DE3218121A1 - Pharmaceutical compositions for tumour treatment - Google Patents

Abstract

The invention relates to pharmaceutical compositions which can be administered intravascularly for the treatment of oncoses and which contain immunostimulants or cytotoxic agents which are encapsulated in liposomes and/or erythrocytes.

Description

The invention relates to medicaments for the treatment of Tumor diseases. Numerous research projects in

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various countries are concerned with the immunological control mechanisms responsible for changes in tumors and metastases and have already contributed a great deal to a better understanding. According to this, the macrophages play a central role in the recognition and selective killing of tumor cells. The predominantly sessile macrophages belong to the phagocytes, i.e. those cells that absorb and digest tissue debris, foreign bodies, microbes and other cells, the amoeboid mobile macrophages are characterized by a large spindle-shaped cell nucleus with numerous vacuoles. It has now been found that in two critical phases of tumor formation and development, namely in the critical early phase of the initial establishment of spontaneously or induced transformed single cells and in the second critical phase of metastasis after dissipation and settlement of the primary tumor cells in secondary organs, the activation of the the natural immune defense, which is also responsible for NK, NC and K leukocytes, only runs through activated macrophages or only works in their presence. According to some studies, macrophages are supposed to be involved even in the specific cytotoxicity of T lymphocytes, which leads to an increase in immunogenicity by converting the antigens to so-called superantigens. A precondition for the unspecific tumoricidity of the macrophages, which can precisely differentiate between transformed and normal cells in syn-, allo- and even xenogenic systems and do not trigger any resistance that is otherwise regularly observed with cystostatics and radiation, is their activation; unactivated macrophages are ineffective. After specific or unspecific stimulation, macrophages become selectively tumoricidal. This property is so pronounced that up to now no tumor cell subpopulations have been observed which would cause the activated pr \ py

Macrophages would develop resistance to im In contrast to the regularly observed resistance of a few tumor cell clones to all cytostatics and irradiation. This fact is of particular importance because today we know that everyone Tumor consists of a large number of subpopulations that differ in phenotype, karyotype, receptor presence, Immunogenicity, antigen structure, metastasis tendency and responsiveness to cytostatics and Distinguish irradiation. Due to the resistance of the subpopulations, tumors can later recur to lead. By selectively killing off the non-resistant tumor cell cones, the resistant ones win Tumor cells even have a selection advantage. This becomes particularly plausible if the findings are Americanized Researchers envisage what subpopulations are within of the primary tumor develop a great phenotypic diversity as soon as they are out of the tumor context e.g. in experiments in the form of monocloning with subsequent colony formation. The cells are then subject to mutual control in the tumor tissue. As research in Israel has shown, the primary tumor also controls the metastases that can "explode" after the primary tumor has been removed. Even Animal experiments show that a low number of tumor cells leads to smaller tumors, the metastasias however, it can be more pronounced. Since the activation of NK cells, which are primarily responsible for the prevention of the Starting with the first transformed cells and later of (lon disseminated tumor cells in the premicrometastases be held responsible for macrophage activation runs, this results in the central role of macropliages in combating tumor cells. It is also already known that macrophage activation is a direct function of immunostimulator accessibility

is, as in the example of the immunopotentiating synthetic polyanions or in the example of bacterial Lipopolysaccharides could be shown.

The present invention is therefore based on the object of improving the accessibility of immunostimulants or cytotoxics. To solve this problem, medicaments are proposed which are characterized in that they contain immunostimulants or cytotoxics encapsulated in liposomes and / or erythrocytes, the transport liposomes or erythrocytes, in the case of cytotoxics, being associated or covalent on the surface bound tumor-specific immunoglobulins must be loaded. The liposomes are discrete cell particles which essentially consist of phospholipids and are in multilamellar form. The liposomes are primarily phagocytosed by macrophages during the so-called "clearance" of the liposomes from the bloodstream. After phagocytosis, the compounds contained in the liposomes are internasilated into the cytoplasm via phagolysosomes. As we already know from studies with liposome-encapsulated muramyl dipeptides or MAF (macrophage-activating factor, these immune-stimulating substances are already practiced in a concentration that is only 1/500 to 1 / 20,000 of the dosage of the same substance in the liposome-free system, A strong stimulating effect on the macrophages. The liposomes can be loaded with various compounds according to the method described in Cancer Research 39> 88l pp J 979, in which case immunostimulants or cytotoxics are used. Instead of the liposomes, autoloomologous heterologous, optionally pretreated erythrocytes, inactivated bacterial strains that can be phagocytosed well or also homo- or heterologous macrophages which

