WO1988006439A2

WO1988006439A2 - Phospholipase a2-resistant liposomes

Info

Publication number: WO1988006439A2
Application number: PCT/US1988/000508
Authority: WO
Inventors: Wolfgang J. Baumann
Original assignee: Regents Of The University Of Minnesota
Priority date: 1987-02-24
Filing date: 1988-02-19
Publication date: 1988-09-07
Also published as: WO1988006439A3

Abstract

A method to inhibit phospholipase A2-type hydrolytic degradation of liposome membranes is disclosed comprising forming a liposome having a bilayer membrane comprising (i) between about 67.5-85 mol-% of a phospholipid of formula (I), and (ii) between about 15.0-32.5 mol-% of a lysophospholipid of formula (II), wherein R2, R1 and R3 are individually (C10-C30) alkanoyl groups including from about 0-4 double bonds.

Description

PHOSPHOLIPASE A₂-RESISTANT LIPOSOMES

Background of the Invention This invention was made with the assistance of National Institutes of Health Grant HL08214. The Government has certain rights in this invention.

Liposomes are fluid-filled vesicles of varying sizes which have phosoholipid bilayer membranes. A liposome may have a single bilayer membrane, in which case it is unilamellar, or it may have multiple bilayer membranes, in which case it is multilameliar. In recent years, liposomes have been widely employed for the delivery of cosmetics, diagnostic reagents and bioactive substances such as herbicides, insecticides and therapeutic drugs. Liposomes are of special interest as vehicles for in vivo drug delivery because they can be designed to deliver and release drugs directly into target tissues. Therefore, general systemic drug delivery can be avoided and the therapeutic ratio of effectiveness over toxicity can be generally increased.

Since their discovery by A. D. Bangham in 1961, liposomes have been loaded with a variety of therapeutic agents. Liposomes are desirable drug delivery vehicles for a number of reasons. The bilayer memorane is generally composed of natural constituents such as phospholipids, which are found in the body, and are nontoxic and nonimmunogenic. Therefore, gaining regulatory approval for their use in human subjects has been far less burdensome than for many other delivery vehicles. In addition, once a liposome has been directed to diseased tissue, it is unique in its ability to adsorb onto the cell surface, whereupon it may be endocytosed, or swallowed up. Adsorbed liposomes can also fuse with calls, thereby releasing their contents directly into the cell in high concentration. Therefore, if liposomes retain their integrity, they can be selectively directed to diseased tissues, where they can release drugs directly into diseased ceils, thereby obviating the need for general systemic exposure to drugs and their inherent toxic effects. Methods of directing liposomes to diseased tissues are known. For example, tissue-specific monoclonal antibodies can be attached tα the liposomes to guide them to target tissues. Although precise delivery techniques are still being perfected, it is likely that new therapies based on such liposome technology will soon be available. A need, however, exists for further development of liposomes which retain their integrity. The liposomal membrane can be formed when phospholipids such as phosphatidylcholine are dispersed in water. Phospholipids are generally amphipathic, meaning they have hydrophobic regions, or "tails", and hydrophilic regions, or "heads". Two fatty acid chains generally containing from 10-30 carbon atoms make up the hydrophobic tail of most naturally-occurring phospholipid molecules. Phosphoric acid bound to any of several water soluble molecules compose the hydrophilic head. In the phospholipid bilayer membrane, the hydrophobic fatty acid tails point into the hydrophobic environment of the membrane's interior, and the polar head groups point outward to interact with water on both the inside and the outside of the vesicle. Although liposomes employed for drug delivery typically range in diameter from 250A to several micrometers, small unilamellar vesicles (SUV) in the range of 250-30θA are particularly desirable for use as drug vehicles because of their size. SUVs appear to exhibit increased partitioning to the bone marrow and also exhibit increased longevity in the circulatory system when delivered intravenously. Smaller vesicles have also been reported to be more effective in subcutaneous injections to deliver drugs to lymph nodes. However, liposomes, including SUVs, are often unstable during long-term storage and upon infusion into mammalian systems. The reason for the lack of physical stability has not been well understood. With regard to stability within mammalian systems, however, it is known that phospholipids are substrates for enzymes such as phospholipase A₂, lecithin-cholesterol acyltransferase (LCAT) and the like, which are found in vivo. Phosphatidylcholine, or lecithin, which is perhaos the most commonly employed phospholipid bilayer constituent, is attacked at the C-2 position by phospholipase A2 which cleaves the acyl group attached to the C-2 carbon of the glycerol moiety. Other enzymes, including LCAT, also catalyze phosohclipase A₂-type cleavage reactions of a variety of phospholipids. When these enzymes attack the phospholipid constituents of the bilayer membrane in vivo, the liposome membrane is destabilized and its contents will leak out.

