AU640298B2 - Agents for inhibiting adsorption of proteins on the liposome surface - Google Patents

Description

PATENTS ACT 19S 6 1 k I'/00/0il Form COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE Class: Int. CI: Application Number: Lodged: Complete Specification Lodged: Accepted: Published:

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*5*S Priority: Related Art: TO BE COMPLETED BY APPLICANT TERUMO KABUSHIKI KAISHA Name of Applicant: Address of Applicant: 44-1 Hatagaya 2-chome, Shibuya-ku, Tokyo, Japan.

.00:6 Got* Actual Inventor/s: Address for Service: Hiroshi YOSHIOKA, Hiroshi GOTO CARTER SMITH BEADLE Patent Trademark Attorneys Qantas House 2 Railway Parade CAMBERWELL VIC 3124 Australia Comp.,le Specification for the invention entitled: AGENTS FOR INHIBITING ADSORPTION OF PROTEINS ON THE LIPOSOME SURFACE The following statement is a full description of this invention, including the best mnethod of performing it known to me: 1. The present invention relates to agents for inhibiting adsorption of proteins on the liposome surface.

Further, che invention relates to agents for preventing liposome agglutination.

o 10 Furthermore, the invention is concerned with liposomes on which adsorption of proteins is inhibited and which are agglutination-free and a method for preparing the same.

Prior Art Use of liposomes as a carrier for water-soluble or at-soluble drugs has widely been attempted (Gregoriadis, et al., Ann. N.Y. Acad. Sci., 446, 319 (1985)). Use of liposomes as artificial erythrocytes by incorporat.ng hemoglobin, the oxygen carrier for animals, in the inner p.0 aqueous space of liposomes has also been attempted (Japanese Patent Application Laid-Open to Public 178521/1987).

Liposome membrane-constituting materials of the liposomes used in these attempts, however, were those composed only of natural or synthetic lipids such as phospholipids and cholesterol.

2 In order to use liposomes as a carrier for drugs it is necessary to introduce the liposomes into blood vessels in the living body. However, the liposomes composed only of lipids which were conventionally employed were encountered with problems of adsorbing plasma-constituting proteins of the living body (for example, albumin, globulin and fibrinogen) which results in mutual agglutination of the liposomes. The problems were considerable especially of the liposomes which particle size exceeds 0.1 um. Particle size of the liposomes generally employed is usually 0.1 um 1 pm. The particle size as it is will be of no obstacle in passing through the blood vessels in the living body because the capillary blood vessels have inner diameter as large as several um. However, if the liposomes are agglutinated by adsorbing plasma-constituting proteins, size of the agglutinates becomes tens of micrometers. If the 4 S* agglutination occurs in the blood vessel, agglutinates of the liposomes will plug the blood vessel to inhibit blood flow possibly causing death of the living body.

Particularly when liposomes are used as artificial erythrocytes, a large dose of liposomes should be administered so that the problem of liposome agglutination in plasma was not negligible. Heretofore, however, there has been developed no technique at all for preventing the agglutination of liposomes in plasma.

In addition, when liposomes are introduced into the living body, antibody protein (immunoglobulin) to the liposome which is an antigen will be adsorbed on the liposomes to produce foreign body recognition in the phagocytes (macrophage) with a result that the liposomes will be included in the macrophage and disappear within a short period of time. Therefore, inhibition of the protein adsorption on liposomes could also delay disappearance of the liposomes in plasma.

It is also noted that hemoglobin concentration in 0 natural erythrocytes is approximately 30%; as volume ratio of erythrocytes to the whole blood (hematocrit) is approximately 50%, hemoglobin concentration in the whole blood is approximately 15%. Accordingly, in the case of artificial erythrocytes which are formed by enclosing hemoglobin in the liposome smaller in particle size than natural erythrocytes, volume ratio of artificial erytirocytes in an artificial erythrocyte suspension will S exceed 50% when hemoglobin concentration in the artificial erythrocyte suspension is 15%, unless an aqueous solution of hemoglobin with a hemoglobin concentration of 30% or more is subjected to liposome formation. Such suspension, which is poorly fluidized, will produce adverse effects upon circulatory dynamism when administered. In this respect, it is desirable to encapsulate a large amount of hemoglobin in the inner aqueous space of liposomes using lipid in an amount as small as possible. In other words, a method for 4 preparing artificial erythrocytes with a high encapsulation efficiency is desirable. By the dialysis method or the reverse phase method, however, it is difficult to form liposomes of an aqueous solution of hemoglobin with a higher hemoglobin concentration (30% or more) and a higher viscosity. Also by the lamina method in which a liposomeforming lipid is uniformly dissolved in an organic solvent, then the organic solvent is removed and an aqueous solution is added to the lamina of the lipid thus formed to a dispersion, the hydration and dispersion cannot easily be accomplished by the addition of an aqueous solution because the liposome-forming lipid after removal of the organic 8io* solvent has been solidified or nearly in loss of fluidity.

When the aqueous solution is an aqueous solution of hemoglobin with a high concentration, proportion of the water combined with the globin protein is high, and amount *9* of the free water available for hydration of the 'ipid is small. Thus, liposome formation at a high efficiency was difficult. Therefore, an object of the invention is to provide agents for inhibiting adsorption of proteins on the liposome surface, agents for preventing liposome agglutination, liposomes on which adsorption of proteins in plasma is inhibited and a method for preparing the same. A further object of the invention is to provide a method for preparing artificial erythrocytes comprising forming liposomes of a highly-concentrated hemoglobin at a high efficiency.