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previously in-vitro the immunostimulator-containing erythrocytes internalized. In this way the immune stimulators can be used in high concentration and pass it on specifically to the target cells and the body's own macrophages.

1. According to the invention, the following are suitable as tumor-specific immunostimulants:

1.1. Membrane fragments and / or membrane extracts of Metastatic cells

1.2. Membrane fragments and / or membrane extracts from primary tumor and metastatic cells that are specifically modified in their surface structure, it being possible for the parent tumor cells to be preopsonized with heterophilic antibodies.

The targeted membrane (cell surface) change can be achieved through the following types of treatment :

1.2.1. Treatment of tumor cells with lectins and protectins

1.2.2.1. with concanavalin A (Con A), which specifically contains the dL-D-Gl uco.se and .s-D-mannose saccharide group binds to the cell surface

1.2.1.2. with WGA (agglutinin from wheat seedlings), which for the terminal N-acetyl-D-glucosamine groups is specific

1.2.1.3 · with SDA (agglutinin from soybeans), that with the N-acetyl-D-QJ actοsamin end groups specific

reacted
1.2.1.4. with the agglutinin from Ricinus communis, which specifically binds the D-gninctose and L-arabinose end groups.

Other interesting plant agglutinins are still extracts from Lotus tetragonolobus, Phaseolus limensis, Dolichos biflorus, Lima beans (PHA) Phytolacca americana, Laburnum alpinum, Vicia cracca, Sophora japonica, solanum tuberosum, Satnbucus nigra, Maraamius oreades and others

Also lectins which are proteolytically split and which have retained their specificity are interesting because they restore contact inhibition and the surface of tumor cells can make them more immunogenic. The protectins are also a very interesting group these animal agglutinins include extracts from Helix pomatia, Helix hortensis, Otala lactea, Salmo sal ar, Salmo irideus etc.

1.2.2. Treatment of the tumor cells (primary tumor and metastatic cells) by enzymes that remove the surface groups split off

1.2.2.1. with RDE (- "receptor destroying enzyme", neuraminidase, sialidase): splitting off the en & daily neuraminic acid units

1.2.2.2. with spec. Glycosidases (J ^ - and ß-glycosidases): Cleavage of terminal saccharide units

1.2.2.3 with spec. Glycosaminidases (Mucopolyaaccharidases Cleavage of terminal glycosamines bziv. Surface surface mucopolysaccharides

1.2.2.4. with proteolytic enzymes (pronase, papain, etc.) Trypsin): splitting off of mucoproteins and lipoproteins from the cell surface

1.2.2.5- evil: light treatment with Elast (in) ase, CoI1agcnase, Phosphatase and RNase.

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1.2-3- Treatment of the tumor cells with periodate

1.2.4. Careful pretreatment of the lyophilized Tumor cells by lipoid solvents

1.2-5 Treatment of tumor cells with iodoacetate or iodoacetamide or N-ethylmaleimide, all of which react with the sulfhydry groups

1.2.6. Treatment of tumor cells with dinitrophenylaminocaproate or TNBS (2, k , 6-trinitrobenzylsulfonic acid)

1.2.7- treatment of tumor cells with (DNCB)

(Dinitrochlorobenzene) or DNBB (Dinitrobromobenzene)

1.2.8. Treatment of the tumor cells with formaldehyde solution or guanidine / urea. The mild treatment of the tumor cells with formalin is therefore of particular interest there, as with diphtheria toxoid, the immunologisi Specificity after the treatment of the cells is not lost and one of one could benefit from another advantage, namely the fact that antiseing against one with Formaldehyde treated protein cross reactivities with other formaldehyde treated proteins (Cells) enter, even if there are none between the native forms of protein cells There is cross-reactivity. The latter property of formally in-treated cell proteins should only be a special case of a more general one Be observed that denatured proteins very often show immunological cross-reactivity. He formally treats this property Tumor cell 1 surface proteins could be a important role in the intended breakthrough of the "smal1-dose" paralysis Tumor-, si) oziell metastaspncel 1 s play.

