HU219353B - Synthetic membrane vesicles as vehicles containing functionally active fusional peptides and pharmaceutical compositions comprising them - Google Patents

Abstract

BACKGROUND OF THE INVENTION The present invention relates to a bilayer phospholipid bladder comprising at least one functionally active fusion peptide and a desired drug or pharmaceutically active agent attached to the membrane and comprising: a) a membrane having a phosphatidylethanolamine and phosphatidylcholine non-viral phospholipidate preferably together with a viral phospholipid; b) at least one functionally active natural or synthetic fusion peptide, wherein the natural fusion peptide is a hemagglutinin primer or monomer, one or both of its subunits, and the HA1 and HA2 glycopeptides, and from an influenza virus, preferably its A-H1N1 subtype, rabdovirus, type III parainfluenza virus, , From Semliki Forest virus or togovirus; c) a bifunctional cross-linking agent, preferably a sulfosuccinimidyl derivative, bound to an amembrane phosphatidyl ethanolamine; and d) at least a single cell specific marker attached to said membrane bound crosslinking agent. The invention also relates to pharmaceutical compositions containing a pharmaceutically active agent, in particular a medicament, comprising a bladder bladder, in particular pharmaceutical compositions for the treatment of viral infections or cancer. ŕ

Description

BACKGROUND OF THE INVENTION The present invention relates to two-layer phospholipid synthetic membrane vesicles (liposomes), particularly those containing a fusion peptide molecule and a cell-specific marker on their surface. The fusion peptide molecule may be in the form of a synthetic or purified peptide, or it may be part of an enveloped viral glycoprotein molecule, such as hemagglutinin, which may be derived from influenza, parainfluenza or Semliki Forest virus. The cell-specific protein may be an IgG antibody, such as a CD4 antibody. The invention also relates to pharmaceutical compositions comprising the vesicles.

When an active agent is administered to a patient, it will normally have to reach the site of administration from the site of administration and, consequently, the route of administration may have an important effect on the circulatory pharmacokinetic profile. Thus, oral administration of the active ingredient, where appropriate, results in the onset of activity of the active ingredient and is somewhat unreliable in the time required to reach the optimum plasma drug level. In contrast, intravenous injection has an immediate effect, and circulating levels can be accurately determined, at a cost to the patient with some pain and discomfort. However, when the active ingredient is injected directly into the systemic circulation, the relationship between the administered dose, the level of the active ingredient and the time of arrival at the target site is by no means straightforward. These parameters are determined by a complex and often competing network of pathways leading to the assembly or inactivation of drug molecules at the target site and drug selection. These pathways include biotransformation in the liver and other tissues, renal or biliary excretion, binding of the drug to fixed or circulating cells or macromolecules, and passive or mediated passage of the drug across the membrane barrier (Creasy, WA (1979): Drug Disposition in Humans, Oxford University Press, New York].

In recent years, many possibilities of interest in influencing the distribution and metabolism of active ingredients in the form of various carriers or delivery systems have become known. Such systems aim to control one or more of the following parameters [Juliano, R. R., Can. J. Physiol. Pharmaco 56: 683-690 (1975)].

(a) the rate of entry of the active substance into a given part of the body;

(b) the distribution and localization of the active substance in the body;

(c) the persistence or rate of metabolism of the active substance.

The major advancement in controlled drug delivery systems has been the development of liposomes, first described by Bangkam et al., Bangkam, A.D. Standish, Μ. M. and Watkins, J.C., J.Mol. Biol. 13, 238-252 (1965)]. Nowadays, the literature attributes an extremely wide variety of benefits to the delivery of certain active substances in liposomes. These can be roughly classified into the following groups.

1. Liposomes can pass through biomembranes and facilitate the transport of active ingredients through normally impermeable barriers. In particular, liposomes facilitate the penetration of the encapsulated compounds into the cell.

2. Liposomes can be engineered to interact with particular tissues, thereby improving the selectivity of the active ingredient and reducing its toxicity.

3. Liposomes can modify the pharmacokinetics of the drug in the correct direction by regulating drug release, distribution, and systemic clearance.

4. Chemically and metabolically labile drugs can be protected by liposomes from deactivation.

Potentially therapeutically active agents may be enclosed in such a manner that the enclosed compound has, at least in vitro, modified properties relative to the unmodified substance. Unfortunately, however, early hopes for a revolutionary approach to chemotherapy have not been fully realized by experimental facts [Fildes, F. J. T. (1981), Liposomes. From physical structure to therapeutic applications. Knight (ed.), Elsevier / North-Holland Biomedical Press].

Better results were achieved by targeting liposomes as vectors (carriers) with specific proteins. If liposome-encapsulated materials are to be more successfully delivered to the selected organ or tissue, such delivery techniques must be devised to avoid liposome assembly at an undesired location (thereby reducing toxicity) and optimizing delivery to the desired cells (thereby the desired effect).

