DD293264A5 - METHOD FOR PRODUCING MEMBRANE ANCHOR COMPOUNDS FOR THE COVALENT LIPOSOMAL IMMOBILIZATION OF HYDROPHILIC AMINOGRUPPENEHOLDING LIGANDS - Google Patents

Abstract

The invention relates to a process for the preparation of membrane anchor compounds for the covalent liposomal immobilization of hydrophilic amino group-containing ligands. The membrane anchor compounds prepared according to the present invention are used to prepare liposome immunoassays and agglutination assays, for the cellular or tissue specific transport of liposomal encapsulated drugs, for the introduction of genetic material into cells, and for the preparation of novel synthetic vaccines based on the use of liposomes as immunoadjuvants. The essence of the invention is that by acylation of cardiolipin with succinic or glutaric anhydride in the presence of 4-dimethylaminopyridine as a catalyst, cardiolipin derivatives of the formula I wherein R1, R2, R3 and R4 represent the alkyl group of a saturated or unsaturated long-chain carboxylic acid and n2 or 3 mean to be produced. Formula I {membrane anchor compound; Cardiolipinderivat; Immobilization, liposomal; Ligand, hydrophilic; acylation}

Description

wherein R ₁ , R ₂ , R ₃ and R _{4 represent} the alkyl group of a saturated or unsaturated, long-chain carboxylic acid and η = 2 or 3.

Field of application of the invention

The invention relates to a process for the preparation of membrane anchor compounds for covalent liposomal immobilization of hydrophilic amino group-containing ligands. Liposomes having covalently immobilized via membrane anchor compounds hydrophilic ligands such as various proteins, u.a. Immunoglobulins, Fab fragments, leucines and enzymes as well as natural and synthetic peptides, membrane receptor-specific sugar derivatives, hormones and the like. be used:

in the immunological-analytical and medical-diagnostic field for the production of liposome immunoassays (LIA) and agglutination assays,

in the medical therapeutic field for the tent or tissue-specific transport of liposomal encapsulated drugs,

for the introduction of genetic material into cells and

in the form of "virosomes" or "immunosomes" in the field of the production of novel synthetic vaccines.

Characteristic of the known state of the art

The most commonly used synthetic membrane anchor compounds have a hydrophobic moiety consisting of only one or two fatty acid residues. Their preparation and use for immobilizing hydrophilic ligands on liposomes is described in the following literature:

Torchilin, V.P., (1987) CRC Critical Reviews in Therapeutic Drug Carrier Systems 2,65-115,

- Machy, P., and Leserman, L., (1987) Liposomes in Cell Biology and Pharmacology, Editions lnserm, John Libbey Eurotext, London,

- Gregoriadis, G., (ed.) (1984) Liposome Technology, Vol.3, CRC Press, Boca Raton.

Membrane anchor compounds having three fatty acid residues (synthetic derivatives of a natural membrane anchor compound from the outer membrane of Escherichia coli) are also known. The use of these compounds as intermediates for the preparation of membrane anchor drug conjugates and their potential utility for liposome preparations is described in DE 3546150 A1.

All previously known membrane anchor compounds for the covalent liposomal immobilization of hydrophilic ligands have the disadvantage that their hydrophobic moiety consists of a maximum of three fatty acid residues. This has the consequence that for achieving a sufficiently high hydrophobicity, the number of bound membrane anchors per ligand (especially for higher molecular weight ligands) must be relatively high, which in turn may affect the specific properties of the ligand.

Object of the invention

The aim of the invention is to improve the covalent attachment of hydrophilic ligands to liposomal membranes by novel membrane anchor compounds. Ligand binding should be gentle and with high binding rates.

Explanation of the essence of the invention

The object of the invention is to propose a process for the preparation of novel membrane anchor compounds for covalent liposomal immobilization of hydrophilic amincgruppenenthaltender ligands, wherein in the ligands to be immobilized with each bound anchor molecule simultaneously four fatty acid residues are introduced and thus per anchor molecule ligands a higher hydrophobicity than when using conventional membrane anchor compounds is awarded. According to the invention the object is achieved in that cardiolipin derivatives of the formula I are prepared, wherein R ₁ , R ₂ , R ₃ and R "is the alkyl group of a saturated or unsaturated long-chain carboxylic acid and η = 2 or 3.