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1-2.9- Treatment of tumor cells with glutaraldehyde 3.2.1ο. Treatment of tumor cells with picrylchloride

1.2.11. Treating tumor cells with some dye those stable with proteins (the cell surface) Make connections, for example: Coomassie R-25o; Amjdoschwarz 1 oB; Nigrosine; Dibromotrisulfofluorescein; Procion Brilliant Blue RS; Fast Green (F.C.F., Food Green 3); Xylene Brilliant Cyanine G (Coomassie Brilliant Blue G 25o) Red W and FDNB

1.2.12. Covalent binding of proteins (flags ") and controlled synthesized polypeptides on primary tumor and preferably on metastatic cell surface structures for the purpose of increasing immunogenicity.

Synthetic polypeptides can be linear or branched, e.g., in the form of a main strand with side chains. They can be built up from a single type of amino acid, e.g. as Poly-d, 1-alanine or polytyrosine or also Combined from 2 or 3 different amino acids, these chains are called heptene to antigens appended, so they can make the immunogenicity of the carrier antigen favorable or unfavorable influence. Poly-d, 1-alanine sets as a hapten the .immunogeneity of the carrier. Polytyrosine chains increase the immunogenicity of the carrier antigen and can even make gelatin immunogenic. In general, diversity increases of the amino acids of a synthetic antigen its immunogenicity. Essential requirements to such an antigen molecule are stable Basic structure, a not too high electrical charge and position of the imraunogßn important Tyrosine at the exposed parts of the side chain

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The co-operative binding of immunogenicity-increasing Proteins on the tumor cell surface can be multiply with the help of affinity chromatography proven techniques such as EDAC, BrCN, N-hydroxysuccinimide ester and BDB (bis-diazobenzidine) take place.

Another possibility is the coupling via hexaazotated pararosaniline. 1.2.13 · Di elimmunogeneity could be increased and the "low-zone" / "1ow-zone" / "smal1-dose" tolerance are breached when primary tumor and preferably metastatic cells with myeloma cells hybridized and cell membrane fragments and extracts of hybridomas formed in this way as liposome encapsulates injected.

We know from the experiments with the (non-encapsulated) Interspecies hybridoma cells that these hybridomas differ from the original tumor cells after inoculation do not address them because they are because of the homoloj or heterologous histocompatibility antigens immunologically repelled on the cell surface will. This is an important consideration when considering breaking the "1ow-zone" / "small-dose" tolerance mentioned above thus the paralysis of the immune apparatus in 2 kriti Sections dor tumor development, in the early phase the establishment of the first transformed cells and in the 2nd extremely criti: see phase of the Establishment of dissorinated mctasin cells in colonion (premiltrometastnsen), whereby a curable primary tumor becomes incurable metastasis-associated tumor. The fact that this minierie turnor cells as easily accessible single

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and micro-aggregates in Her Blutbnhn dor immunological surveillance can miss ^l: eats hardly mentioned except by the "low zone" / "small-dose" -Paralyse interpret. The decisive factor here is likely to be the "shedding" of the (tumor (neo) antigens) (TATA ₁ TSTA) into the bloodstream, ie the persistent, even excretion of small amounts of (weak) immunogens, the unstable ones and, especially in the form of immune complexes, slightly from the tumor cell membrane detachable tumor antigens.