Summary of the Invention *0 *be 2008 .0 8 15 8s 8 86' As a result of extensive studies in order to achieve the above-mentioned objects we have found that adsorption of proteins in plasma on the surface of liposomes can be prevented by incorporating a specific agent for inhibiting adsorption of proteins into lipid layer of the liposome, eventually preventing agglutination of the liposomes each other and further facilitating hydration of the lipid even when artificial erythrocytes are prepared with an aqueous solution of hemoglobin at a high concentration thereby enabling formation of liposomes of a highly-concentrated hemoglobin at a high efficiency. The pres(,it invention was completed on the basis of the above findings.

Accordingly the invention provides liposomes containing thereon agents for inhibiting adsorption of proteins on the surface thereof.

The liposomes are preferably characterized by a hydrophilic macromolecular chain moiety bound at a first end to an external liposome membrane constituting lipid and having the second end externally extending from the liposome surface.

The hydrophilic moiety may be covalently bound at the second end to a hydrophobic moiety wherein the degree of polymerization for the hydrophilic moiety may be 5 to 1000 moles.

The hydrophilic moiety is most preferably polyethylene glycol.

mwspe#3848 92 1 2 -6- 0I see *see 0606 *0 0 .0 :2

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15 4 S S 4 *0 0 4 0505 The hydrophobic moiety is preferably a phosphilipid, most preferably phosphatidylethanolamine.

The liposomes most preferably contain hemoglobins in the inner phase.

The invention further provides a method for preparing liposomes on which adsorption of proteins is inhibited which comprising the addition of hydrcphilic macromolecular compound activated so as to bind with a liposome membrane-constituting lipid to a liposome suspension and allowing to react in such a way that one end of the hydrophilic macromolecule is bound with the liposome membrane-constituting lipid and the other end is extended externally from the liposome surface.

Preferably the liposome suspension is formed from an aqueous solution of a liposome forming phospholipid and an aqueous solution of hemoglobin wherein the mixture of the two solutions is blended to form liposomes containing hemoglobins therein.

The excess phospholipid and hemoglobin is then removed from the formed liposomes preferably by centrifugation or filtration. Finally, a solution of polyethylene glycol bound phospholipid is added to the liposome encapsulated hemoglobin resulting in liposomes bound only on their outer surface with the protein adsorption inhibition agent.

In a particularly preferred form the liposomes are formed by mixing a liposome membrane forming lipid with a phospholipid having a reaction-active functional group and the hydrophilic macromolecular compound is a mwspe13848 92 12 0see 0 0

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e g -7polyethylene glycol activated at one end. The polyethylene glycol bound phospholipid may be added to the liposome suspension.

Detailed Description of the Invention The agents for inhibiting adsorption of proteins on the liposome surface or the agents for preventing agglutination of liposomes in the present invention are compounds which have a hydrophobic moiety at one end and a hydrophilic macromolecular chain moiety at the other end.

As preferred examples of the hydrophobic moiety are mentioned alcoholic radicals of a long chain aliphatic alcohol, a sterol, a polyoxypropylene alkyl or a glycerin fatty acid ester and phospholipids. As preferred examples C.

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mwspe#3848 92 1 2 -8of the hydrophilic macromolecular chain moiety are mentioned polyethylene glycols.

Especially preferable in the invention are nonionic surface-active agents of PEG addition type in which a polyethylene glycol (called PEG hereinbelow) and an alcoholic radical of the hydrophobic moiety are bound by ether bond or PEG-bound phospholipids in which PEG and a phospholipid are covalently bound.

The polyethylene glycol-bound phospholipid in the 10 invention is a molecule of such a structure that polyethylene glycol (PEG) is covalently bound with the hydrophilic moiety (polar head) of a phospholipid which contains one or more PEG chains per molecule. The end of the PEG chain that has not been bound with the phospholipid may also be hydroxyl group or an ether with a short chain such as with methyl or ethyl or an ester with a short chain 9e 4 such as with acetic acid or lactic acid.

In order to achieve the objects of the invention, PEG chain length in the PEG-bound phospholipid molecule is desirably in the range of 5 1000 moles, more preferably 200 moles in terms of the average degree of polymerization. Below the above-defined range, the effect of preventing agglutination of liposomes in plasma will hardly be produced. Beyond the above-defined range, watersolubility of the PEG-bound phospholipid will be too high to be readily fixed inside the liposome membrane, -9- In order to produce a covalent bond between PEG and a phospholipid a reaction-active functional group is necessary at the polar moiety of the phospholipid. The functional group includes amino group of phosphatidylethanolamine, hydroxyl group of phosphatidylglycerol, carboxyl group of phosphatidylserine and the like; the amino group of phosphatidylethanolamine is preferably used.

For the formation of a covalent bond between the reaction-active functional group of a phospholipid and PEG 10 are mentioned a method employing cyanuric chloride, a method •employing a carbodiimide, a method employing an acid anhydride, a method employing glutaraldehyde and the like.