Therefore, a need exists for liposomes, especially SUVs, which are physically stable and which resist phospholipase A₂-type cleavage. An additional need exists for a method to physically stabilize and to prevent the hydrolytic degradation of liposomal phospholipid bilayers by phospholipase A₂-type cleavage.

Summary of the Invention The present invention provides a method to inhibit the phospholipase A₂-type cleavage of a liposome membrane comprising: forming a liposome having a bilayer membrane comprising (i) between about 67.5-85 mol-% of a phospholipid of the formula (I):

and (ii) between about 15-32.5 mol-% of a lysophospholipid of the formula (II):

wherein, in formulas I and II, R₁, R₂ and R₃ are individually (C₁₀-C₃₀)alkanoyl groups including from 0-4 double bonds, and preferably are individually (C₁₂-C₂₂)-n-alkanoyl groups, including 0-1 double bonds. Preferably R₁ is identical to R₃. For example, in one embodiment of the invention, the phospholipase A₂ (PLA₂) hydrolysis of small unilamellar vesicle (SUV) liposomes having membranes comprising a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and about 20-30 mol-% 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC) was completely innibited.

In liposomes containing phospholipid (I) and lysophospholipid (II), the initial rate of PLA₂ hydrolysis of phospholipid I correlates with the ratio of lysophospholipid (II) to phospholipid (I) in the outer layer of the bilayer membrane. The PLA₂ hydrolysis of the phospholipid I in the liposome membrane is substantially innibited when the outside lysophospholipid (II)-to-phospholipid (I) ratio reaches about 0.275-0.35, preferably about 0.3-0.325. The ratio of phospholipid I to lysophospholipid II in the bilayer membrane is preferably about 3-4:1.

The present method is particularly useful for the stabilization of liposomes which are preferably prepared by the sonication of aqueous dispersions of a mixture of the membrane components of formula I and II to equilibrium, e.g., for about 20 min. to 2.0 hours under the conditions set forth herein below in part B of the Example. These liposomes typically are unilamellar spherules having an outside diameter of about 250-300A and a membrane thickness of about 35-45A. It was also found that phospholipase A₂ hydrolysis of these membranes is stimulated by lysophospholipid II at concentrations of up to 12.5 mol-%.

The present invention also provides a method of stabilizing a liposome membrane comprising forming a liposome having a bilayer membrane having an outer layer and an inner layer comprising: (a) between about 67.5-85 mol-% of a phospholipid (PL) of the formula:

and

(b) between about 15-32.5 mol-55 of a lysophospholipid (LPL) of the formula:

wherein R₁, R₂ and R₃ are individually (C₁₀-C₃₀) bonds; by sonicating a mixture of (a) and (b) in water for a period of time effective to equilibriate (a) and (b) in the bilayer membrane so that the ratio of LPL to PL in the outer layer is about 0.275-0.35:1, and optimal stability is achieved. In a preferred embodiment of the present method, the ratio of PL to LPL in the bilayer membrane is about 3-4:1. Preferably, the membrane includes about 20-30 mol-% of the LPL, and most preferably the ratio of LPL to PL in the outer layer is about 0.3-0.325:1.0.