The proposal according to the invention to increase the cross-reactivity through targeted alteration of the tumor cell surface, be it through controlled enzymatic or chemical treatment or covalent coupling of immunogenity-increasing proteins and synthetic polypeptides, be it through hybridization (interspecies cell hybrids), and through this xenogenization "sleeping" To stimulate inactive clones of the small lymphocytes ("memory cells") to proliferate and cell mediating filter cytotoxicity, receives strong impulses in the latest findings of immunogenetics, according to which, as shown in the model of the 3-1'L tumor, inoculated tumor cells only in those mouse strains Metastasis were capable, which had an identical H-2 haplotype. Differences as small as, for example, differences in the H-2 ^k , h-2 ^d , H-2 ^a or H-2 ^S haplotype of the MHC of the mouse test; mme were sufficient to prevent the metastasis from attacking.

2. As unspecific immune stimulants are suitable liposome and erythrocyte encapsulates according to the invention from:

2.1. Interferon (e.g. Recombinant Interferon A, generally: fibroepithelles leukocitic and immune-induced, i.e. by antigen and / or

Mitogen Contact Induced Interferon) 2.2. Interferon inducers

2.2.1. Polyribonucleotides (stabilized with CM cellulose and polylysine)

2.2.1.1-single-strand homopolymerization poly rl.

2. 2. 1 . 2. Double stranghoraopol ymere P ⁰ ^ -Y rl: rC

poly rA: rU

2.2.1.3-Alternating copolymers: poly rI: rC / poly rA: rU 2.2.1.4 Homopolymer-Copolymers: poly rl: poly rC: rG

2.2.2. Polydeoxyribonucleotide (stabilized with CN cellulose and Polylsin)

2.2.2.1 Homopolymer e poly dA: dT

2.2.2.2 Polydeoxyribonucleotide analogs poly sI: sC

(Thiophosphate instead of phosphate

2.2-3. Polyribonuclcotid-Aggregate poly rl: rC / DEAE-

Dextran

poly rl: rC / poly-L-Ly s in

(The binding to poly-L-lysine increases the stability)

2.2.4. Low molecular interferon inducers Tilorone (bis-diethylamino-ethylfluorenone), cationic dyes such as acridine and quinacrine, propanediamine, mercaptoalkylamines, BL-2o8o3 » MA-56, AET, U-25 / l66, PMA (= phorbol myristate acetate), Cimetidines.

2.2.5 * Other immunostimulators, some of which are via interferon induction boost the immune system

2.2.5-1 Bacteria and their extracts (lipopolysaccharides, Lipid A, endotoxins, toxoids) BCG, Brucella abortus, Bruceila melitensis, Corynebacterium parvum, Haemophilus influenzae, Klebsieila aerogenes, Listeria monocytogenes and others

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2.2 · 5 · 2. Lectins and protectins, partly proteolytic pre-split ConcanavalIn A, WGA, SBA, PHA, PNA, Phytohemagglutinins (lectins) from Phaseolus limensis, Lotus tetragonolobus, Dolichos biflorus, Phytolacca americana etc. as well as protactins from Helix hortensis, Helix pomatia, Otala lactea, Salmo salar, SaI mo irideus etc.

Combinations of 2.2.3.I. are particularly advantageous. and 2.2-3.2. e.g. from BCG and Con A as well as from 2.2.3-2. and neuraminidase pretreated 2.2.3.1 e.g. PNA (= peanut agglutinin) and neuraminidase pretreated BCG. The effect can be achieved by simultaneous injection of unchanged or in their surface structure specifically modified (primary tumor or metastasis) cells are reinforced. Basic polymers (polylsin, protamine sulfate) are also of interest. Instead of BCG, the Methanol extract from BCG (MER) or the purified active substance from BCG (MDP, Muramyl dipeptide) be used.

2.2.5 3 Plant extracts Lentinan and Pachymaran or Carboxymethyl pachymaran

2.2-5 ^ KLH (Keyhole-limpet-hemocyanin, Leatil, Levatnisol, Zymosan, TNP-KLH (Trinitrophenyl-KeyhoIe-Iimpet-hemocyanin)

2.2.5-5 Statolon, pyran copolymers (maleic acid divinyl ether copolymers), COAM (after Claes), Levan, Triton WR I339

2.2.5.6. Thymus hormone (thymus extract)

2.2.5 * 7 · 13-cis-retinoid, water-soluble selenium compounds (e.g. Na selenite) and li salts

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2.2.5.8. Oxazolone (a T-cell stimulator)

2.2.5.9. Ca (PO ^) ₂ , BeSO ^, Vit.A / bentonite

102.5.lo. Hiu I & II, Wax C, US-treated bacteria

3. According to the invention, it is advisable to support active phagoxytosis directly or indirectly the liposome encapsulation of:

3.1. Prostaglandin inhibitors (indomethaxin, aspirin)

3.2. Inhibitors of the enzymes superoxide dismutase and catalase.

The local withdrawal of ascorbic acid, glutathione and vitamin E could have a similar effect as electron acceptors.

