiose, lactose.

The carbohydrates are added in a molar ratio of at least 1 mol of carbohydrate to 2 mol of lipid in the starting mixture. A higher mole fraction of carbohydrate is preferred.

After the addition of said stabilizers, the vesicles are dried. The vesicle dispersion is frozen, for example up to the temperature of the liquid nitrogen - 196 ° C. Drying takes place until no decrease in weight can be observed. The stabilized liposomes can in this state under oxygen occlusion z. Under inert gas atmosphere, e.g. Nitrogen or under vacuum at low temperatures, preferably at -20 ° C and with exclusion of light for a long period of time (i.e., in principle arbitrarily long) are stored.

The resuspension is conveniently carried out only in distilled water by shaking. The necessary ion and buffer concentrations are already set during the preparation of the vesicles. By adding the amount of water or additional buffer with resuspension, the lipid and active ingredient concentration can be varied. The resuspended vesicles are ready for use immediately and correspond in their parameters, in particular vesicle size, largely the starting material. If the further presence of the stabilizers proves undesirable, they may be removed by suitable methods, e.g. Dialysis be removed again. Most carbohydrates (stabilizers) are biodegradable. Substantial advantages of the method according to the invention are that the vesicles retain their original size, do not fuse, and the once included agent remains trapped even during and after drying or resuspension. In the stabilization of the liposomes according to the invention, the carbohydrates replace the water bound to the polar head group of the amphiphiles, maintain the structure of the bilayer and prevent phase separation.

A particular advantage of the method according to the invention is that even vesicles S 50 nm 0 can be stabilized. This result was surprising, since until then drying of uni- or otigolamellar liposomes while preserving their structure and the included active ingredient was not possible.

embodiments

The following examples illustrate the invention without limiting it.

example 1

a) One disperses 25 mg of predried egg phosphatidylcholine (egg PC) and 62 mg trehalose dihydrate (molar ratio 1/4) in 1 ml of distilled water or buffer z. B. 0.15 M sodium acetate buffer pH 5.5 by shaking. To form small unilamelfaren liposomes, the dispersion of a 1-2 bath sonication at T = 30 ° C is subjected. Remaining large unilameltar liposomes or multilamellar liposomes can be separated by ultracentrifugation (1 h, 180000 g). The characterization of the vesicles is carried out, for example, by means of high-resolution ³¹ P, 1 H-NMR and quasi-elastic light scattering. The Liposomendispersiort contains almost exclusively small unilamellar liposomes with a diameter kIeiner50 nm. The liposome dispersion is frozen in a heart of the rotary evaporator in liquid nitrogen and then lyophilized. The sample is stored for 24 h in a desiccator and then resuspended in 1 ml of distilled water and shaken. For NMR stabilization, some D ₂ O is added to the aqueous medium. The sample is used immediately for ³¹ P NMR studies. In the ³¹ P-NMR spectrum as well as before freeze-drying and resuspension, a narrow high-resolution signal can be seen. This comes from small unilamellar vesicles with a diameter smaller than 100 nm (Mops, A. [1984] Dissertation A, Leipzig). The addition of water-soluble displacement reagents z. B. Pr ³ ^ in the outer water phase allows the separation of the ^3l P-NMR signals of the phospholipids of the outer and inner monolayers. From the intensity ratio, the determination of the mean vesicle radius is possible. The vesicle radius before and after freeze-drying is less than 25.0 nm. The Pr.sup.- ions simultaneously serve as a permeability indicator and thus provide information about the closed nature of the membrane. The SUVs (egg PC / trehalose, 1: 4) are after lyophilization for the Pr ^ ions and are closed, as are the vesicles after sonication (see Hentschel, M., et al., 11985) Biochim Acta 812, 447-452).

b) Analogously to Example 1 a) is dispersed 25 mg of egg PC in 1 ml of distilled water by shaking and performs the bath sonication. To complete liposome dispersion, add 62 mg of trehalose dihydrate, incubate for about 1 h, and dry the vesicles as in 1 a). After 24 h, a ³¹ P-NMR spectrum is recorded again. As a result, there are small unilamellar vesicles smaller than 50 nm in diameter.