Some studies have used the process of incorporating liposomes with aggregated immunoglobulins to optimize delivery to phagocytic cells (Ismail, G., Boxer, L.A. and Bachner, R.L., Pediatr. Res., 13, 769-773 (1979), Finkelstein, MC, Kuhn, SH, Schieren, H., Weissmann, G. and Haffstein, S. (1980): Liposomes and Immunobiology (Tom, H.H. and Six). , HR Editors), 255-270. Elsevier / North-Holland Publishing Co., Amsterdam],

Further improvements have been described by Gregoriadis and Neerunjun, Biochem. Biophys. Gap. Commun., 65, 537-544 (1975)], which enhanced the delivery of liposomes by binding to cell-specific IgG antibodies. The uptake of liposomes increased 3-25-fold when liposomes obtained with different IgG immunoglobulins were developed for different cell strains. However, this procedure showed that the targeted liposomes did not fuse with the cell membrane, preventing efficient delivery of the drug to the cells [Leserman, L., Weinstein, J.N., Blumenthal, R., Sharrow, SO and Texy, WD, J. Immunol. 122: 585-591 (1979)].

Liposomes have been shown to make greater progress in the production of drug delivery systems by viral proteins2

The liposome membranes were prepared with proteins such as hemagglutinin from influenza virus (Almeida, J. D., Brand, C. M., Edwards, D. C. and Heath, T. D., 1975, 2, 899-901). The efficacy and specificity of early viral interaction (adsorption and invasion) with host cells can be transferred to liposome carriers by incorporation of appropriate viral proteins into liposome membranes. Indeed, incorporation of the Spike protein of Sendai virus spike into the liposome bilayer results in at least a 100-fold increase in the uptake of diphtheria toxin A fragment by mouse L cells compared to liposomes containing fragment A without the above proteins [Uehida, T., Kim, J., Yamaizumi, M., Miyahe, Y. and Okada, Y., Cell. Biol., 80, 10-20 (1979).

The major disadvantage of the above procedure is the lack of fully active influenza hemagglutinin fusion peptides. Influenza A viruses penetrate their host cells by membrane fusion. Once bound to the cell surface, the viral particles enter the cell and are transported to endosomes and lysosomes. The acidic environment of these organs activates fusion between the virus and the host cell membrane. The advantage of a low pH η of bladder-cell fusion is that the bladder contents are released upon entry into the cell cytoplasm (endosome pH about 5.2). Recognition of this low pH-induced fusion of influenza virus glycoproteins has led to numerous attempts to develop an efficient drug delivery system with influenza hemagglutinin-targeted liposomes.

Kawasaki, K., et al., Biochemica et Biophysica Acta, 733, 286-290 (1983)] with a 1.6: 0.4: 1 molar ratio of influenza hemagglutinin glycoprotein in egg white phosphatidylcholine / spin-labeled phosphatidylcholine / cholesterol. bladders were used. The appropriate protein: lipid ratio formulations (1:44 and 1: 105 mol / mol) contained vesicles of 100-300 nm in diameter and high density of spikes on the surface, but with some disadvantages.

1. Due to their high cholesterol content and residual detergent content, their endocytosis by phagocytic cells was reduced. Kawasaki et al. Themselves report only 66% activity, but according to a recently developed improved assay, activity is only 1% (!) After 7 minutes (see Example 10 below). This may be due to the fact that Triton X-100 used by Kawasaki et al. Reacts with hemagglutinin and cannot be completely removed by dialysis.

2. To be able to study in more detail the role of viral membrane components in the fusion reaction, it is necessary to be able to manipulate these components. To this end, there is a need for a method by which viral spike proteins can be isolated and reconstituted, and reconstructed virosomes having full biological fusion activity can be obtained. Despite their efforts, Kawasaki et al. Did not disclose the reconstitution of an influenza virus envelope with full biological fusion activity.

3. No binding of the target liposomes containing the pharmacologically active substance to specific cells could be detected.

Most other methods for reconstituting the envelope are based on solubilizing the viral membrane with detergent and then removing the detergent from the supernatant after settling the internal viral proteins and genetic material. Detergents with high critical micelle concentrations, such as octylglucoside, can be effectively removed by dialysis. Reconstitution using octylglucoside has been described by Semliki Forest Virus (SFV) (Helenius, A., Sarvas, M. and Simons, K., Eur. J. Biochem., 116, 27-35 (1981)); bladder disease (VSV) [Eidelman, O .; Schlegel, R., Tralka, T.S., and Blumenthal, R., J. Bioi. Chem. 259: 4622-4628 (1984)]; influenza virus (Huang, RTC, Wahu, K., Klenk, HD and Rou, R., Virology, 104, 294-302 (1980)) and Sendai virus (Harmsen, MD, Wilschut, J., Scherphof, G.). , Hulstaert, C. and Hoekstra, D., Eur. J. Biochem., 149, 591-599 (1985). However, in no case was a well-reconstructed virus envelope constructed. For example, the protein: lipid ratio of the SFV virosome was different from that of the viral membrane, and the VSV virosome showed no biological fusion activity.