Therefore, the present invention also is directed to a liposome comprising a bilayer membrane having an outer layer and an inner layer, wherein said liposome has an outer diameter of about 250-300A, wherein said bilayer membrane has a thickness of about 35-45A, wherein said membrane comprises (i) between about 67.5-35 mol-% of a phospholipid of the formula:

and (ii) between about 15-32.5 mol-% of a lysophospholipid of the formula:

wherein R₁, R₂ and R₃ are individually (C₁₀-C₃₀) alkanoyl groups including from about 0-4 double bonds; and wherein said membrane is resistant to phosoholipase A₂ hydrolysis. As used herein, the terms "resistant" to phospholipase A₂ (PLA₂) or A₂-type cleavage or hydrolysis, or to "inhibit" said cleavage or hydrolysis, indicates that the membrane phospholipid (I) is not hydrolyzed, and the present membranes retain essentially ail of their contents under the assay conditions described hereinbelow. For example, liposomal membranes initially containing 2.5 mol-35 of a lysophospholipid of formula II became leaky after one hour of phospnolipase A₂ hydrolysis. In contrast, in liposomal membranes containing 20-25 mol-% of a lysophospholipid of fo rmula II, PLA₂ hydrolysis of the phospholipid matrix was completely inhibited as determined by ³¹P-NMR. These liposomes maintained an intact ion barrier for at least four days.

As used herein, the term "phospholipase A₂-tyρe hydrolysis or cleavage" refers to cleavage of the C₂-ester linkage of a membrane phospholipid of formula I at the glycerol moiety. This cleavage can be accomplished in vivo or in vitro by various enzymes of the phospholipase A₂-type or by lec ithin-choles terol acyltransferase (LCAT), and other esterases.

Although cholesterol and cholesterol derivatives have previously been employed to stabilize liposomes, the present invention represents an imp rov ement over that technique in that (a) substantially greater stabilization can be achieved over that possible using cholesterol; and (b) in the case of the administration of the liposomes to a human, the potentially deleterious effects of large amounts of cholesterol are avoided. The liposome membranes exemplified herein are essentially cholesterol-free. However, small amounts of up to about 5 mol-%, preferably up to about 2 mol-% of cholesterol, alone or in combination with other phospholipid-compatible steroids, may be employed in the present membranes. At these concentrations, for example, at about 0.1-1 mol-%, such steroids can exhibit a useful stabilizing effect, and exhibit a preference for the inner layer of the membrane.

Brief Description of the Drawings Figure 1 graphically depicts the outside-to-inside ratio (R_o/i) of 1-palmitoyl lysophospnatidylcholine (lysoPC) versus the mol-% of lysoPC in POPC vesicles. The R_o/i is determined by two methods, 1) from the integral values of the relevant ³¹P NMR peaks and 2) by actually cutting and weighing the respective peaks. The error between the two methods was ± 5%. It can be seen from Figure 1 that the R_o/i value of lysoPC decreases sharply between about 5 and 10 moI-% of lysoPC and then approaches a value of about 5.3 for higher lysoPC concentrations. This indicates that increasing the mol-% of lysoPC decreases the asymmetric distribution of lysoPC over the two halves of the bilayer, and that lysoPC always shows a preference for the outer layer of the membrane.

Figure 2 graphically depicts the ratio of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) versus lysoPC on the outer monolayer. It can be observed from Figure 2 that this ratio falls off sharply with increasing mol-% of lysoPC and finally remains constant between 20-25 mol-% lysoPC.

Figure 3 graphically depicts the ratio of lysoPC/POPC in the outer monolayer versus the mol-% of lysoPC.

Figure 4 graphically, depicts the amount of POPC hydrolyzed in the first 30 min. versus the mol-% of lysoPC originally present in the POPC/lysoPC vesicles. The amount of POPC hydrolyzed at 30 min. is calculated from the difference in NMR peak integral values between 0 and 30 min. As shown in Figure 4, the amount of POPC hydrolyzed at 30 min. correlates very well with the ratio of lysoPC tα POPC in the outer monolayer (Figure 3).