The method employing cyanuric chloride (2,4,6-trichloro-striazine) is preferably used for binding the amino group of phosphatidylethanolamine with PEG. For example, treatment of monomethoxypolyethylene glycol and cyanuric chloride by 0* 4 known reaction procedures affords 2-O-methoxypolyethylene glycol-4,6-dichloro-s-triazine (activated PEGL) or 2,4-bis- (O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2) Inada, et al., Chem. Lett., 7, 773-776 (1980)). Binding of these with the amino group by a dehydrochloric acid condensation reaction yields a phospholipid with PEG covalently bound with the polar head of phosphatidylethanolamine. In the above reaction there is contained one PEG chain in one phospholipid molecule when employing activated PEG1 and two PEG chains with activated 10 PEG2. Phospholipids bound with PEG v-a amide bond is also produced by reacting monomethoxy PEG with succinic anhydride to introduce a carboxyl group into the end of the PEG and reacting the product with phosphatidylethanolamine in the presence of a carbodiimide.

In order to prepare a liposome with the PEG-bound phospholipid of the invention contained in the lipid layer, a PEG-bound phospholipid may uniformly be mixed with a liposome-forming lipid in advance, and the lipid mixture may be treated by a conventional method to form liposomes. The liposome-forming lipids as herein referred to contain as the

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66 main component phospholipids obtained from natural materials such as egg yolk and soybean or those which are produced by Sooo.! organic chemical synthesis used alone or in combination.

0 Representative are phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. In 4 addition, sterols such as cholesterol and cholestanol as a membrane-stabilizing agent, phosphatidic acid, dicetyl phosphate and higher fatty acids as a char3ed substance and other additives may be added. Mixing ratio of the PEG-bound phospholipid with the liposome-forming lipid is 0.1 mol%, preferably 0,5 20 mol% and more preferably 1 mol% in terms of the molar ratio to the phospholipid of the main component. Below the above-defined range, the effect of preventing agglutination of liposomes in plasma will not be sufficiently high. Beyond the above-defined range, 11 solubilizing capacity of the PEG-bound phospholipid will cause instabilization of the liposomne.

In effecting in advance uniform mixing of the liposome-forming lipid with the PEG-bound phospholipid, for example, the two may be dissolved in a volatile organic solvent and then the organic solvent removed by evaporation.

If a fat-soluble drug is to be contained in the liposomes, it may be mixed with the liposome-forming lipid during the above procedures. Formation of liposomes from the mixed 10 lipids thus obtained may be carried out according to a liposome formation method usually employed. For example, any of such methods as shaking, sonication and French pressure cell may be employed. Liposomes of particle sizes between 0.1 um and 1 um are produced allowing for carrying a sufficient amount of a water-soluble drug or physiologically active substance in the inner aqueous space, provided that the above-mentioned PEG-bound phospholipid is used within 9 S the above-defined ranges. The PEG-bcund phospholipid is contained in the lipid layr- of liposomes thus obtained, but the content is not necessarily the same as that based upon the proportion originally mixed with the lipid. If water solubility of the PEG-bound phospholipid is high, part of it will possibly be eluted into the aqueous phase outside the membrane. Although the form of the PEG-bound phospholipid present in the lipid membrane of liposome is not clear, it is believed that the hydrophobic moiety of the PEG-bound 12 phcspholipid is present in the hydrophobic region of the liposome membrane, and the hydrophi.ic PEG chain i7 present from the hydrophilic region in the membrane over to the aqueous medium outside the membrane. It follows therefore that the PEG chain of the PEG-bound phospholipid in the liposome obtained by the method of the invention is present in both of the outer aqueous phase and the inner aqueous space of the liposome.

The PEG-bound phospholipid of the invention need 10 not necessarily give a clear solution when dissolved in water. However, if the PEG-bound phospholipid of the invention is uniformly dissoled in water, the liposome of the invention may also be prepared by an alternative method.

As a matter of fact, liposomes containing the PEG-bound phospholipid in the lipid layer may also be prepared as follows: To a suspension of liposomes carrying a watersoluble or fat-soluble drug or the like (which have been prepared by a conventionally employed liposome formation .method) is added the PEG-bound phcspholipid of the invention either as it is or in aqueous solution. In this case, the PEG-bound phospholipid appears to be in dispersion in the form of micelle-like molecular aggregates in the aqueous solution. When liposomes are co-existent in the dispersion, the hydrophobic moiety in the PEG-bound phospholipid molecule is fixed in the hydrophobic region in the liposome membrane by hydrophobic interaction thereby taking a 13 structure in which the hydrophilic PEG chain is exposed on the surface of liposomes on the side of the outer aqueous phase only.

Addition of the PEG-bound phospholipid in aqueous solution may be made at the critical micelle concentration or higher. At a lower concentration, however, amount of the phospholipid adsorbed on the liposome will not be sufficient to maintain the effect of preventing agglutination of liposomes in plasma. At a too high concentration, the 10 liposome will be so unstable as eventually to cause leakage s of the water-soluble drug or the like carried in the inner aqueous space. Therefore, the concentration is preferably 0.01 20%, more preferably 0.05 20% in terms of the concentration in the liposome suspension.

Liposomes containing the PEG-bound phospholipid in the lipid layer can also be prepared by an alternative method. As a matter of fact, liposomes containing a phospholipid with a reaction-active functional group are prepared by a conventional method, and subsequently a PEG activated at one end is added to the outer solution of the liposomes to allow for binding with the phospholipid. For example, liposomes containing 1 50 mol% of phosphatidylethanolamine in the whole phospholipid are prepared, activated PEG2 in a basic buffer solution (pH 9 or higher) is added at a concentration of 1 20% and the mixture is allowed to react at room temperature for 1 24 14 hours. There is formed a structure in which the hydrophilic PEG chain is exposed on the surface on the side of the outer aqueous phase of the liposomes.