Another method described in the literature is to remove flu virus spikes from virus particles using bromelain. The resulting hemagglutinin is coupled to liposomes [Doms, R., Helenius, A. and White, J., J. Biol. Chem. 260 (5) 2973-2981 (1985). The main disadvantage of this procedure is the limited biological activity of such blisters resulting from the cleavage by bromelain.

Although the viral envelope resulting in the formation of phospholipid vesicles containing functional fusion peptides known in the art, or the generation of active bilayer phospholipid liposomes (without fusion peptide), or the generation of cell-specific markers (e. G., Antibodies), combination of virosomes.

This is because of the difficulty in properly reconstituting viral phospholipid membranes containing a functionally active fusion protein (e.g., hemagglutinin) and stable attachment of a cell-specific marker, such as another protein, such as an antibody.

Thus, it is an object of the present invention to overcome the technical problems by providing vesicles which deliver the desired active ingredient or pharmaceutically active agent to specific cells, such as macrophages, T4 helper cells, brain cells, and other cells or specific organs, completely bound to these cells by phagocytosis or endocytosis. into the cells and immediately after endocytosis, the desired active ingredient is delivered to the cytoplasm of the desired cell.

The above technical problem was solved by viral hemagglutinin liposomes. More specifically, the find3

The aim of the present invention has been achieved by the use of bilayer phospholipid vesicles containing cell-specific markers on their membranes and containing at least one pharmaceutically active substance within the membrane and having at least 90% biological activity by Luescher & Glueck, 39 ].

It has been found that by using a nonionic detergent, preferably octaethylene glycol monododecyl ether, by properly removing the detergent (using a microcarrier), and in particular by diluting the viral phospholipid with additional phospholipid, -choline to reduce the protein concentration in the membrane) avoids precipitation of electrostatically charged neighboring molecules (i.e., fusion peptides and antibodies) in the membrane.

Embodiments of the invention and features of improved bladders are described below.

Preferably, the membrane cholesterol content of the bladder membrane is less than 10%, more preferably less than 2%, and / or the membrane detergent content is less than 10 ppb, more preferably less than 1 ppb (1 ppb = ^10-9 parts per liter).

The blisters preferably have a diameter of less than 100 nm, more preferably less than 80 nm.

The above blisters carry at least one cell-specific marker, preferably a monoclonal antibody.

The above blisters contain at least one fusion peptide, preferably a trimer or monomer of hemagglutinin, or one or both of its cleavage subunits, HA1 and HA2 glycopeptides, fusion peptides isolated from natural sources, or synthetic "fusion peptides".

Preferably, the hemagglutinin is derived from influenza virus, preferably subtype A-H1N1, rhabdovirus, parainfluenza virus or togavirus.

Preferably, at least one end of the amino acid sequence of the fusion peptide has one cysteine residue, preferably three cysteine residues.

The phospholipid of the above bladders is derived from at least one of the influenza virus, preferably its A-H1N1 subtype, rhabdovirus, parainfluenza virus and togavirus, and is combined with 2 to 100 times, preferably 5 to 15 times, phosphatidylcholine.

Preferably, the vesicles are characterized by:

the membrane phospholipid consists of 70-95% by weight of phosphatidylcholine and 5-30% by weight, preferably 10-20% by weight of phosphatidylethanolamine;

5-10% by weight, preferably 6-8% by weight of crosslinker, preferably a sulfosuccinimidyl derivative and at least one cell specific fusion peptide.

The membrane comprises bilayer phospholipid vesicles carrying hemagglutinin and a cell-specific marker and containing at least one pharmaceutically active agent, wherein the hemagglutinin comprises at least one fusion peptide and the hemagglutinin comprises a viral strain with a nonionic detergent and an ultracentrifug monododecyl ether, which does not react with hemagglutinin, is added to the supernatant after ultracentrifugation, and the detergent is removed by repeated treatment of the solution with a polystyrene bead microcarrier; in the solution by sonication.

The detergent solution is preferably used at a concentration of 10-250 pmol / l, preferably 80-120 pmol / l, and the detergent solution is preferably removed from the solution using 1-2 g, preferably 1.5 g of polystyrene bead microcarrier per 100 mg of detergent.

The bladders described above are useful for the preparation of pharmaceutical compositions for AIDS. For such use, the vesicles contain at least one of dextransulfate, ribonuclease dimer and lysozyme dimer.

The above bladders can also be used for the preparation of pharmaceutical compositions against carcinomas. For such use, the blisters contain at least one of imidazolecarboxamide, hydroxycarbamide, adriplastin, endoxane, fluorouracil and colchicine.