Detailed Description of the Invention Membrane components of formulas I and II are formally derivates of glyceryl-3-phosphoryl choline, or "dilysophosphatidyl choline", which has been di-(1,2-) and mono(1-)acylated, respectively, with fatty acids, e.g., (C₁₀-C₃₀)alkyl-CO₂H. Compounds of formulas I and II are commercially available, e.g., from Sigma Chemical Co., St. Louis, MO and from Avanti Polar Lipids, Inc., Birmingham, AL. Commercially-available compounds of formula I include alpha-lecithin, 1- palmitoyl-2-oleoyl-phosphatidylcholine (PC); 1-stearoyl-2-arachidonoyl-PC; 1,2-diarachidoyl-PC; 1,2- bicaproyl-PC; 1,2-dieIaidoyl-PC; 1,2,-diheptanoyl-PC; 1,2-dilauroyl-PC; 1,2-dilinoleoyl-PC; 1,2-dimyristoyI-PC; 1,2-dioleoyl-PC; 1,2-dipentadecanoyl-PC; 1,2-dipalmitoyl-PC; 1,2-distearoyl-PC; 1,2-diundecanoyl-PC; 1-palmitoyl-2-elaidoyl-PC; 1-palmitoyl-2-linoleoyl-PC; 1-stearαyl-2-oleoyl-PC; 1-oleoyl-2-palmitoyl-PC; 1-oleoyl-2-stearoyl-PC. (C₁₀-C₃₀)-fatty acid esters of glyceryl-3-phosphαrylchαline wherein the fatty acids contain 1-2 triple bonds can also be used.

The corresponding compounds of formula II can be prepared from compounds of formula I by hydrolysis with phospholipase A₂, e.g., as disclosed by L. L. M. van Deenen and G. H. de Haas, Biochim. Biophys. Acta, 70, 538 (1963), the disclosure of wnich is incorpo ra ted by reference herein. Glyceryl-3-phosphocholine can also be selectively mono- or di- esterified by techniques well known in the art of organic synthesis. For example, see I. T. Harrison et al., Compendium of Qrganic Synthetic Methods, Wiley-Interscience, New York, NY (1571), at pages 280-286.

As described in detail hereinbelow, small unilamellar vesicle (SUV) liposomes (250-300A O.D.) can be prepared by dispers ing films comprising phospholipids of formulas I and II in water or aqueous buffer, by sonication of the mixture to equilibrium under an inert atmosphere at about 1-5ºC. In a preferred embodiment, the lipid dispersion is sonicated for about 20 min. to 2.0 hours under nitrocen with a Branson tip sonicator model 350, at a power setting of 4. It will be appreciated that other sonication devices may be used to achieve similar results and that equilibrium may be reached in less than 20 min. at a higher power setting. Small unilamellar vesicles can also be prepared by extruding a lipid mixture through a micropαre filter having a pore size of about 250-350A.

Larger liposomes can be prepared by extruding the lipid mixture through a larger micrαoαre filter, e.g., of 500, 800 or 1000A. The ootimal mol-% of lysopnospholipid required in order to achieve total inhibition of PLA₂ hydrolysis of the membrane can be determined by empirical trials In accord with the detailed example described hereinbelow. It has also been found that preferred amounts of lysophospholipid (II) act to physically stabilize SUVs, as well as to protect them against enzymatic degradation. These stabilizing effects appear to be due to the fact that the lysophospholipid molecule spacially occupies a "cone-shaped" space in the outer layer, thereby fulfilling the geometric packing requirements of highly curved vesicle surfaces. Thus, the lysophosoholipid molecules strengthen the walls of SUVs which have highly curved membrane surfaces and act to relieve their internal stress. It is also believed that they make the surfaces impermeable to enzymes such as PLA₂ by blocking access to the C-2 carbon of the glycerol moiety.

When liposomes are sonicated until their constituents reach "equilibrium", they have reached a point of stable equilibrium of the mol-% ratios of LPL to PL in the outer layer of the membrane, and of LPL in the outer layer to LPL in the inner layer. When this equilibrium has been reached, optimum stability with respect to PLA₂-type hydrolysis is exhibited.