The non-ionic surface-active agent of polyoxyethylene ether addition type as referred to in the invention is a non-ionic surface active agent having a molecular structure that contains a polyoxyethylene chain as the hydrophilic moiety and in which the polyoxyethylene chain is bound with an alcoholic radical of the lipophilic (hydrophobic) moiety by ether bond. It includes, for example, polyoxyethylene alkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene polyoxypropylene block polymers, *k 4 S polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters and the like, "d Among non-ionic surface active agents of polyoxyethylene addition type, a non-ionic surface active S agent of polyoxyethylene ester addition type that has a molecular structure in which the polyoxyethylene chain is bound with the lipophilic moiety by ester bond is contained in the lipid layer of liposomes will produce a low effect in inhibiting adsorption of proteins in plasma and preventing agglutination of liposomes.

In order to achieve the objects of the invention the polyoxyethylene chain length in the non-ionic surface active agent of polyoxyethylene ether addition type is desirably in the range of 5 1000 moles, more preferably 40 moles in terms of the average degree of polymerization of ethylene oxide. Below the above-defined range, the effect of preventing agglutination of liposomes in plasma will hardly be developed. Beyond the above-defined range, water solubility of the non-ionic surface active agent will become too high to be readily fixed in the liposome membrane.

Among a variety of non-ionic surface active agents of polyoxyethylene ether addition type, polyoxyethylene alkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene U polyoxypropylene alkyl ethers and polyoxyethylene glycerin fatty acid esters are particularly effective in producing liposomes of high protein adsorption-inhibitory and agglutination-preventive effects when contained in the lipid layer of liposomes.

Polyoxvethylene alkyl ethers have a structure in which a polyoxyethylene and a saturated or unsaturated 9 aliphatic alcohol are bound by ether bond. Aliphatic alcohols having 8 22 carbon atoms are preferably employed.

The polyoxyethylene sterol ethers are compounds having a molecular structure in which a polyoxyethylene and a sterol are bound by ether bond. The sterol includes animal sterols (zoosterols) such as cholesterol and cholestanol, plant sterols (phytosterols) such as sitosterol 16 and stigmasterol and fungal sterols (mycosterols) such as ergosterol and zymosterol. Although it is not necessary to specifiy nature of the sterol in the polyoxyethylene sterol esters, those which have the same structure in the side chain as that of cholesterol are preferably used.

The polyoxyethylene polyoxypropylene alkyl ethers have a molecular structure in which a polyoxypropylene is added to a saturated or unsaturated aliphatic alcohol by ether bond, and to the end hydroxyl group of the polyoxypropylene is further added a polyoxyethylene by ether p.

r bond. Average degree of polymerization for the S" polyoxypropylene is preferably 2 8, and aliphatic alcohols

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having 8 22 carbon atoms are preferably employed.

The polyoxyethylene glycerin fatty acid esters have a molecular structure in which a polyoxyethylene is added to the free hydroxyl group of a glycerin fatty acid S. ester (mcnoglyceride or diglyceride). Either saturated or unsaturated fatty acids having 8 22 carbon atoms are preferably employed.

In order to prepare a liposome containing the nonionic surface active agent of polyoxyethylene ether addition type in the lipid layer according to the invention, a nonionic surface active agent of polyoxyethylene ether addition type may uniformly be mixed with a liposome-forming lipid in advance, and the lipid mixture may be treated by a conventional method to form liposomes. The liposome-forming 17 lipid as herein referred to contains as the main component phospholipids obtained from natural materials such as egg yolk and soybean or those which are produced by organic chemical synthesis used alone or in combination.

Representative are phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. In addition, sterols such as cholesterol or cholestanol as a membrane-stabilizing agent, phosphatidic acid, decetyl phosphate and higher fatty acids as a charqe-providing 1C substance and other additives may be added. Mixing ratio of e the non-ionic surface active agent of polyoxyethylene ether .e addition type with the liposome-forming lipid is 0.5 0* moles, preferably 1 5 moles of ethylene oxide unit per mole of the phospholipid of the main component, For t 0 example, when dipalmitoylphosphatidylcholine (molecular weight 752) as the phospholipid and polyoxyethylene qu. phytostanol ether with an average degree of polymerization of 25 for ethylene oxide (molecular weight ca. 1500) as the non-ionic surface active agent of poiyoxyethylene ether addition type are used, molar ratio of the non-ionic surface active agent of polyoxyethylene ether addition type is a 0.02 0.8 moles, preferably 0.04 0.2 moles per mole of the phospholipid, and weight ratio is 0.04 1.6 parts by weight, preferably 0.08 0.4 parts by weight of the nonionic surface active agent of polyoxyethylene ether addition type per part by weight of the phospholipid. Below the 18 above-defined range, the effect of preventing agglutination of liposomes will not be sufficient. Beyond the abofedefined range, the liposomes will be unstable due to solubilizing capacity of the non-ionic surface active agent of polyoxyethylene ether addition type.

In order to effect in advance uniform mixing of a surface active agent of polyoxyethylene ether addition type with a liposome-forming lipid, for example, the two may be dissolved in a volatile organic solvent, and then the 10 organic solvent removed by evaporation. If a fat-soluble drug is to be contained in the liposome, it may be mixed with the liposome-forming lipid during the aL ve procedures.

