DK175799B1 - Process for the Preservation and Process for the Preparation of Liposomes and a Lyophilized Composition and a Preserved Composition - Google Patents

Description

DK 175799 B1

The present invention relates generally to liposomes and more particularly to a method for the preservation of liposomes containing biologically active molecules. This method is useful in applications such as in vivo drug delivery and prescribing of diagnostic agents. Furthermore, the invention relates to a lyophilized composition useful for storing encapsulated material.

Liposomes are unilamellar or multilamellar lipid vesicles enclosing a fluid compartment. The walls of the vesicles are formed by a bimolecular layer of one or more lipid components with polar heads and non-polar tails. In an aqueous (or polar) solution, the polar heads in one layer are oriented outwardly and extend into the surrounding medium, and the non-polar tail portions of the lipids are interconnected, thus providing a polar surface and a non-polar nucleus in the wall of the vesicle. Unilamellar liposomes have such a bimolecular layer, whereas multilamellar liposomes generally have a plurality of substantially concentric bimolecular layers.

Liposomes are recognized as useful for encapsulating drugs and other therapeutic agents and for carrying these agents to in vivo sites.

Eg. the disclosure to U.S. Patent No. 3,993,754 to Rahman et al., Issued November 23, 1976, an improved chemotherapeutic method by which an antitumor drug is encapsulated in liposomes and then injected. In the description of U.S. Patent No. 4,263,428 to Apple et al., Issued April 21, 1981, discloses an anti-tumor drug which can be more effectively delivered to selective cell sites in a mammalian organism by incorporating the drug into liposomes of uniform size. Drug administration via liposomes may have reduced toxicity, altered tissue distribution, increased drug efficacy, and an improved therapeutic index.

I DK 175799 B1 I

I 2 I

Liposomes have also been successfully used to introduce various I

chemicals, biochemicals, genetic materials and the like. in living cells in vitro I

In and as carriers of diagnostic agents. IN

I * M

A number of methods for preparing liposomes are known, of which

many have been described by Szoka and Papahadjopoulos, Ann. Rev. Biophv- I

I say Bioenq. 9: 467-508 (1980). Within the patent literature, I have also become

Several liposome encapsulation methods have been described, in particular in

In the relief of the U.S. Patent No. 4,235,871 to Papahadjopoulos et al., Issued 25. I

In November 10, 1980, and in the description to U.S. Patent No. 4,016,100 to Suzuki et

In eel., Issued April 5, 1977. I

Although encapsulation of therapeutic agents and biologically active compounds in I

In liposomes have significant commercial utility, have a greater I

In 15 difficulties which have been shown by the commercial use of liposome ind

In enclosure, their stability has been long term. Although liposome structures, you can

In maintaining intact under certain storage conditions, these conditions are often inappropriate

In or not available. The present process provides a solution for H

In this problem. IN

I 20

It is therefore an object of the present invention to provide an I

In commercially feasible method for the preservation of liposomes. IN

It is another object of the present invention to provide an I

25 commercially feasible method for preserving liposomes at I

With the help of freeze-drying, the resulting liposomes after rehydration I

You can retain as much as 100% of their original encapsulated material

In rial. - I

It is yet another object of the present invention to provide I

In a method for preserving liposomes by a carbohydrate H

In the context of being able to preserve the structure and function of biologics

In geese membranes. IN

DK 175799 B1 I 3 i

It is yet another object of the present invention to provide a method for preserving liposomes by means of a preservative such as trehalose, which is present either as an encapsulated material inside the liposome or outside in the solution during freeze-drying 5 or both. .

It is yet another object of the present invention to provide a lyophilized composition which, after rehydration, retains up to 100% of the initially encapsulated material.

10

Further objects and advantages of the present invention will become apparent from the following description, claims and accompanying drawings.

In one aspect of the present invention, it comprises a method for preserving liposomes, thereby providing initial liposomes with lipid membranes, which liposomes have an initial amount of a water-soluble encapsulated material, which encapsulates any of the carbohydrates trehalose, maltose and sucrose, contacting these initial liposomes with an aqueous solution containing any of trehalose, maltose and sucrose and lyophilizing the initial liposomes in the aqueous solution to form lyophilisates containing at least 80% intact liposomes.