Example 2

a) 25 mg of dry egg PC and 62 mg of trehalose dihydrate (molar ratio 1/4) are dispersed in 1 ml of distilled water, 50 μΙ0.1 M Pr (NO ₃ I ₃ are added and sonicated, a ³¹ P NMR spectrum is recorded 100 μΙ of a 0.1 M EDTA stock solution are added and another spectrum is taken in. Complex formation of the EDTA with the Pr ^ ions in the outer but not in the inner water phase clearly gives rise to two ³¹ P NMR signals

differ that the phospholipids of the outer monolayer - not displaced - and that of the inner phospholipids, shifted by the interaction with Pr ³⁺ . The SUVs with the adjusted concentration gradient, Pr ³⁺ only inside, EDTA excess outside, are freeze-dried and resuspended after 24 h in 1 ml of distilled water. The ³¹ P-NMR spectrum corresponds to that before freeze-drying. The existing concentration gradient is maintained during the freeze-drying / resuspension, b) Analogously to Example 2a) was dispersed 25 mg egg PC in 1 ml of distilled water are 50μΙ 0.1 M Pr (NO ₃ J _3, performs the bath sonication, and outputs The EDTA addition takes place immediately before freeze-drying In contrast to 2 a), no concentration gradient outside / inside is detectable in the ³¹ P-NMR spectrum after freeze-drying / resuspension. By adding more Pr ³⁺ , the integrity of the bilayer after resuspension is demonstrated. The radius of the vesicles is unchanged less than 25 nm.

Example 3

As in Example 1 a, 25 mg of egg PC and 62 mg of trehalose dihydrate are dispersed in 1 ml of distilled water. The dispersion is added to before sonication calcein in a form suitable for fluorescence concentration of 5 x 10 ~ ^s M. After sonication, the fluorescence in the outer water phase is quenched by an excess of Co ²⁺ (5 x 10 ^-4 M), the sample is split, a control sample is measured immediately, the actual sample is lyophilized, and after 24 h the sample is resuspended and A difference in the intensities before and after freeze-drying is not detected and the calcein concentration gradient is thus retained.

Example 4

25 mg of egg PC and 62 mg of sucrose are dispersed in 1 ml of distilled water in the same way as in Example 2a, and 50 μl 0.1 M Pr (NO ₃ I ₃ solution are added and sonicated before freeze-drying, the concentration gradient is increased by adding 100 μl 0.1 EDTA The ³¹ P-NMR spectrum after freeze-drying and resuspension indicates the maintenance of the concentration gradient.

Example 5

In each case, 25 mg of egg PC and 58 mg of glucose or 58 mg of fructose (molar ratio 1/10) are dispersed in each case in 1 ml of distilled water and the bath sonication is carried out analogously to Example 1 a. Subsequently, the sample is freeze-dried and resuspended after 24 h in 1 ml of distilled water. The ³¹ P-NMR spectrum is recorded first without and then in the presence of Pr ³¹ . After resuspension, there are closed, small unilamellar vesicles with a diameter of less than 50 nm.

Example 6

50 mg of egg PC, 25 mg of cholesterol and 3.5 mg of dicetyl phosphate (molar ratio 10/10/1) and 124 mg of trehalose dihydrate are dispersed in 1 ml of distilled water by shaking overnight. The dispersion is sonicated alternately under a nitrogen atmosphere in an ice bath for 4 minutes for 4 minutes using a proboscis sonicator (Brownson B12). The sample is then centrifuged for 1 h at 100,000 g. The SUVs in the supernatent are frozen in a dry ice / alcohol cold bath (T = -70 ° C) and dried in a freeze dryer for 3 days. The sample is resuspended with 1 ml of distilled water, Pr ^{3+ is} added and the ³¹ P-NMR spectrum is recorded. There are closed, small unilamellar vesicles with a diameter smaller than 50nm.