Formation of liposomes from the mixed lipids obtained S* may be carried out according to a conventioral liposome formation method. For example, any of such methods as shaking, sonication and 'rench pressure cell may be employed. Liposomes with particle sizes of 0.1 I um can be produced allowing for carrying a sufficient amount of a water-soluble drug or physiologically active substance in the inner aqueous space, provided that the "bove-mentioned non-ionic surface active agent of polyoxyethylene ether addition type is used within the above-defined ringe. The non-ionic surface active agent of polyoxyethylene ether addition type is contained in the lipid layer of liposomes thus obtained, but the content is not necessarily the same as that basec u'pon the proportion originally mixed with the 19 lipid. If water solubility of the non-ionic surface active agent of polyoxyethylene ether addition type is high, part of it will possibly be eluted into the aqueous phase outside the membrane. Although the form of the non-ionic surface active agent of polyoxyethylene ether addition type present in the lipid membrane of liposomes is not clear, it is believed that the hydrophobic moiety of the molecule of nonionic surface active agent of polyoxyethylene ether addition type is present in the hydrophilic region of the liposome Q membrane and the hydrophilic polyoxyethylene chain is present from the hydrophilic region in the membrane over to the aqueous medium outside the membrane. I' follows oo ~therefore that the polyoxyethylene chain of the non-ionic surface active agent of plyoxyethylene ether addition type obtained by the method of the invention is present in both of the outer aqueous phase and tne inner aqueous space of the liposome.

The non-ionic surface active agent of polyoxyethylene ether addition type of the invention need not necessarily give a clear solution when dissolved in

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water. However, if the non-ionic surface active agent of polyoxyethylene ether addi' 'n type of the invention is uniformly dissolved in water, the liposome of the invention may also be prepared by an alternative method. As a matter of fact, liposomes containing the non-ionic surface active agent of polyoxyethylene ether addition type of the 20 invention in the lipid layer may also be prepared as follows: To a suspension of liposomes carrying a watersoluble or fat-soluble drug or the like (which have been prepared by a liposome formation method generally employed) is added the non-ionic surface active agent of polyoxyethylene ether addition type of the invention either as it is or in aqueojs solution. In this case, the nonio,,ic surface active agent of polyoxyethylene ether addition type is dispersed in the form of micelles in the aqueous solution. When liposomes are co-existent in the dispersion, the hydrophobic moiety in the non-ionic surface active agent molecule of po3yoxyethylene ether addition type is fixed in the hydrophobic region in the liposome membrane by hydrophobic interaction thereby taking a structure in which the hydrophilic polyoxyethylene chain is exposed on the surface of liposomes on the side of the outer aqueous phase only.

Addition of the non-ionic surface active agent of polyoxyethylene ether addition type in an aqueous solution may be made at the critical micelle concentration or higher.

At a lower concentration, however, the amount adsorbed on the liposome will not be sufficient to maintain the effect of preventing agglutination of liposomes in plasma, At a too high concentration, the liposome will be so unstable as eventually to cause leakage of the water-soluble drug or the like carried in the inner aqueous space. Therefore, the 21 concentration is preferably 0.01 more preferably 0.1 2% in terms of the concentration in the liposome suspension.

When artificial erythrocytes are prepared, mixing ratio of the non-ionic surface active agent to the liposomeforming lipid is preferably 0.5 30% by weight. Below the above-defined range, formation of hemoglobin liposomes will hardly be achieved at a high efficiency. Beyond the abovedefined range, solubilizing capacity of the non-ionic surface active agent will unstabilize the artificial i 'A erythrocytes formed.

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The liposome-forming lipid used in the invention *0 •Oe* is phospholipids obtained from natural materials such as egg yolk and soybean or those which are produced by organ.i chemical synthesis. They are used as the main component either alone or in combination. Representative are phosphatidylcholine (lecithin), sphingomyelin, phosphatidylethanolamine and phosphatidylserine. In addition, sterols such as cholesterol and cholestanol as a membrane-stabilizing agent, phosphatidic acid, dicetyl phosphate and higher fatty acids as a charge-providing ~substance and other additives may also be added.

If the phospholipid contains an unsaturated bond, there occur such special problems that lipid peroxides generated by peroxidation reaction of the unsaturated bond may be toxic, and the enclosed hemoglobin is liable to oxidative degradation. Therefore, hydrogenation products to 22 the unsaturated group are preferably used. For example, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin and the like are mentioned as hydrogenated natural phospholipid readily available. When such a hydrogenated natural phospholipid is employed as the main component, the phase transition temperature is as high as about 50"C. In general, liposomes are hardly formed unless the operation is carried out at the phase transition temperature or higher.

However, hemoglobin will be heat degraded if formation of *Q hemoglobin liposomes is operated at 40*C or higher. If Se sterols are contained in the liposome-forming lipid, there is no definite phase transition temperature for the whole r/ S lipid mixture, and artificial erythrocytes can be prepared e satisfactorily even when operated at a temperature below the phase transition temperature of the lipid main component.

Higher fatty acids are preferably employed as a char-e- 0 providing substance which is usually contained in order to prevent mutual agglutination of the formed artificial erythrocytes. Adequately, mixing ratios in these liposomeforming lipids are 0.2 1 part by weight of sterols and 0.05 0.2 parts by weight of higher fatty acids per part by weight of the phospholipid.

In order to prepare a mixture of a non-ionic surface active agent and a liposome-forming lipid the two may uniformly be dissolved in a volatile organic solvent capable of uniformly dissolving the non-ionic surface active 23 agent and the Iliposome- forming lipid and then thie orqanic solvent removed by such a method as evaporation, freezedrying or spray-drying.