In another aspect of the present invention, the method comprises the preparation of liposomes, which method is characterized by that set forth in claim 2. Preferred weight ratios of total preservative to lipid are from ca. 0.1: 1 to 3.0: 1. A particularly preferred weight ratio is approx. 1.0: 1.0.

The present invention also encompasses a lyophilized composition such that when the resulting liposomes, when reconstituted by rehydration, retain substantially all of the initially encapsulated material.

Such a lyophilized composition can be prepared by the process described above.

I DK 175799 B1 I

I I

In the manner described above. The composition is characterized by I

In the claim 5.

In addition, the present invention comprises preserved compositions I

5 characterized in that they comprise: lyophilisates having a lipid component

In part, a disaccharide component and an initial amount of an encapsulated I

In a component wherein the disaccharide component comprises trehalose and is to I

Present in a weight ratio to the lipid component of from ca. 0.1: 1 to approx. IN

In 3.0: 1, the lipid component forms lipid membranes with an inner surface and an I

10 outside, the disaccharide component is present in the lyophilisates on both inner

On the inside and outside of the lipid membranes, and at least the majority of I

In the initial amount of encapsulated material is present in the lyophilisates on I

In the interior of the lipid membranes. IN

The present invention relates to a method of preserving I

In liposomes containing biologically active molecules, using an I

In any of the carbohydrates trehalose, maltose and sucrose. Forward

In the process, either lyophilization of liposomes involves the presence of I

You can see any of the carbohydrates trehalose, maltose and sucrose

In 20 se, or lyophilization of liposomes containing any of

In the carbohydrates trehalose, maltose and sucrose together with encapsulated. IN

In medications, or both. IN

Trehalose is a naturally occurring sugar that is found in high concentrations

In 25 trations in the organisms that are able to survive dehydration. Trehalose I

You are particularly effective in preserving the structure and function of dry biology.

happen membranes. The liposomes that are lyophilized in the presence of I

In trehalose, and which further contains encapsulated trehalose, exhibit particularly in I

In good restraint to the encapsulated material. That is, when liposo-

For 30 mer, trehalose is exposed both internally and externally during lyophilization

In gene, they can retain as much as 100% of the initially encapsulated I

In content by rehydration. This is in sharp contrast to the liposomes that I

B1 is lyophilized without any preservative and shows extensive fusion of liposomes with loss of content to the surrounding medium.

Representative phospholipids used to form the liposomes that can be used in the present process include phosphatidylcholine, phospatidylserine, phosphatidic acid and mixtures thereof. Both natural and synthetic phospholipids can be used successfully.

The encapsulated biologically active or therapeutic material is preferably water-soluble. Examples of suitable therapeutic agents by which this method of preservation can be successfully carried out include sympathomimetic drugs such as amphetamine sulfate, epinephrine hydrochloride or ephedrine hydrochloride; antispasmodics such as atropine or scopalamin; bronchodilators such as isoprotemol; vasodilators such as dilthiazene; hormones such as insulin and antineoplastic drugs such as adriamycin. Suitable biologically active molecules include, e.g. RNA, DNA, enzymes and immunoglobulins.

Small unilamellar vesicles (SUVs) are prepared as starting material prior to encapsulation of trehalose and can be prepared by any of the methods available. Suitable methods include injection of the lipid into an organic solvent in water, extrusion from a "French pressure cell" and sound treatment. The material to be captured can be added at any stage during the preparation of the small unilamellar vesicles, but in practice it is most convenient to mix the small unilamellar vesicles with an aqueous solution to the material to be captured immediately prior to preparation of the large unilamellar vesicles. Preferred weight ratios of the encapsulated to the lipid are about 1.0: 1.0.

30 Large unilamellar vesicles (LUVs) with enhanced entrapment effect can then be prepared by either freeze drying or rotary evaporation. An example of a rotary evaporation process, and a method particularly effective in the present process.