Example 7

The same quantities are used as in Example 6 and 3 mg of bleomycin sulfate are added. The further processing corresponds to Example 6. After centrifugation, the entrapped bleomycin is removed by gel chromatography or dialysis. The further processing corresponds to Example 6. Re-gel chromatography of the freeze-dried and resuspended vesicles does not give free bleomycin,

Example 8

100mg of egg PC, 40mg of cholesterol and 12mg of phosphatiolic acid, 7mg of daunorubicin hydrochloride and 240mg of sucrose are dispersed in 4.5mL of 0.01M Tris buffer and shaken overnight. Production of SUV by sonication, subsequent centrifugation, gel chromatography or dialysis correspond to Example 7. Re-gel chromatography of the freeze-dried and resuspended vesicles does not give free daunorubicin.

Example 9

To a solution of 300 mg of egg PC and 150 mg of cholesterol in 16 ml of chloroform and 8 ml of diisopropyl ether are added 8 ml of physiological saline containing 650 mg of sucrose and 75 U aL-arabinofuranosidase, produces in a bath sonicator a stable emulsion and these in a known Reconstituted by evaporation of the organic solvent under reduced pressure in a rotary evaporator. The non-entrapped enzyme is removed by centrifugation and multiple washing of the pellet with sucrose-containing saline. The vesicles were then frozen and freeze-dried as indicated in Example 6. Resuspending with distilled water gives vesicles. which still contain 95% of the originally included a-L-arabinofuranosidase and correspond in size (electron microscopy, negative contrast) of the non-lyophilized starting preparation (mean diameter 250 nm).

Claims (9)

A process for the stabilization of vesicles and Aufvesikulärer based delivery systems by addition of hydrophilic substances, lyophilization and storage under inert gas atmosphere and / or at low temperatures, characterized in that either during construction or after formation of small unilamellar vesicles (SUV), large unilamellar vesicles (LUV) and "reverse phase-evaporation vesicles (REV) consisting of a lipid, a lipid mixture, a mixture of lipid and surfactant, a synthetic amphiphile, a mixture of lipids and synthetic amphiphiles and / or surfactants, the water bound to the polar head group thereof substitutes membrane-stabilizing carbohydrates, introduces active ingredients, then dries, partially dries or lyophilized, optionally stores and resuspends. 2. The method according to claim 1, characterized in that are used as membrane-stabilizing carbohydrates monosaccharides, disaccharides, tri- and oligosaccharides, water-soluble polysaccharides or mixtures of said saccharides. 3. The method according to claim 1 and 2, characterized in that one uses the carbohydrates D-glucose, D-fructose, sucrose, maltose, α, α-trehalose, cellobiose or mixtures thereof. 4. The method according to claim 1 to 3, characterized in that the addition of the membrane-stabilizing carbohydrates in the molar ratio of at least, preferably more than 1 mol of carbohydrate to 2mol lipid occurs. 5. The method according to claim 1, characterized in that the membrane-stabilizing carbohydrates are biodegradable. 6. Vesicle and delivery system stabilized on a vulvar basis according to claim 1, comprising as active component pesticides, agrochemicals, fertilizers, dyes, chelating agents, contrast agents for imaging methods such as computed tomography, magnetic resonance tomography or NMR spectroscopy and for non-medicinal use vitamins, enzymes, monoclonal antibodies , Hormones, genes, viruses and cell organelles. * 7th Process according to Claim 1, characterized in that the storage of the vesicles and the vesicular administration systems takes place on a vesicular basis with exclusion of oxygen and under a protective gas atmosphere. 8. The method according to claim 1, characterized in that the storage of the vesicles and the vesicular administration systems at low temperatures, in particular at -20 ⁰ C to - 196 ° C. 9. The method according to claim 1, characterized in that one of the vesicles and delivery systems aufvesikulärer basis of the intended use adapted lipid and active ingredient concentration and sets the resuspension by means of distilled water or buffer, the desired concentration optionally after removal of the carbohydrate.