In order to form artificial erythrocytes from the mixed lipid obtained, said mixed lipid may be hydrated and dispersed in an aqueous solution of hemoglobin. Whereas the hydration and dispersion may be effected merely by mechanically mixing the two, it is desirable to add high pressure -delivery treatment using such a machine as a French pressure cell. H-emoglobin concentration in the aqueous solution of hemoglobin is preferably 30 60%. Below the above-defined range, encapsulation efficiency of the hemoglobin will be low. Beyond the above-defi.ned range, viscosity of %he aqueous solution of hemoglobin will be so much increased that the hydration and dispersion wi.ll be difficult even when a non-ioni~c surface act~ve agent is added.

lin the method for preparing atf~a erythrocytes according to the invention in which a liposotneforming lipid with hydrogenated phospholipids, sterols or higher fatty acids mixed and an aqueous solution of hemoglobin in the above-defined range are used, there are produced almost none of the artificial erythrocytes with particle sizes of 0.01 0.03 pim having very low hemoglobin encapsulation efficiency, but for the most part, artificial erythrocytes with particle sizes of 0.1 prn or larger having 24 high hemoglobin encapsulation efficiency.

In the lipid layer of the artificial erythrocytes thus obtained is contained the non-ionic surface active agent content of which is not necessarily be the same as that based upon the initial mixing ratio with the lipid. In case where water solubility of the non-ionic surface active agent is high, part of it will possibly be eluted into the aqueous phase outside the membrane.

The invention will be described in more detail %GoQ below with reference to Examples and Comparative Examples.

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.o Example In 20 ml of dichioromethane were dissolved 630 mg of hydrooenated egg yolk lecithin, 317 mg of cholesterol, 53 mg of my Atic acid and 150 mg of polyoxyethylene phytostanol ether (average degree of polymerization for ethylene oxide 25, BPSH 25 manufactured by Niko Chemicals The organic solvent was removed by evaporation. To the mixed lipid thus obtained was added 20 ml of 50% aqueous solution of hemoglobin. The mixture was ble ided by shaking followed by French pressure cell under a pressure of 250 2 kg/cm The treatment was repeated ten times, and the liquor obtained from the treatment through French pressure cell was 1:10 diluted with physiological saline solution and subjected to centrifugal separation (17,000 r.p.m. for min.). The liposome precipitates were subjected to 25 add:iional centrifugal washing with two portions of 140 ml of physiological saline solution. The liposome precipitates after the washing were suspended in physiological saline solution to a hemoglobin concentration of Average particle size of the liposomes thus obtained was 0.2 pm.

With 0.1 ml of the liposome suspension was mixei 0.5 ml of citrate-containing human plasma. The mixture was observed under optical microscope (x 400) to find almost none of liposome agglutinates exceeding 1 um in size.

0'.10 Example 2 *0S In 20 ml of dichloromethane were dissolved 630 mg of hydrogenated egg yolk lecithin, 31/ mg of cholesterol and 53 mg of myristic acid. The organic solvent was removed by 3 ovaporati.on. To the mixed lipid thus obtained was added ml of 50% aqueous solution of hemoglobin. The mixture was blended by shaking followed by French pressure cell under a S /m2 pressure of 500 kg/cm The treatment was repeated ten times, and the liquor obtained was 1:10 diluted with S* physiological saline solution and subjected to centrifugal separation (17,000 r.p.m. for 30 min.). The liposome precipitates were subjected to additional centrifugal washing with two portions of 140 ml of physiological saline solution. The liposome precipitates after the washing were suspended in physiological saline solution to a hemoglobin concentration of Average particle size of the liposomes thus obtained was 0.2 pm. With 0.1 ml of the liposome 26 suspension was mixed 0.5 ml of citrate-containing human plasma. The mixture was observed under optical microscope (x 400) to find that the liposomes were completely agglutinated to agglutinates exceeding 50 pm in size.

To 1 ml of the above-mentioned liposome suspension adjusted to a hemoglobin concentration of 5% was added 9 ml of physiological saline solution containing 2% polyoxyethylene oleyl ether (average degree of polymerization for ethylene oxide 20). The mixture was 0 allowed to stand at room temperature for 30 min., 1:10 diluted with physiological saline solution and subjected to O centrifugal separation (17,000 r.p.m. for 30 min.). The

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S liposome precipitates were subjected to additional centrifugal washing with two portions of 140 ml of physiological saline solution. The liposome precipitates after the washing was suspended in physiological saline solution to a hemoglobin concentration of With 0.1 ml of the liposome suspension was mixed 0.5 ml of citratecontaining human plasma. The mixture was observed under 20 optical microscope (x 400) to find almost none of the

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liposome agglutinates exceeding 1 um in size.

Example 3 Investigations were made exactly in the same way as in Example 1 except that 150 mg of polyoxyethylene polyoxypropylene cetyl ether (average degree of polymerization for ethylene oxide 20 and for propylene oxide 27 8) was used in place of the polyoxyethylene phytostanol used in Example 1. There were produced the same results as in Example 1.

Example 4 Investigations were made exactly in the same way as in Example 1 except that 150 mg of polyoxyethylene glyceryl distearate (average degree of polymerization for ethylene oxide 30) was used in place of the polyoxyethylene phytostanol used in Example 1. There were produced the same results as in Example 1.