Accordingly, the present invention relates to viral haemagglutinin virosomes comprising a liposome ideal for endocytosis, and to biologically fully active cell-specific markers and fusion peptide, preferably a viral haemagglutinin glycoprotein or derivative thereof having inducing immediate fusion of the virosome with the desired cell after endocytosis.

In another embodiment of the invention, an appropriate crosslinking agent adsorbed to a specific liposome is used in admixture with a specific antibody that targets the appropriate receptor of the desired cell and induces an endocytosis mechanism that binds the crosslinking agent to its biological activity. completely preserves.

These drug delivery vesicles are characterized by the presence on their surface of fully active viral glycoproteins or derivatives and biologically active specific antibodies capable of binding to the desired cell, which are introduced into these cells by phagocytosis or endocytosis; and release the virosome contents into the cytoplasm of these cells. Due to the fully active fusion peptides of the invention, the active compounds are released immediately after phagocytosis, thus avoiding their undesirable long residence in the endocytosis, which would lead to non-specific degradation of the therapeutic agents contained in the viral hemagglutinin vesicles of the invention. In a pH = 5 environment, the flu fusion peptides on the surface of the vesicles are activated in the same way as in the case of live influenza viruses. The bladder4

The contents of the kits are released in the cytoplasm, similar to the flu virus and the released nucleoprotein.

The term "liposome" refers to medium-sized bilayer phospholipids formed by controlled detergent removal. The initial size of the blisters, which develops when detergent is removed, also depends on the detergent and phospholipid used and, in some cases, the method and rate of detergent removal.

The process for the preparation of the bladders which are particularly suitable for phagocytosis of the present invention comprises the following steps:

1. dissolving one or two phospholipids in a nonionic detergent;

2. forming blisters by removing the detergent with bead polystyrene microcarrier (wet size 0.30-0.84 mm);

3. using a defined mechanical movement during detergent removal;

4. sonication of the desired 50-100 nm diameter of the blisters.

In particular, the blisters can be produced by:

a) phosphatidylethanolamine and phosphatidylcholine non-viral phospholipids and optionally viral phospholipids, preferably in combination with the fusion peptides, in the presence of a nonionic, hemagglutinin-inactive detergent, preferably octaethylene glycol monododecyl ether;

b) adding the desired pharmaceutically active substance to the solution,

c) removing the detergent by repeatedly treating the solution with a microcarrier, preferably a wet polystyrene bead microcarrier having a pore size of 0.84-0.30 mm,

d) ultrasound of the bladders,

e) adding a bifunctional cross-linking agent, preferably a sulfosuccinimidyl derivative, which binds to the bladder membrane and granulating the blisters;

f) adding fusion peptides associated with the membrane-bound cross-linking agent to the granulated blisters, unless dosing is carried out in step a); and

g) adding cell-specific markers associated with the membrane-bound cross-linking agent to the granulated blisters.

Another embodiment of the invention relates to vesicles in which the phospholipid comprises 70-95% by weight of phosphatidylcholine and 5-30% by weight of other phospholipids such as phosphatidylethanolamine. The cholesterol content is preferably below 1%.

By "fusion peptide" is meant viral spike glycoproteins containing the fusion peptide. In one embodiment, the invention relates to either the whole hemagglutinin trimer of one of the viral surface spikes or one or both of its cleavage subunits, the HA1 and HA2 glycopeptides, which contain the functional fusion peptide. The fusion peptide is isolated or synthetically produced.

In a particular embodiment, these polypeptides comprising the fusion peptide are influenza hemagglutinins, in particular its A-H1N1 subtype.

The term "cross-linking agent" refers to an organic heterofunctional molecule that is capable of binding to the surface of the blister of the present invention and of binding to polypeptides. In a preferred embodiment of the invention, this molecule is an organic bifunctional molecule containing a carboxyl group and a thiol group, in particular a sulfosuccinimidyl (S) derivative such as S-4- (p-maleimidophenyl) butyrate, S-acetate , S-2- (m-azido-o-nitrobenzamido) ethyl-1,3'-dithiopropionate, S-6- (4'-azido-2'-nitrophenylamino) hexanoate, S- (4azidophenyldithio) propionate, S-2- (p-azidosalicylamido) ethyl 1-1,3'-dithiopropionate, S-2- (biotinamido) ethyl, 3'-dithiopropionate, S-6 - (biotinamido) hexanoate, S-3- (4-hydroxyphenyl) propionate, S- (4-iodoacetyl) aminobenzoate, S-4- (N-maleimidomethyl) cyclohexane-1- carboxylate, S-2,2,5,5-tetramethylpyrroline-1-oxy-HCl.

The term "cell-specific" protein or marker refers to a protein which is capable of binding to a cross-linking agent on the surface of the vesicles, and of binding to a cell receptor by inducing the endocytosis mechanism. In a preferred embodiment of the invention, this molecule relates to a cell receptor specific antibody, in particular a monoclonal antibody.