EXAMPLE Small Un ilamel la r POPC/lvsoPC Vesicles

A. Methods and Materials 1-Palmitoyl-2-oleoyl phosphatidylcholine

(POPC) and 1-palmitoyl lysophosphatidylcholine (lysoPC) were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). D₂O (99.8% D) and CDCl₃ (99.8% D) were obtained from KOR Isotopes (Cambridge, MA). Praseodymium chloride (99.955) and calcium chloride were obtained from Aldrich Chemical Company (Milwaukee, WI). Snake venom phospholipase A₂ (Ophiophagus hannah) was obtained from Miami Serpentarium (Miami, FL) and was used as such. A single spot on thin-layer chromatography confirmed the purity of POPC and lysoPC (developing solvent, chloroform/methanol/water, 65:35:8).

B. Preparation of SUV

Chloroform/methanol (1:1 v/v) solutions of the two lipids were mixed in a 25 ml round-bottomed flask. The solvents were removed on a rotary evaporator at 25°C and the lipid film was thoroughly dried under vacuum for 6.0 hr at 25°C. The lipid film was dispersed in D₂O to permit NMR observations, and vortexed for 20 min. The resulting lipid dispersion was sonicated under nitrogen with Branson tip sonicator model 350, at a power setting of 4. Clearing was usually achieved within 30 min. The sonication vial was kept in an ice-water bath at 4°C during sonication. Metal particles from the probe and any unbroken lipid aggregates were removed by high-speed ultracentrifugation for 1.0 hr. at 105,000 × g on a Beckman model L5-75 uitracentrifuge. Inorganic phosphorus assay of the sediment after centrifugation, by the method of C. H. Fisks and Y. Subbarow, J.B.C., 66 , 375 (1925), showed less than 0.5% of phosphorus, indicating that all o f the lipid had been converted into SUVs. C. Characterization and Hydrolysis of SUV

The SUV prepared were characterized by negative staining electron microscopy. Sixty umoles of POPC and different amounts of lysoPC were present per 1 ml of vesicle solution. Vesicles were prepared with

2.5, 10, 15, 17.5, 20 and 25 mol-% of lysoPC.

³¹P NMR spectra were recorded on a Varian FT 80A spectrometer, operating at 32.2 MHz for phosphorus. Eight hundred transients were collected for each sample using a sweep-width αf 4KHzwith 8 K data points. All spectra were recorded under proton noise-decoupled conditions at 37°C.

Extra vesicular ionic concentrations of 6 mM Ca²⁺ and 3 mM Pr³⁺ were obtained by adding small volumes (~20 ul) of D₂O stock solutions of the metal ions directly into the NMR tube. The required amount of phospholipase A₂ (PLA₂)(0.7 mg of protein/ml) was introduced into the NMR tubes by pipetting out small volumes of a D₂O stock solution (about 7 ul) to the vesicular solutions already containing the required metal ions.

The hydrolysis of SUV was carried out in the

NMR sample tube and was followed by ³¹P NMR. In a typical experiment, 1.5 ml of the vesicle solution was taken in the NMR tube and the required amounts o f Ca²⁺, Pr³⁺ and PLA₂ were added. ³¹P NMR spectra were recorded at 30, 60 , 90 , etc. minutes after adding PLA₂. From the peak integral values, the amount of POPC hydrolyzed and tne amount of lysoPC produced were calculated.

In another experiment, 3.0 ml of the vesicle solution with Ca²⁺, Pr³⁺, and PLA₂ were taken in a screw-cap bottle immersed in a water bath maintained at 37°C (same temperature as that of the NMR probe). Two hundred ul of the vesicle solution was drawn out at 30, 60, 90, etc. minutes, and the lipids were extracted by the method of E. G. Bligh and W. J . Dyer, Canad. J . Biochem. Physiol., 37, 911 (1959). Organic and aqueous phases of the extraction were separately analyzed by thin-layer chromatography. The organic phase showed spots corresponding to POPC, lysoPC and fatty acids in the POPC system (developing solvent systems for POPC and lysoPC, chloroform/methanol/water, 65:35:8; and chloroform/diethyl ether/acetic acid, 70:30:2, for fatty acids).