Comparative Example 1 Investigations were made exactly in the same way aS in Example 1 except that 150 mg of polyoxyethylene 005060 moncstearate with an average degree of polymerization for ethylene oxide of 25 was used in place of the polyoxyethylene phytostanol ether used in Example 1. The liposomes were completely agglutinated. The agglutinates exceed 50 um in size.

In addition the same results were also produced with polyoxyethylene distearate (n=10 or 140).

e Comparative Example 2 When polyoxyethylene monostearate was used in place of the polyoxyethylene oleyl ether used in Example 2, the liposomes were completely agglutinated. The agglutinates exceed 50 pm in size.

Example 28 In 20 ml of dichloromethane were dissolved 1.81 g of hydrogenated egg yolk lecithin, 0.913 g of cholesterol, 0.153 g of myristic acid and 0.142 g of polyoxyethylene phytostanol (average degree of polymerization for ethylene oxide 25, BPSH 25 manufactured by Nikko Chemicals as a non-ionic surface active agent. The organic solvent was removed by evaporation. To the mixed lipid thus obtained was added 20 ml of 50% aqueous solution of hemoglobin. The mixture wa, blended by shaking followed by French pressure 't0O cell under a pressure of 250 kg/cm 2 The treatment was repeated ten times, and the liquor obtained was 1:10 diluted with physiological saline solution. The dilu'ion was filtered through a filter with a pore size of 0.45 um and *06 *0 then subjected to centrifugal separation (17,000 r.p.m. for 30 min.). The liposome precipitates were subjected to additional centrifugal washing with two portions of 140 ml of physiological saline solution. As the artificial erythrocytes that will be low in efficiency of hemoglobin encapsulation are not precipitated due to their low specific gravity and removed during the above operations. The artificial erythrocyte precipitates after the washing were suspended in physiological saline solution to a hemoglobin concentration of Average particle size of the artificial erythrocytes thus obtained was 0.2 um. The entire lipid concentration in the artificial erythrocyte suspension was 33 mg/ml, and recovery ratio of the 29 hemoglobin was 12%.

When exactly the same operations as above were conducted but without adding a non-ionic surface active agent, there were produced artificial erythrocytes with an average particle size of 0.2 pm. The entire lipid concentration in the artificial erythrocyte suspension adjusted to a hemoglobin concentration of 5% was 39 mg/ml, and recovery ratio of the hemoglobin was 7%.

Example 6 10 In 50 ml of dehydrated chloroform were dissolved 150 mg of dipalmitoylphosphatidylethanolamine and 2.5 g of *ego activated PEG2 (average molecular weight of PEG 5,000 x 2,

**S

manufactured by Seikagaku Kogyo To the solution was added 2 g of sodium carbonate, and the mixture was allowed to react overnight at room temperature. After confirming completion of the reaction by disappearance of the ninhydrin color reaction the reaction mixture was filtered, and hexane was added to the filtrate for purification by reprecipitation. The purified product was dried in vacuo to obtain a PEG-bound phospholipid.

I In 20 ml of dichloromethane were dissolved 630 mg of hydrogenated egg yolk lecithin, 317 mg of cholesterol, 53 mg of myristic acid and 150 mg of the above-obtained PEGbound phospholipid. The organic solvent was removed by evaporation. To the mixed lipid thus obtairnd was added ml of 50% aqueous solution of hemoglobin. The mixture was 30 blended by shaking followed by French pressure cell under a 2 pressure of 250 kg/cm The treatment was repeated ten times, and the liquor obtained was 1:10 diluted with physiological sillJne solution and subjected to centrifugal separation (17,000 r.p.m. for 30 min.).

The liposome precipitates were subjected to additional centrifugal washing with two portions of 140 ml of physiological saline solution. The liposome precipitates after the washing were suspended in physiological saline o 010 solution to a hemoglobin concentration of Average particle size of the liposomes thus obtained was 0.2 tm.

With 0. 1 ml of the liposome suspension was mixed 0.5 ml of *see citrate-containing human plasma. The mixture was observed under optical microscope to find almost none of liposome agglutinates exceeding I uim in size.

Example 7 te, as In 20 ml of dichloromethane ,ere dissolved 630 mg of hydrogenated egg yolk lecithin, 317 mg of cholesterol and S• 33 mg of myristic acid. The organic solvent was removed by evaporation. To the mixed lipid thus obtained was added ml of 50% aqueous solution of hemoglobin. The mixture was blended by shaking followed by French pressure cell under a 2 pressure of 500 kg/cm The treatment was repeated ten times, and the liquor obtained was 1:10 diluted with physiological saline solution and subjected to centrifugal separation (17,000 r.p.m. for 30 min.). The liposome "1 I 31 precipitates were subjected to additional centrifugal washing with two portions of 140 ml of physiological saline solution. The liposome precipitates after the washing were suspended in physiological saline solution to a hemoglobin concentration of Average particle size of the liposomes thus obtained was 0.2 pm. With 0.1 ml of the liposome suspension was mixed 0.5 ml of citrate-containing human plasma. The mixture was observed under optical microscope (x 400) to find liposomes completely agglutinated. Size of 10 the agglutinates exceeded 50 um.

To 1 ml of the above-prepared liposome suspension adjusted to a hemoglobin concentration of 5% was added 9 ml of physiological saline solution containing 1% of the PEGcombined phospholipid obtained in Example 6. The mixture was allowed to stand at room temperature for 30 min., then 1:10 diluted with physiological saline solution and subjected to centrifugal separation (17,000 r.p.m. for min.). The liposome precipitates were subjected to additional centrifugal washing with two portions of 140 ml of physiological saline solution. The liposome precipitates •after the washing were suspended in physiological saline solution to a hemoglobin concentration of With 0.1 ml of the liposome suspension was mixed 0.5 ml of citratecontaining human plasma. The mixture was observed under optical microscope (x 400) to find almost none of the liposome agglutinates exceeding 1 um in size.