The encapsulation of peroxidase, furthermore of peroxide (persulphate = persulphate = peroxodisulphate, hypohalide), alone or combined with halide, and of the catalase inhibitor aminotriazole, with the aim of supporting and / or supporting _{the tumoricidal effect emanating from the macrophage via HO 0, is advantageous.} to strengthen. Interesting in this regard are the latest research results, according to which the H _Q 0 reducing and thus inactivating glutathione protects the tumor cell from cell lysis by the macrophages and this protective effect is reversed by suppressing the glutathione replication by means of BCNU-mediated specific inhibition of the enzyme glutathione reductase involved can. 3-3. Dibutyryl-CAMP, ionophore A 23 I87 and mitogens (general), which regulate the

2 +
intracellular Ca stimulate lymphocyte proliferation.

Since the mitogen-stimulated lymphocyte transformation, which initiates the actual mobilization of the small lymphocytes ("memory cells"),

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can only take place in the presence of (extra / .ßl lular) Ca ions and the mitogens de Cn influx in di <; Induce lyraphocytes, and since this Ca influx is accelerated by dibutyryl-cGMP (and inhibited by dibutyryl-cAMP), liposome encapsulation of dibutyryl-cGMI ^{1 should be} considered as one of the possibilities for stimulating the lymphocytes to proliferate. Another possibility arises from the encapsulation of ionophore A 23187, which promotes lymphocyte proliferation.

Since, according to modern theories, phagocytosis, exotyzos and lysosome-phagosome fusion are monitored by the intracellular Ca concentration, the merabran-mediated signals via Ca influx or mobilization of intracellular Ca deposits raise the basal Ca level short of Io M Raise Io -Io M and thereby enable the functioning of the intracellular act in / myosin mechanism in the microfilarent and microtubule system (cytoskeleton), a transport of Ca ions vZ.D. as CaCl, encapsulated in liposomes) lead to macrophage stimulation through the cell membrane by bypassing the membrane ion barrier, whereby the effect can be protracted by choosing a favorable Ca depot form. The effect of the above-mentioned ionophore A231Ö7 is likely to be due to the mobilization of the intracellular Ca depot; Interestingly, even extremely low concentrations of the aforementioned ionophore (Io) can make the cell (e.g. the macrophage) largely independent of extracellular Ca and Mg.

Since substances with an affinity for hydrophobic regions of the macrophage membrane are the same more permeable for

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make (divalent) ions, it can be advantageous the liposome surface with proteins rich in non-polar side chains or those that are through special Forms of denaturation have acquired a strongly hydrophobic character.

Per
3. h. Since low CAMP levels were found in tumorgression and high CAMP levels in tumor regression and since CAMP-CGMP regulation of cell proliferation is assumed, local (intracellular) stimulation of adenyl cyclase can have a tumor-inhibiting effect, hence liposome encapsulation of theophylline, caffeine, catecholamines (ine 1st epinephrine, norepinephrine and isoproterenol) and other cAMP stimulators (the first two methylxanthines increase the cAMP level via phosphodiesterase inhibition, while the catecholamines via adenyl cyclase stimulation or direct cAMP encapsulation.

') . 5. Liposome encapsulation of thioglycol lat (macrophage activator).

k. According to the invention, stimulation of liposome phagocytosis ("liposome clearance") or liposome-membrane fusion by:

kl polyethylene glycol (PAG = PEG)

PEG enhances the Ag / Ab (antigen / antibody) reaction. This fact is of particular interest because the PEG (in a relatively high concentration of ^ι ίο%) is used in cell fusion (cell hybridization) according to Köhler and Milstein.