The aqueous phase of the extraction was blown down under nitrogen and redissolved in a known volume of water. It was spotted on a thin-layer chromatography plate along with choline, phosphocholine and glycerophosphocholine standards. It was then developed in an n-propanol/aqueous ammonium hydroxide (1:1) solvent system. No spots corresponding to choline, phosphocholine, and glycerophosphocholine were observed in our samples. This demonstrated the fact that the PLA₂ did not contain any phospholipase C or phospholipase D.

All experiments reported here were repeated at least twice and the standard deviation from experiment to experiment was less than ± 5% . D. Results

³¹P NMR spectra of the POPC vesicles containing various mol-% of lysoPC, Pr³⁺ (3 mM), Ca²⁺ (6 mM), and snake venom PLA₂ recorded at regular intervals indicate that the intensity of the lysoPC peak in the outer monolayer increases at the expense of outer monolayer POPC peak. The spectra also show a decrease in the intensity of the inner monolayer POPC peak without any change in its chemical shift value. This decrease is inversely proportional tα the amount of lysoPC initially present in the vesicles. In other words, the decrease in the intensity of inner monolayer POPC peak is fast in vesicles containing low mol-55 αf lysoPC. This decrease also indicates a rapid flip-flop of POPC from the inner tα the outer monolayer without causing any changes in the barrier properties of the vesicle.

The spectra also indicated that POPC vesicles containing 2.5 mol-% lysoPC became leaky after 1 hr. in contrast, vesicles containing 20-25 moI-% of lysoPC maintained an intact ion barrier beyond 4 days. The downfield movement of inner monolayer signals (towards the outer monolayer signals) is taken as a measure of the loss αf the permeability barrier of tne vesicles. The time at wnich the permeability barrier is lost is found to increase with increasing mol-% of lysoPC. Also, just before the collapse of the ion barrier in each case, a signal corresponding to lysoPC in the inner monolayer is detected which could be due to flip-flop of lysoPC from outer to inner monolayer, compensating the flip of POPC from inner to outer monolayer discussed above. As a result of this, increasing amounts of lysoPC are accumulated in the inner monolayer. Such a high accumulation of lysoPC in the inner monolayer could create some defective sites because of the cone shape of lysoPC. This leads to a diffusion of Pr³⁺ ions to the intravesicular solution, resulting in a down field shift of the inner monolayer peaks.

Surprisingly, a low mol-% of lysoPC (0-12.5 mol-%) stimulates, while a higher mol-% (20-25 mol-%) of lysoPC inhibits the PLA₂ hydrolysis of POPC vesicles. As shown in Figure 1, the measured values of R_o/i of lysoPC are 15 (at 5 mol-% lysoPC) and 5.3 (at 15 mol-% lysoPC). These correspond to 93% and 84% of lysoPC in the outer monolayer. This also indicates that a decrease in the asymmetry of lysoPC occurs when increasing mol-% of lysoPC is incorporated into the vesicles.

Figure 2 demonstrates that the ratio of POPC/ lysoPC in the outer monolayer also decreases with increasing mol-% of lysoPC. If the asymmetric distribution (with a higher number of lysoPC molecules in the outer monolayer) of lysoPC decreases with increasing mol-% of lysoPC (as discussed above and shown in Figure 1), the decreased number of lysoPC molecules in the outer monolayer should manifest itself as a decrease in the ratio of POPC/lysoPC in the outer monolayer. Figure 3 depicts a plot of the lysoPC/POPC ratio in the outer monolayer.

Figure 4 is a plot of the amount of POPC hydrolyzed by PLA₂ in the first 30 min. in POPC vesicles containing various amounts of lysoPC. It can be seen that the amount of POPC hydrolyzed falls off linearly with increasing mol-% of lysoPC up to 17.5 mol-% lysoPC and that no hydrolysis takes place between 20-25 mol-% lysoPC. In pure POPC vesicles (containing no lysoPC initially), about 25% of outer monolayer POPC is hydrolyzed in the first 30 min. In Figure 4, this value would correspond to a lysoPC content of about 12.5 mol-%. Hence, it is believed that lysoPC up to 12.5 mol-% (in POPC vesicles) stimulates PLA₂ activity whereas lysoPC above 12.5 mol-% inhibits PLA₂ activity and the inhibition is virtually 100% above about 20 mol-% lysoPC.