The invention will now be illustrated by way of examples and with reference to the drawings.

Example 1

A process for the preparation of phosphatidylcholine synthetic membrane vesicles having virally fusion peptides that are fully active in the influenza virus-derived hemagglutinin trimer and containing dextran sulfate as the antiviral agent.

Figure 1 illustrates the essence of the process; circles represent a liquid solution or suspension; squares represent a solid sediment or precipitate. The procedure involves mixing a 2700 mg dry solids fusion buffer (FB) and a hemagglutinin suspension containing 46 mg hemagglutinin (HA) and 31.1 pmol viral phospholipid (VPL) and subjected to ultracentrifugation (UC1). The resulting pellet is dissolved in solution III, which contains a fusion buffer (FB) and octaethylene glycol monodecyl ether (OEG) as a mild detergent. Next, ultracentrifugation (UC2) was used to swallow supernatant IV containing FB, HA, VPL and OEG to which was added antiviral dextran sulfate (DS) to obtain solution V. Meanwhile, a solution of phosphatidylcholine VI (PC) VI is evaporated in a vacuum rotary flask to form a thin PC layer on the inner surface of the flask. Solution V is added to this layer, after dissolution, solution VII is swallowed three times with polystyrene bead microcarrier (PBM) to remove OEG, and ultrasound (US) of solution VIII is obtained to obtain blisters of the desired size.

HU 219 353 Β re; diluting the resulting suspension IX with NaCl solution (X) to obtain the desired drug suspension (DR).

The following is a detailed description of the general description above.

A fusion buffer solution (Solution I) was prepared containing 7.9 mg / ml sodium chloride, 4.4 mg / ml trisodium citrate dihydrate, 2.1 mg / ml 2-morpholinoethanesulfonic acid monohydrate (MES) and 1,2. mg / ml of N-hydroxyethylpiperazine-N'-ethanesulfonic acid in water, the solution was adjusted to pH 7.3 with 1N sodium hydroxide.

Influenza virus (A / Shanghai / 16/89 (H3N2) strain) is grown in the allantoin cavity of hen eggs and purified twice by sucrose gradient ultracentrifugation according to Skehel and Schild (Virology 44, 396-408 (1971)). The purified influenza virus suspension (II) contains 266 pg of influenza virus hemagglutinin per ml, totaling 31.1 pmoles of viral phospholipid, determined as follows.

Viral phospholipids are described by Folch et al., Folch, J., Lees, M., and Sloane, SGH; J. Bioi. Chem. 226: 497-509 (1971)]. For analysis of phospholipids, the lower organic phase was evaporated and the residue was subjected to either phosphate determination [Chen, PS, Toribara, TY and Warner, H .; Anal. Chem. 28, 1756-1758 (1956)] or dissolved in a small amount of chloroform-methanol, the solution was analyzed for phospholipid content by thin-layer chromatography. For this purpose, silica gel plates (Merck) were used and the developing agent was a mixture of chloroform, methanol, acetic acid and water (25: 15: 4: 2 by volume). Each phospholipid was visualized by exposure to iodine and identified by reference to R _r values relative to reference samples. Proteins were determined by a modified Lowry method [Peterson, GL; Anal. Biochem., 83, 346-356 (1977)].

A total of 173 ml of solution II was mixed with an equal volume of fusion buffer I. Sedimentation of the diluted influenza virus solution was performed by ultracentrifugation at 100,000 x g for 10 minutes.

To the influenza virus pellet is added 15.3 ml of detergent solution containing 54 mg / ml (= 100 pmol / ml) octaethylene glycol monododecyl ether (OEG) in the above fusion buffer. This corresponds to 18 pg = 33.3 nmoles of OEG per 1 pg hemagglutinin. Within 10 minutes the sediment completely dissolved. The solution is ultracentrifuged at 100,000 x g for 1 hour. The remaining supernatant, containing the reconstituted influenza hemagglutinin trimer and the non-essential neuraminidase residue and other ingredients (solution IV), contains 3000 mg of dried antiviral dextran sulfate active ingredient [Lüscher, M. and Glück, R., Antiviral Research, 14 39-50] so that solution V was swallowed.

A 10 mg / ml solution of phosphatidylcholine (SIGMA) in chloroform / methanol (2: 1) was prepared. 28 ml of this solution VI were carefully evaporated in vacuo in a rotary evaporator to form a thin layer of phospholipid (280 mg) on the inner surface of the round bottom flask.

Solution V is then added to the phospholipid layer in the round-bottomed flask. After shaking the flask for at least 15 minutes while the phosphatidylcholine is completely dissolved, the resulting solution VII is transferred to a glass vessel together with 4.8 g of a polystyrene bead microcarrier (wet 25-50 mesh) (PBM).