DK 175799 B1 I

they, are illustrated in Deamer, D.W., "A Novel Method for Encapsulation of I

Macromolecules in Liposomes ”in Gregoriadis, G. (editor) Liposome Tech- I

noloov (1984). The method comprises providing a polar solution

containing initial liposomes and a quantity of the material to be incorporated

5 capsules. Essentially all solution is removed and the resulting liposomes I

is recovered by hydration of the concentrated mixture. This progress I

manner is also discussed in the description to U.S. Patent No. 4,515,736 to Deamer, et al

eel. The resulting vesicles can then be made more uniform by filtration

centrifugation or gel penetration chromatography. IN

Trehalose can be added at any stage during the preparation of the large unilamel I

laryngeal vesicles, but a much improved preservation is achieved when trehalose is to I

present on both sides of the phospholipid annex. Trehalose is therefore added for I

stepwise before the large unilamellar vesicles are prepared so that trehalose H

15 are trapped in the interior. The preferred weight ratio of total trehalose to lipid I

is of the order of approx. 0.1: 1 to approx. 3.0: 1, a particularly preferred one

weight ratio is approx. 1.0: 1.0. The large unilamellar vesicles are then frozen in aircraft

then nitrogen and freeze-dried. In certain cases, such as when lipids are used, I

that are sensitive to damage due to the presence of oxygen, it is you

It is desirable to seal the dry preparations under vacuum. Rehydration out- I

is simply passed by adding water to the dry mixture. IN

Although in a preferred embodiment of the present invention, the liposomes

Substance is subjected to trehalose, it should be understood that a wide variety of preservatives

25 can be substituted for trehalose, including carbohydrate compounds which I

consists of at least two monosaccharide units. In particular, sucrose and maltose are eg

down options. IN

The following examples illustrate certain aspects and embodiments of the I

30 and does not intend to limit the scope of I

the invention defined in the appended claims. IN

7 DK 175799 B1

Example 1

A phospholipid mixture consisting of ca. 40 mg of dipalmitoyl (phosphatidylcholine and phosphatidic acid in a 95: 5 molar ratio were sonicated for optical clarity in a bathtub processing apparatus. Large unilamellar vesicles were prepared by lyophilizing in a 50 mM solution of isocitronic acid in water as the compound excess isocitric acid was removed by dialysis Trehalose (2.0: 1.0, trehalose: phospholipid weight ratio) was added either before or after freeze drying to provide some large unilamellar vesicles with only trehalose exterior and some vesicles with In the citric acid was analyzed by adding isocitrate dehydrogenase and NADP to the exterior of the vesicles according to the method of Plaut, et al (editors), Methods of Enzymoloav. Volume 5 (New York, Academic Press) .

The isocitrate which was outside the vesicles was oxidized by isocitrate dehydrogenase resulting in reduction of NADP to NADPH, which rate and amount can be determined fluorometrically. Total isocitric acid in the vesicles was analyzed after the addition of Triton X-100 (octylphenoxypolyethoxyethanol, a detergent and emulsifier prepared by Rohm & Haas Co., 20 Philadelphia, PA, "TRITON" is a registered trademark of Rohm & Haas Co.) , which releases the trapped isocitric acid to the surrounding medium. The isocitronic acid trapped in the vesicles was analyzed both before and after freeze drying and rehydration, thus providing an estimate of the effect by which the captured isocitrate is retained. As shown in Table 1, the results show that over 60% of the captured isocitrate was retained when the vesicles were lyophilized with trehalose both outside and inside the vesicles. When trehalose was present only outside, there was still a significant increase in the effect of retention, but to a lesser extent than that in the case where trehalose was present on both sides of the lipid membrane.

Estimation of the lipid concentration at the time of freezing showed that it had no significant effect on retention of the captured material after freeze drying.