The asymmetric distribution of lysoPC (in POPC vesicles) is very high, particularly at low mol-% of lysoPC (see Figure 1). In other words, most of the total lysoPC is accommodated in the outer monolayer (particularly at 2.5 and 5 mol-% of lysoPC). A much higher concentration of lysoPC in the outer monolayer may create some organizational defects that act as sites for incorporation of hydrolytic enzymes in the bilayer. With the decreasing asymmetry in the distribution of lysoPC, the concentration of total lysoPC in the outer monolayer decreases with a consequent decrease in the number of organizational defects.

This, in turn, is reflected in the apparent inhibition αf the enzyme activity by lysoPC at higher concentrations.

Finally, between 20-25 mol-% lysoPC, the head group packing in the outer monolayer is so tight that the enzyme cannot penetrate tα the glycerol C-2 carbon to catalyze any hydrolysis. This corresponds to POPC/lysoPC ratio of about 3.2 in the outer monolayer. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification ⁴ (11) International Publication Number : WO 88/ 064 A61K 9/50 A3 (43) International Publication Date: 7 September 1988 (07.09.

) (21) International Application Number: PCT/US88/O05O8 (81) Designated States: AT (European patent), BE (Eu pean patent), CH (European. patent), DE (Europe

(22) International Filing Date: 19 February 1988 (19.02.88) patent), FR (European patent), GB (European tent), IT (European patent), JP, LU (European tent), NL (European patent), SE (European patent

(31) Priority Application Number: 017,369

Published

(32) Priority Date : . 24 February 1987 (24.02.87) With international search report

Before the expiration of the time limit for amending t

(33) Priority Countr : US claims and to be republished in the event of the receipt_. amendments.

(71) Applicant: REGENTS OF THE UNIVERSITY OF

MINNESOTA [US/US]; Morrill Hall, 100 Church (88) Date of publication of the international search report: Street SE, Minneapolis, MN 55455 (US). 3 November 1988 (03.11.8

(72) Inventor: BAUMANN, Wolfgang, J. ; 2806 W. Oakland

Ave., Austin, MN 55912 (US).

(74) Agent: FREED, Robert, C; Merchant, Gould, Smith, Edell, Welter & Schmidt, 1600 Midwest Plaza Building, Minneapolis, MN 55402 (US).

(54) Title: PHOSPHOLIPASE A₂-RESISTANT LIPOSOMES

R₁CH2-CH ( R2 )-CH₂0PQ ( H₂ )2N⁺ CCH3 ) 3 (j.) i _ 0

R CH -CH ( 0H ) -CH20P0 C CH₂ )2N⁺ CH ) 3 (II)

(57) Abstract

A method to inhibit phospholipase A -type hydrolytic degradation of liposome membranes is disclosed comprisin forming a liposome having a bilayer membrane comprising (i) between about 67.5-85 mol-% of a phospholipid of formul (I), and (ii) between about 15.0-32.5 mol-% of a lysophospholipid of formula (II), wherein R₂, Rj and R₃ are individuall (C₁₀-C₃₀) alkanoyl groups including from about 0-4 double bonds.

FOR THE PURPOSES OFINFORMAHON ONLY

Codes used to identify States party to the PCT on the frontpages ofpampMetspubishinginternationalappli- cations under the PCT.

AT Austria FR France ML Mali

AU Australia GA Gabon MR Mauritania

BB Barbados GB United Kingdom M Malawi

BE Belgium HU Hungary NL Netherlands

BG Bulgaria IT Italy NO Norway

BJ Benin JP Japan RO Romania

BR Brazil KP Democratic People's Republic SD Sudan

CF Central African Republic ofKorea SE Sweden

CG Congo KR Republic of orea SN Senegal

CH Switzerland LΓ Liechtenstein SU Soviet Union

CM Cameroon LK Sri Lanka TO Chad

DE Germany, Federal Republic of LU Luxembourg TG Togo

DK Denmark C Monaco US United States of America π Finland MG Madagascar

Claims

WHAT IS CLAIMED IS:

1. A method to inhibit the phospholipase A₂-type cleavage of a liposome membrane comprising forming a liposome having a bilayer membrane comprising a mixture of (i) between about 67.5-35 mol-% of a phospholipid (PL) of the formula:

and (ii) between about 15.0-32.5 mol-% of a lysophospholipid (LPL) of the formula:

wherein R₁, R₂ and R₃ are individually (C₁₀-C₃₀) alkanoyl groups including from about 0-4 double bonds.