The vessel is then shaken so that its contents move from side to side twice per second. After 1 hour, the suspension is aspirated with a thin pipette and transferred to a new container with 2.4 g of polystyrene bead microcarrier of the same size. Shake for 30 minutes. This operation is repeated twice. After the final step, the resulting solution VIII was removed from the beads, placed in a water bath and treated with an ultrasound device (Bransonic, Branson Europe BV, frequency: 50 kHz ± 10%). Ultrasound shocks of 10 seconds were repeated twice, with a 10-second pause to obtain medium-sized blisters of about 50-100 nm in diameter. The resulting solution IX is then diluted 1: 100 with physiological saline (solution X).

Example 2

A method for producing synthetic membrane vesicles containing an antiviral antibody adsorbed to influenza virus hemagglutinin trimers and CD4 monoclonal antibodies.

The blisters were prepared as described in Example 1 with the following modifications: Instead of 10 mg of phosphatidylcholine, only 9 mg of phosphatidylcholine and 1 mg of phosphatidylethanolamine (cephalin) were dissolved per ml in chloroform / methanol.

After the hemagglutinin vesicles were prepared according to Example 1, 20 mg of sulfosuccinimidyl 4- (N-maleimidophenyl) butyrate (SMPB) dissolved in 2 ml of water was added as a crosslinking agent. The mixture was gently shaken for 1 hour at room temperature and the bladders were pelleted by ultracentrifugation at 100,000 g for 10 minutes.

To the pellet was added 8 ml of Anti-leu 3A (Becton & Dickinson). The resuspended material is shaken gently for one hour every 5 minutes for several seconds at room temperature. Finally, the material was pelleted again to remove unbound antibodies by ultracentrifugation at 100,000 x g for 10 minutes. The pellet was suspended in 1500 ml of physiological saline.

Example 3

The procedure of Example 2 is repeated with the following modifications.

Instead of dextran sulfate, 1000 mg of imidazole carboxamide and 1000 mg of hydroxyurea are added to solution IV (see Example 1), as described by Brunner and Nagel, Springer Verlag, 2nd edition, Internistische Krebstherapie, p. 1979)] for the treatment of melanomas.

After addition of the crosslinking agent and further processing according to Example 2, 1 mg of monoclonal antibody (VES), which is a monoclonal antibody or R24, is added as described by Houghton, A.N., et al., Proc. Nat. Ac. Se., 82, 1242

(1985)] or 0.5 mg of L55 + 0.5 mg of L72 as described by Iric, R. S. et al. (Chain 1989, 786-787) to give 1000 mg of the humane substance having the following composition:

mg hemagglutinin 250 mg phosphatidylcholine mg phosphatidylethanolamine (cephalin) mg cross-linking agent (Sulfo-SMPB) mg monoclonal antibody

1000 mg imidazole carboxamide

1000 mg of hydroxyurea

Up to now, these drugs have been administered at doses about five times that of the indicated doses of only 200 human doses.

Example 4

The following modifications are performed as described in Example 2.

Instead of dextran sulfate, at least 1000 mg of at least one of the following drugs is used: adriplastin, endoxan, fluorouracil, as described by Brunner and Nagel (Intemistische Krebstherapie, Springer-Verlag,

309 (1979)] and colchicine (SIGMA) (see Example 1).

After addition of the cross-linking agent and further processing as described in Example 2, 1 mg of JDB1 monoclonal antibody (VÉS) was added to Strelkauskas, A. J., Cancer-Immunol. Immunother., 23, 31 (1986)] to provide 1000 human doses of anti-mammary carcinoma:

mg hemagglutinin 250 mg phosphatidylcholine mg phosphatidylethanolamine (cephalin) mg cross-linking agent (Sulfo-SMPB) mg monoclonal antibody

1000 mg of adriplastin

1000 mg of endoxane

1000 mg of fluorouracil

Example 5

The procedure of Examples 1 and 2 is repeated with the following modifications.

Instead of the influenza hemagglutinin trimer, monomers containing a fusion peptide are used. The hemagglutinin-2 monomer containing the fusion peptide was obtained by chemical reduction of the S-H bridges by chemical reduction with D4-dithiothreitol (DTT, Sigma) followed by gel chromatography (Sephadex G 50) of the hemagglutinin-1 peptide. The purified hemagglutinin-2 monomer suspension contains 180 µg of the monomer containing the fusion peptide.

Example 6

The following modifications are carried out as in Example 2.

The crude fusion peptide was used in place of the influenza hemagglutinin trimer. The flu fusion peptide used to make synthetic membrane vesicles is swallowed by chemical synthesis. Any of the amino acid sequences shown in Figure 2 can be used. An arrangement containing at least one, preferably three cysteine residues at one end of the sequence has been found to be useful for fusion activity.