32 Example 8 The same procedures as in Example 7 were repeated except that hydrogenated soybean lecithin containing 30 mol% of phosphatidylethanolamine was used in place of the hydrogenated egg yolk lecithin to obtain hemoglobincontaining liposomes. To 1 ml of a suspension of the aboveprepared liposomes adjusted with 0.1 M borate buffer solution (pH 10) to a hemoglobin concentration of 5% was added 100 mg of activated PEG2. The mixture was allowed to 0'.10 react overnight at room temperature. The reaction mixture was 1:10 diluted with physiological saline solution and subjected to centrifugal separation (17,000 r.p.m. for min.). The liposome precipitates were subjected to additional centrifugal washing with two portions of 140 ml of physiological saline solution. The liposome precipitates after the washing were suspended in physiological saline o* C solution to a hemoglobin concentration of With 0.1 ml of the liposome suspension was mixed 0.5 ml of citrate- 0 containing human plasma. The mixture was observed under optical microscope (x 400) to find almost none of the liposome agglutinates exceeding 1 um in size.

Example 9 To a solution of 50 g of monomethoxy PEG5000 (manufactured by Union Carbide; in 250 ml of 1,2dichloromethane were added 5 g of succinic anhydride and 4 ml of pyridine. The mixture was boiled under reflux for 4 4 33 days. The reaction mixture was filtered, subjected to evaporation and dissolved in 100 ml of distilled water. The aqueous phase was washed with ether and then extracted with 100 ml of chloroform. After evaporated the residue was recrystallized from ethyl acetate to give monocarboxyterminated PEG. In 30 ml of chloroform were dissolved 725 mg of the PEG, 100 mg of dipalmitoylphosphatidylethanolamine and 30 mg of dicyclohexylcarbodiimide. The solution was allowed to react overnight at 50 0 C. The reaction mixture S10 was subjected to re-precipitation with 300 ml of hexane.

There was. obtained phospholipid bound via amide bond with PEG. The same results as in Examples 6 and 7 were produced in an experiment using the phospholipid.

The claims form part of the disclosure of this specification.

a 9 a g O

Claims (14)

1. A liposome characterized in containing on the outer surface constituting lipid thereof, an agent for inhibiting adsorption of proteins.

2. A liposome according to claim 1 wherein said agent a Lydrophilic macromolecular chain moiety bound at a .rst end to said outer surface constituting lipid and 'iaving a second end externally extending from the said our-.- surface of said liposome.

3. A liposome according to claim 2 wherein the hydrophilic moiety may be covalently bound at said second end to a hydrophobic moiety.

4. A liposome according to claim 3 wherein said hydrophilic moiety has a degree of polymerization from 5 to 1000 moles. A liposome according to any one of claims 1 to 4 wherein the hydrophilic moiety is polyethylene glycol.

6. A liposome according to any one of claims 3 to wherein the hydrophobic moiety is a phospholipid.

7. A liposome according to claim 6 wherein the phospholipid is phosphatidylethanolamine.

8. A liposome according to any one of claims 1 to 7 wherein said liposomes contain hemoglobin in the inner phase.

9. A method of preparing liposomes characterized by adding to a liposome suspension, a hydrophilic macromolecular compound activated so as to bind with the liposome membrane constituting lipid of said suspension, such that one end of the hydrophilic macromolecule is mwspeII3848 92 1 2 9 06 1551 IA 0o 0 2 4* S. p ~C 20 bound with the liposome membrane constituting lipid and the other end is extended externally from the liposome surface wherein said liposomes are inhibited from adsorption of proteins.

10. A method according to claim 9 wherein the liposome suspension is formed from an aqueous solution of a liposome forming phospholipid and an aqueous solution of hemoglobin, blended to form liposomes containing hemoglobins in the internal phase.

11. A method according to claims I or 10 where the hydrophilic macromolecule is polyethylene glycol bound phospholipid.

12. A method according to any one of claims 9 to 11 wherein the hydrophilic macromolecule 17 added to the liposome suspension after formation of liposome vesicles such that only the outer phospholipid layer of said liposomes is bound with said hydrophilic macromolecular compound.

13. A method according to claim 9 where the liposomes are formed by mixing a liposome membrane-forming lipid with a phospholipid having a reaction-active functional group and the hydrophilic macromolecular compound is a polyethylene glycol activated at one end.

14. A method according to claim 11 where polyethylene glycol bound phospholipid is added to the liposome suspension. A method according to any one of claims 1 to 14 substantially as hereinbefore described. tnwspeI38948 92 1 2 36 DATED this 2 January 1992 CARTER SMITH BEADLE Fellows Institute of Patent Attorneys of Australia Patent Attorneys f or -the Applicant: TERUMO KABUSHIKI KAISHA rnwspeOI848 9 92 1 2 4 55 37 ABSTRACT A liposome charac~t'~rized in containing on the outer surface constituting lipid the~eof, an agent for inhibiting adsorption of proteins. '0 Me *00 00 9. 0060 C 9 *OSS 00.0 6 S OOOSSO 6 00 S je C0 000) C0 I 0 0 00 36 0 rnWSP"113848

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