I DK 175799 B1 I

I I

In Table 1 I

I Progress A I

In the way of Concern Treha- Trehalose I

preparation tration lose / g% Reten- I

I of LUVs of lipid lipid exterior inner ion I

(mg / ml) I

I FT * 10.8 o - - 0 I

I FT 11.1 0.08 - + 0 I

In FT 10.8 1.78 + 42

I FT 11.1 1.78 + + 61 I

In 5 * FT = freeze drying H

In Example 2 H

I 10 Small unilamellar vesicles were prepared by sound treatment of 43 mg of egg I

In gephosphatidylcholine in 4 ml of water. Large unilamellar vesicles were prepared H

I by rotary drying of the phospholipid in the presence of 32 mg of treha-H

In loose and 13 mg isocitronic acid. The weight ratios of phospholi- I

pid: trehalose: isocitric acid was approx. 4: 3: 1. Excess isocitric acid and treha- I

In 15 loos was removed by dialysis against distilled water and the amount of isocitron I

acid trapped in the vesicles is determined by the enzyme assay which is I

described in Example 1. Trehalose was added to the dialyzed liposomes I

To obtain a final weight ratio of phospholipid: trehalose of I

In 1.0: 1.4 and the sample was freeze-dried. The sample was then rehydrated with distillate

In clay water and the remaining isocitric acid in the liposomes was determined by I

In enzyme analysis. The lyophilized vesicles retained 75% of their origin- H

In equal content. IN

9 DK 175799 B1

Example 3 A phospholipid mixture of palmitoyloleoylphosphatidylcholine (90%) and phosphatidylserine (10%) was hydrated to 10 mg / ml and small unilamellar vesicles were prepared by sonication. Large unilamellar vesicles were prepared by rotary drying in the presence of isocitronic acid, which served as the encapsulated molecule. Essentially, the same methods as previously described in Examples 1 and 2 were used. without trehalose. As can be seen in Table 2, the results show that 100% of the captured isocitronic acid is retained when large unilamellar vesicles are lyophilized and rehydrated under the established conditions. As shown in the previous examples, trehalose is preferably present both outside and inside to optimize the retention of the encapsulated.

Example 4 20

One of the damaging conditions that is believed to occur during freeze-drying is the close association of the large unilamellar vesicles with one another, which leads to fusion and outflow of the vesicular contents. The fusion has been analyzed by resonance energy transfer, which is a fluorescence method, which depends on energy transfer from one excited probe ("donor probe") to another probe ("acceptor probe"). The acceptor probe fluoresces as the energy transfer occurs. For the transfer to occur, the two probes must be in close proximity to each other. Thus, probe mix mixing can be used as an assay for fusion of the vesicles during freeze drying. Large unilamellar vesicles were prepared with donor probe in one preparation and acceptor probe in another, and the two preparations were mixed before freeze-drying. After freeze-drying and rehydration, the probe mixture was measured to obtain the results given in Table 3. The results show that with

I DK 175799 B1 I

I I

In increasing trehalose concentration, there is a decrease in probe mixture. Furthermore, I-

the presence of trehalose alone within the liposomes substantially diminishes I

In probe mix. Thus, the use of trehalose tends to reduce

In re vesicle fusion. * I

I I

In Table 2 I

In Progress I

In way of g Treha- Trehalose I

In preparation lose / g% Reten- I

I of LUVs lipid exterior inner tion H

I RD * 0.06 + o I

I RD 3.2 + + 100 I

I RO 0 - .0 I

I RD 3.9 + 26 I

In RD 0.11 + +22 I

In RD 0.19 ++ 49 I

In RD 0.33 ++ 69 I

I RD 0.63 ++ 76 I

I RD 0.91 ++ 86 I

I RD 1.76 ++ 99 I

In RD * = rotation drying I

11 DK 175799 B1

Table 3, Procedure for g Treha- Trehalose Preparation loose / g% Sample of LUVs Lipid External Internal Mixture RD * 0.05 - + 72 RD 0.15 + + 39 RD 0.25 + +29 RD 0.50 + + 12 RD 0.95 + +8 FT ** 0, - - 93.0 FT 0.4 + + 79.0 FT 0.8 + + 59.0 FT 1.2 + + 54.0 FT 1, 6 ++ 38.0 FT 2.0 ++ 15.0 * RD = rotary drying 5 ** FT = freeze drying

Fusion between palimtoyloleoylphosphatidylcholine:

Phospatidylserine (90:10) large unilamellar vesicles, as analyzed by resonance energy transfer between fluorescence probes.