A 4 pg / ml solution of one of these synthetic fusion peptides is used.

The blisters containing any of the above fusion peptides are prepared as follows.

9 mg of phosphatidylcholine and 1 mg of phosphatidylethanolamine were dissolved in 1 ml of a 2: 1 mixture of chloroform and methanol. 28 ml of this solution were carefully evaporated in vacuo on a rotary evaporator; Thus, a thin layer of phospholipid is formed on the inner surface of a round-bottomed flask.

Dissolve dextran sulfate in 15.3 ml detergent solution containing 100 pmol of octaethylene glycol monodecyl ether per ml of fusion buffer and add to the phospholipid layer in a round-bottomed flask. After shaking for at least 15 minutes and completely dissolving the phospholipid layer, the solution is transferred to 4.8 g of a polystyrene bead microcarrier, 25-50 mesh wet, in a glass container.

The vessel is then shaken to move its contents from one end to the other twice per second. After 1 hour, the suspension is aspirated into a thin pipette and transferred to a new vessel with 2.4 g of polystyrene bead microcarrier of the same size as above. Shake for 30 minutes. This procedure is repeated twice. After the final step, the resulting solution is removed from the beads, placed in a water bath and treated with an ultrasound device (Bransonic, Branson Europe BV, frequency: 50 kHz ± 10%). Repeat the 10 second ultrasound beats twice, with a 10 second pause. This results in medium-sized vesicles having a diameter of about 50-100 nm.

To the above suspension was added 2 ml of water containing 20 mg of sulfo-SMPB (Pierce). The mixture was gently shaken for 1 hour at room temperature and the bladders were pelleted by ultracentrifugation at 100,000 x g for 15 minutes.

2 ml of the solution containing the synthetic fusion peptide are added to the pellet. The suspended material is shaken gently for 1 hour at room temperature every 5 minutes for a few seconds. Finally, the solid was pelleted again to remove unbound fusion peptides by ultracentrifugation at 100,000 x g for 10 minutes. The precipitate was resuspended in 200 ml of physiological saline.

Example 7

The following modifications are performed as described in Example 6.

To the pellet containing pellet was added 2 ml of a solution containing 2 µg of synthetic fusion peptide and 100 µg of a solution containing 100 µg of Anti-Leu 3A. The suspended material was allowed to stand for 1 hour at room temperature for 5 minutes7

Shake gently for several seconds. The material was then pelleted again to remove unbound fusion and cell-specific peptides by ultracentrifugation at 100,000 x g for 10 minutes. The pellet was suspended in 200 ml of physiological saline.

Example 8

1-4. The blisters of Examples 1 to 5 are prepared with a fusion peptide or hemagglutinin from rabdoviruses, parainfluenza viruses or togaviruses.

a) Rabdovirus rabies (rabies virus) is produced in human diploid cells. The pooled supernatant (supernatants) containing 107 rabies virus per ml was purified and concentrated by sucrose density gradient ultracentrifugation. The purified rabies virus suspension contains 210 pg rabies hemagglutinin per ml, and the suspension is further processed as described in Example 1.

b) Type III parainfluenza virus is grown in a hen's egg allantoin cavity and purified twice by sucrose gradient centrifugation as described in Example 1. The purified parainfluenza virus suspension contains 245 pg / ml of parainfluenza virus haemagglutinin per ml, and the suspension is further processed as in Example 1.

c) The togavirus rubella (measles, rubella virus) is cultured in human diploid cells and purified as described in step a). The purified rubellavirion suspension contains 205 µg / ml of rubellavirus haemagglutinin, further processed as in Example 1.

Example 9

Antiviral drugs were prepared as described in Example 1. Hu-PBL-SCID mice were treated with ribonuclease dimer for 14 days, alternatively with phosphate buffer solution (blank) for 10 days, or with dextran sulfate or ribonuclease dimer at three dose levels (prepared according to PCT / US90 / 00141) or liposomes, liposome-bound ribonuclease dimer, the latter two prepared as in Example 1 above. Treatment was commenced with co-infection with 100 TCID ₅₀ HIV-Ijub virus. Hu-PBL-SCID mice were assayed for viral infection (virus isolation, PCR determination of provirus sequences) 2, 4 and 6 weeks after viral infection. The test results show the percentage of animals from which the virus was isolated after each treatment:

phosphate buffer solution (blank) 80% ribonuclease dimer 0.001 mg / kg 92% ribonuclease dimer 0.1 mg / kg 30% ribonuclease dimer 10.0 mg / kg 63% dextran sulfate 10 mg / kg 33% dextran sulfate 10 mg / kg in liposomes 14% in ribonuclease dimer liposomes 25%

The results show that the majority of huPBL-SCID mice treated are protected against HIV infection

12 hours after injection of 0.1 mg / kg ribonuclease dimer, 10 mg / kg dextran sulfate, liposomes containing ribonuclease dimer or liposomes containing dextran sulfate. Low protection is seen with the use of 10 mg / kg ribonuclease dimer, which may be due to the fact that the ribonuclease dimer suppresses immunity at higher dose levels. No protective effect was observed when using 0.001 mg / kg ribonuclease dimer. However, when the ribonuclease dimer is used in liposomes according to the invention, the successful rate is improved from 63% to 25% despite the high dose. Further improvement is expected from providing optimal dosage levels enclosed in liposomes.