2. The method αf claim 1 wherein R₁, R₂ and R₃ are individually (C₁₂-C₂₂)-n-alkanoyl groups including about 0-1 double bonds.

3. The method of claim 1 wherein R₁, R₂ and R₃ are individually oleoyl, palmitoyl, stearoyl or myristoyl.

4. The method of claim 3 wherein R₁ and R₃ are palmitoyl and R₂ is oleoyl.

5. The method of claim 1 wherein the membrane includes about 20-30 mol-% of the lysophospholipid.

6. The method of claim 5 wherein said liposome has an outside diameter of about 250-300A and a membrane thickness of about 35-45A.

7. The method of claim 6 further comprising forming said liposome by sonicating the mixture in water for a period of time effective to equilibrate constituents in the bilayer membrane.

8. The method of claim 7 wherein the bilayer membrane has an outer layer and an inner layer; and wherein the ratio of LPL to PL in the outer layer is about 0.275-0.35:1.0.

9. A metho d of stabilizing a lioosome membrane comprising forming a liposome having a bilayer membrane having an outer layer and an inner layer comprising:

(a) between about 67.5-85 mol-% of a phospholipid (PL) of the formulas

and

(b) between about 15-32.5% mol-% of a lysophospholipid (LPL) of the formula:

wherein R₁, R₂ and R₃ are individually (C₁₀-C₃₀) alkanoyl groups including from about 0-4 double bonds; by sonicating a mixture of (a) and (b) in water for a period of time effective to equilibriate (a) and (b) in the bilayer membrane so that the ratio of LPL to PL in the outer layer is about 0.275-0.35:1.0.

10. The method of claim 9 wherein the ratio of PL to LPL in the bilayer membrane is about 3-4:1.

11. The method of claim 10 wherein the membrane includes about 20-30 mol-% of the LPL.

12. The method of claim 9 wherein R₁, R₂ and R₃ are individually oleoyl, palmitoyl, stearoyl or myristoyl.

13. The method of claim 12 wherein R₁ and R₃ are palmitoyl and R₂ is oleoyl.

14. A liposome comprising a bilayer membrane having an outer layer and an inner layer, wherein said liposome has an outer diameter of about 250-300A, wherein said bilayer membrane has a thickness of about 35-45A, wherein said membrane comprises: (i) between about 67.5-85 mol-% of a phospholipid (PL) of the formula:

where in R₁, R₂ and R₃ are individually (C₁₀-C₃₀) alkanoyl groups including from about 0-4 double bonds; and wherein said membrane is resistant to phospholipase A₂ hydrolysis.

15. The liposome of claim 14 wherein R₁, R₂ and R₃ are individually (C₁₂-C₂₂)-n-alkanoyl groups including about 0-1 double bonds.

16. The method of claim 14 wherein R₁, R₂ and R₃ are individually oleoyl, palmitoyl, stearoyl or myristoyl.

17. The method of claim 16 wherein R₁ and R₃ are palmitoyl and R₂ is oleoyl.

18. The method of claim 14 wherein the membrane includes about 20-30 mol-% of the lysophospholipid.

19. The method of claim 18 wherein the ratio of LPL to PL in the outer layer of the membrane is about 0.275-0.35:1.0.

20. The liposome of claim 14 wherein said membrane further comprises an amount of up to about 5 mol-% of a steroid comprising cholesterol which is effective to stabilize the membrane.

21. The liposome of claim 14 wherein the ratio of PL to LPL in the bilayer membrane is about 3-4:1.