10

Example 5

A further experiment, identical to that described in Example 3, was performed first with maltose and then with sucrose as a preservative. The results are listed in Tables 4 and 5. As can be concluded from these tables, both maltose and sucrose provide good retention of the encapsulated material after freeze drying.

DK 175799 B1 I

Table 4 I

Progress H

way to Maltose * H

Preparation g Maltose% Reten- H

of LUVs / g lipid exterior inner thion H

RD 0.05 + 3

RD 0.15 + +41 I

RD 0.25 ++ 88 I

RD 0.49 ++ 95 I

RD 0.64 ++ 100 I

Table 5 I

Progress H

way to Sucrose H

Preparation of sucrose%

of LUVs / g lipid exterior inner thion H

RD 0.07 - + 20 I

RD 0.35 ++ 57 I

RD 0.49 ++ 89 I

RD 0.83 ++ 86 I

RD 1.15 ++ 91 I

Claims (11)

A method for preserving liposomes, characterized in that initial liposomes with lipid membranes are provided, which liposomes have an initial amount of a water-soluble encapsulated material, which encapsulates any of the carbohydrates trehalose, maltose and sucrose, contacting these initial liposomes with an aqueous solution containing any of trehalose, maltose and sucrose, and lyophilizing the initial liposomes in the aqueous solution to form lyophilisates containing at least 80% intact liposomes. 15 Process for the preparation of liposomes, characterized in that initial liposomes with lipid membranes are provided, which have an initial amount of a water-soluble encapsulated material, which encapsulates any of the carbohydrates trihalose, maltose and sucrose. contacting these initial liposomes with an aqueous solution containing any of trehalose, maltose, and sucrose, lyophilizing the initial liposomes in the aqueous solution to form lyophilisates containing at least 80% intact liposomes, and contacting the lyophilisates with an aqueous solution to form rehydrated liposomes. 30 I DK 175799 B1 I I I The method according to claim 2, characterized in that the rehydrated I 1 liposomes encapsulate up to approx. 100% of the initial amount of the encapsulated! In material. I I 'I I 5 4. A process according to claim 1 or 2, characterized in that the weight ratio of the encapsulated carbohydrate to the lipid of the liposomes is from approx. I I 0.05: 1 to approx. 0.10: 1st IN A lyophilized composition useful for storing encapsulated material, characterized in that it is prepared by a method I, comprising: II providing initial liposomes having an initial amount I of one herein. encapsulated material comprising a trehalose or I maltose amount, II contact the initial liposomes with a trehalose or maltose II amount in aqueous solution, II 20 the initial liposomes be lyophilized in the presence of trehalose or HI maltose. formation of lyophilisates. IN The composition according to claim 5, characterized in that the encapsulated material comprises a water-soluble therapeutic agent or a biological agent. I 25 active active compound. IN The composition of claim 6, characterized in that the encapsulated material comprises a macromolecule. IN The composition of claim 7, characterized in that the encapsulated material comprises a sympathomimetic drug, an antispasmodic drug, a vasodilatory and / or an antineoplastic drug, RNA, DNA, an enzyme or an immunoglobulin. Preserved composition, characterized in that it comprises 15 lyophilisates having a lipid component, a disaccharide component and an initial amount of an encapsulated component, the disaccharide component comprising trehalose and present in a weight ratio to the lipid component of from 0.1: 1 to approx. 3.0: 1, the lipid component forms lipid membranes having an inside and an outside, the disaccharide component is present in the lyophilizers on both the inside and outside of the lipid membranes, and at least most of the initial amount of encapsulated material is present in the the lyophilisates on the inside of the lipid membranes. Composition according to claim 9, characterized in that the trehalose in the disaccharide component is present in the lyophilisates on both the inside and the outside of the lipid membranes and is effective in retaining most of the initial amount of encapsulated material on the inside of the lipid membranes during reconstitution of the lipid membranes. lyophilisateme. 20 Composition according to claim 9 or 10, characterized in that the encapsulated material comprises a water-soluble therapeutic agent, a biologically active compound or a diagnostic agent, and that the lyophilisates can be recovered as liposomes encapsulating at least the majority of the content. by mixing the lyophilisates with an aqueous solution. i I 30