The same conclusion is reached when mice with poor human PBL transplants at the end of the experiment are excluded from the analysis, although analysis of human immunoglobulin levels and β-globin PCR results indicate that treatment with either dimeric ribonuclease liposomes or dextran-sulfate-intermediate for the survival of human cells. The exclusion of hu-PBL-SGID mice on the basis of human function allows some distinction between direct antiviral activity and immunomodulatory activity.

Example 10

The fusion assay described by Lüscher and Glück (1990, Antiviral Research, 14, 39-50) compared

The bladders prepared according to Example 1 were prepared with reconstructed influenza vesicles according to the method of Kawasaki et al., In model membranes.

Figure 3 shows the kinetics of increased fluorescence of R18-labeled influenza A virus with DOPC-cholesterol liposome. The increase in fluorescence is expressed as% FDQ, as calculated by Lüscher & Glück (see above).

The initial fusion rate is obtained from the tangent of the fusion curve at time 0 at the start of the fusion (dashed line in Figure 3). Curve 2 corresponds to the fusion activity of the blisters produced by the method of Kawasaki et al.

Claims (13)

PATENT CLAIMS CLAIMS 1. Double-layer phospholipid vesicles, comprising at least one functionally active fusion peptide and a desired drug or pharmaceutically active substance attached to the membrane, characterized in that the vesicles contain: a) a membrane comprising non-viral phospholipid phosphatidylethanolamine and phosphatidylcholine, preferably in combination with viral phospholipid; b) at least one functionally active natural or synthetic fusion peptide, wherein the natural fusion peptide is a hemagglutinin trimer or monomer, one or both of its cleavage subunits and a glycopeptide of HA1 and HA2, and a influenza virus, preferably its A-H1N1 subtype, rhabdovirus8; EN 219 353 Β, Parainfluenza virus type III, Semliki Forest vermin or toga virus; c) a bifunctional crosslinking agent, preferably a sulfosuccinimidyl derivative, bound to the phosphatidylethanolamine of the membrane; and d) at least one cell-specific marker bound to the above membrane-bound cross-linking agent. 2. The vesicle according to claim 1, characterized in that their membrane cholesterol content of ⁰ to 10 wt / below, preferably 1 wt ⁰ / below. The blisters according to claim 1 or 2, characterized in that they have a diameter of less than 100 nm, preferably about 80 nm. 4. Bladders according to any one of claims 1 to 4, characterized in that they contain an antibody, preferably a monoclonal IgG antibody, as a cell-specific marker. 5. The blisters according to any one of claims 1 to 4, characterized in that they contain as a fusion peptide a synthetic fusion peptide having at least one cysteine unit, preferably three cysteine units, at least at one end of its amino acid sequence. 6. Double-layer phospholipid vesicles according to any one of claims 1 to 3, characterized in that their membrane contains viral phospholipids in combination with 2-100 fold, preferably 5-15 fold, of exogenous non-viral phospholipid. 7. Double-layer phospholipid vesicles according to any one of claims 1 to 3, characterized in that they have a membrane of 70-95% by weight of phosphatidylcholine and 5-30 ⁰ /, preferably 10-20 wt ⁰ / phosphatidylethanolamine. 8. Double-layer phospholipid vesicles according to any one of claims 1 to 3, characterized in that they contain 5-10% by weight, preferably 6-8% by weight of cross-linking agent, based on the total weight of the membrane. The bilayer phospholipid vesicles of claim 1, wherein at least one fusion peptide is of synthetic origin and is bound to the membrane via a membrane-bound cross-linking agent. 10. Double-layer phospholipid vesicles according to any one of claims 1 to 5, characterized in that the pharmaceutically active substance is selected from the group consisting of imidazole carboxamide, hydroxycarbamide, adriblastine, endoxane, fluorouracil, colchicine, dextran sulfate, ribonuclease dimer or lysozyme. They contain. 11. A pharmaceutical composition for controlled delivery of a pharmaceutically active substance, in particular a medicament, characterized in that at least one of claims 1-10. A blister according to any one of claims 1 to 6. The pharmaceutical composition for the treatment of viral infections or cancer according to claim 11, characterized in that at least one of claims 1-10. A blister according to any one of claims 1 to 6. Pharmaceutical composition according to claim 11 or 12, characterized in that it contains drug-containing blisters containing 0.001 to 10 mg / kg body weight of the desired drug.