Liposomes combined with PEG can facilitate phagocytosis and thus internalization of the liposome content give rise to phagocytic cells. The optimal concentration and degree of polymerization (molecular weight)

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of the PEG would have to be found out empirically (Orientation value: 6% PEG solution, MG 6000). A 1% PEG solution accelerates considerably the Ag / Ak reaction and shortens the time required Incubation period.

A 12.5% PEG solution precipitates selectively the short-chain proteins and peptides bound to antibodies in a homogeneous ("soluble-phase") RIA.

k.2 polyvinylpyrrolidone

4.3- polyvinyl alcohol

kk polyvinyl acetate

k.5 ' hydroxyethyl starch

Ί.6. Dextran, dextran sulfate, polysucrose,

Dextran (poy) phosphate xnd phthalic acid ester Dextran (e.g. as a 1% solution) and polysucrose (e.g. as a 1% solution) increase rosette formation (binding of SRBC and T lymphocytes); rosettes become rosettes even with minimal SRBC loading (opsonization) with immunoglobulins formed and the sensitivity increased. From the hematological-immunological work From here one knows the good tolerance of the under 3-6. listed substances.

As is well known, dextran is used to sediment leukocytes and used as a blood expander.

Phthalic acid esters are used for gradient centrifugation of cells used.

4t «7 · Small addition (when used locally) of

Ammonium sulfate
Ammonium sulfate facilitates Ag / Ak contact

^; *-8th. Cholesterol

The free and the (on the C -hydroxyl group) esterified cholesterol can play an important role

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Influence the incorporation of Liposon-.en. It should be in MuI ti! Amellar vesicles be integrated (cholesterol-containing liposonivesicles). Interesting Kare its effect on fusion and Phagocytosc differently constructed Liposomes in the presence of PEG or DMSO / glycine / glycerine monooleate. The complement binding in the cytolysis by T-lymphocytes only takes place if the membrane content of free cholesterol exceeds 35 mole percent, while antibody binding is independent of CJiol esterol is held in the cement membrane.

^l i. <). AET: AET also increases the sensitivity of the Ag / Ab reaction and, when combined with liposomes, can accelerate the phagocytosis and internalization of the liposome-oncapsulated material.

4.1ο. Small addition of substances that increase Ag / Ak contact.

The experience of immunochemistry stems from the fact that the Ag / Ak reaction (precipitate line, halo line through Formaldehyde (lo ^ solution), tannin (1% solution), ß-N'aphtoquinone-'l-sulfonate and ninhydrin (each l? oig) is intensified.

Possibly small addition of cell membrane permeability increasing substances DMSO and glycerine increase the cell membrane permeability. Both are good cryoprotectants. Own

Cryobiological investigations have shown that also the above mentioned PEG ausRPzcichnete kryoprc.t t-kt i VO has properties.

H . 1 2. Low supply /. of sorni pol aron compounds (acetamide, formamide).

Billows of the semi-polar character that between can convey lipophobic and lipophilic signals,

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are. Acetamid and Form.inii.d Lm Zusair.mcnhniiy; with Intercssso's liposome research. ¹ 1 .13 · Little addition of (a) cationic (b) anionic and (c) η i t ion i se he? π tone si don. Because of their strong presence in the cell membrane and their rigidity, traces of surfactants (above the surface of substances) are of central interest in the study of liposome-cell membrane interaction.

Particularly interesting are compounds in which basic groups with long aliphatic Chains (with 12 or more carbon atoms) are combined, as immunological studies have shown that compounds of this type such as quaternary ammonium compounds, substituted guanidines and Benzamidine have a clear adjuvant effect. This adjuvant effect is linked to the ability in Linked to active cell membranes influence. Indeed, many adjuvants are known to be capable of lysosomes dissolve in amoeboid macrophages as from Saponin, Vitamin A, Endotoxin · 'i.13 ·! Examples of cationic surfactants