• Ki CU -O Ui ;;

Ky "CU - Ü C ti O

1 ti

H ₅₅ C Op-Ui ,.

i-l -.- 'l-f-O-Ci

ϊ-.a; - L O- 0 Cl i '.. ^:

i \ i |. -CU ' ¹ J-CM-;

The synthesis is carried out by acylation of cardiolipin with succinic or glutaric anhydride and 4-dimethylamino-pyridine (4-DMAP) as a catalyst. The membrane anchors synthesized according to the invention can, activated by Carbodiimidmethode, in a known manner either as a lipid component in preformed liposomes (immobilization A) or by hydrophilization of hydrophilic ligands in dimethyl sulfoxide-containing aqueous solution and subsequent incorporation of the hydrophobized ligands in reverse phase evaporation vesicles (REV) in the process liposome formation (immobilization method B) for covalent immobilization of hydrophilic amino-containing ligands to the surface of liposomes.

embodiments

example 1

Synthesis of O-succinyl cardiolipin

25 mg of dry cardiolipin, 71.5 mg of succinic anhydride and 6.22 mg of 4-DMAP are incubated in 3 ml of dry pyridine under an inert gas atmosphere, stirred magnetically. After about 10-15 hours, the conversion of the cardiolipin is complete.

Pyridine is evaporated and the residue taken up in 3 ml of chloroform. The chloroform phase is shaken first with 0.1 N HCl and then with 0.1 M NaHCO ₃ solution (R _F = 0.36 on silica gel plates, d = 0.25 mm, eluent: chloroform / methanol / 25% ammonia = 130 / 64/15). The substance was identified as O-succinyl-cardiolipin by using succinic anhydride because after determining the succinic acid content on the basis of the ¹⁴ C label and the phosphate content by ashing, the theoretically expected ratio of succinic acid: phosphate of 1: 2 was found. The yield based on the phosphate determination was 57%.

Example 2

Immobilization of α-chymotrypsin on the surface of preformed liposomes containing O-succinyl cardiolipin (immobilization method A)

200μΙ ultrasound vesicles (4.8 χ 10 " ³ M total lipid, 60mol% egg lecithin, 35mol% cholesterol, 5mol% O-succinyl cardiolipin) in physiological saline solution are adjusted to pH 3.5 with 2mg 1-ethyl After 5 minutes, 100 μl of an α-chymotrypsin solution in 0.1 M borate buffer pH 8.5 (0.78-31.25 mg of α-chymotrypsin / ml) are pipetted in. The batch is stirred at 4 ° C. The reaction is complete after 16 hours The separation of the proteoliposomes from unbound a-chymotrypsin is via Sephorose CL-4B.

At a concentration of a-chymotrypsin in the coupling mixture of 10mg / ml, a result of 1.44 χ 10 ~ ² moles of a-chymotrypsin / mol total lipid and from 360μg / .mu.mol total lipid is achieved. The loading of the liposomes with protein can be controlled by varying the total lipid and protein concentration and the anchor content of the liposomes.

Example 3

Hydrophobization of α-chymotrypsin with O-succinyl-cardiolipin and binding of the hydrophobisierton enzyme to phospholipid double-polyol layers (immobilization method B)

0.58μπιοΙ dry O-Succinylcardiolipin be suspended in 100μΙ Dimethyhulfoxid and mixed with 500μΙ physiological saline. After a brief ultrasound treatment, the pH is adjusted to 3.5 and 20 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide are added, and after 5 minutes, 4.6 mg of α-chymotrypsin in 500 μl of 0.1 M borate buffer pH 8.5 are obtained After incubation overnight, the hydrophobized protein of non-hydrophobized protein and

unbound anchor on a Sepharose CL-4B mini column (6 x 2cm) separated. The molar ratio of cardiolipinankena-chymotrypsin was determined to be 4: 1 with about 2 anchor molecules covalently attached.

For the purpose of binding the hydrophobized protein to phospholipid bilayers of reverse phase evaporation vesicles (Scoka, F., Papahadjopoulos, D .; Proc. Nat. Acad. Ser. USA75 [1978] 4194), 1 ml of the aqueous solution of the hydrophobized protein with 3 ml Lecithin and cholesterol (2: 1, mohrnol) containing diethyl ether mixed in a 25ml round cuvette. Thereafter, the diethyl ether is removed under reduced pressure.

Up to 1.59 χ 1C ~ ³ mol protein / mol lipid liposome formation is possible, whereby the offered protein is completely incorporated into the vesicles.

Claims (1)

A process for the preparation of Mfcinbranankerverbindungen for covalent liposomal immobilization of hydrophilic amino-containing ligands, characterized in that by acylation of cardiolipin with succinic or glutaric anhydride in the presence of 4-dimethylaminopyridine as a catalyst cardiolipindarivans of the formula I, L. \ " LU-'J-Li I ₂