Substances are benzyldimethyl-alkylammonium chloride (Ilyamine · 35oo, Benzalkonium Chloride); Benzyldiisobbutylphenoxyethoxydimethyl ammonium chloride (Benzethonium chloride, Ilyamine I622, Phemerol); Cety1trimethylammoniumhromid (CTAB, hexadecy1-trimethylammonium bromide);

N-Benzyl-N, N-dimethy LN- ( h- 1, 1,3, 3-th trarai- thy lbuty 1) -

ethoxy ^
phenoxy / ethyl) ammonium chloride; N-Denzy LN, N-

dimethyl-N-Cl- (1,3,3-tetramethylbuty1) -tolyloxyethoxyethyl) ammonium chloride

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Ί.13.2 Examples of anionic · surface-active Substances are butane-1-sulfonic acid-Na-Snlz; Dodecane-1-sulfonic acid sodium salt; Hexadecane-1-sulfonic acid sodium salt; Octadecane-1-sulfonic acid sodium salt; Tetradecane-1-sulfonic acid Na salt; Sulfuric acid decyl ester Na salt; Sulfuric acid octyl ester Na salt; Octadecyl sulfuric acid sodium salt

Ί.13 · 3 · Examples of partially or non-ionic surface-active Substances: nonylphenol polyethylene glycol ether; Methyl tetrapropylenebenzenesulfonate; Octylphenol polyethylene glycol ether; Polyethylene glycol monolauryl ether; Polyethylene glycol monostearyl ether; Polyethylene glycol monostearate.

The combined addition of PEG and polyethylene glycol monolauric ether is particularly interesting in the study of liposomes (or PEG and its monostearyl ether or PEG and its derivative monostearate), i.e. the Combination of liposome phospholipids with PEG and the nonionic surfactants derived from PEG. Surfactants could be combined with formamide and / or acetamide.

't.l't. Liposome encapsulation or phosphatide (phospholipid) admixture to the system mineral oil emulsifier mannitol oleate or lanolin) in incomplete or Complete (i.e. with killed mycobacteria) Freund's adjuvant could have the adjuvant effect increase during macrophage sensitization. Here, too, PEG, AET, surfactants, specifically cationic and nonionic in the smallest doses bring further advantages. Quaternary (ammonium) bases are also of particular interest, since they are already used as Adjuvants can work.

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^ .15 Liposome encapsulation or adsorption on colloidal suspension of aluminum hydroxide or Aluminum phosphate or bentonite / montmorillonite, in combination with PEG, AET, tensides, mineral oil / emulsifier etc. are also from Vor · t ei 1.

5 · Further development according to the invention of multilnmellürcn Liposome vesicles

5.1- In the further development of liposomes in order to increase the interaction between liposome and cell membrane and Combinations of the abovementioned combinations also make the internalization of the liposome content easier Phosphoglycerides with other compounds such as phosphate tidylethanolamine (cephalin), Phosphatidyliriositol, Diphosphatidykgkycerol (Cardiolipin), Sphingosin (4t-Sphingosin (^ - Spingenin), Dihydrosphingosine (sphinganine), phytosphingosine (Ί-hydroxysphinganine), 't, 8-sphingadiene, sphingomyelin, Cerebrosid (Gluccocerebrosid, Galactocerebrosid and its sulfate ester, the SuIfatid, Di-, Tri and Tetrahexoside, acid glycosphingol ipid (ganglioside) i.a. In addition to the mentioned lysolecithin (lysophosphatidylcholine), others can Lysophosphoglycerides (e.g. lysophosphatidylethanolamine or lysophosphatidylserih) as well as synthetic Products containing LPA, i.e. the lysophospholipid analogs, such as l-octadecyl-glycero-3-phosphocholine, 1-octadecyl-2-methyl-glycero-3-phosphocholine, l-stearyl-2-methylglycero-3-phosphocholine, Hexadecylphosphorylcholine and l-dodecylpropanediol-3-phosphocholine and the corresponding derivatives (analogs) of lysophosphatidylserine, lysophosphatidylethanolamine and of lysophosphatidylinositol can be used. 5-2. If necessary, these compounds can also be used in Combination with bile acids ("Chylomicron" effect ") as well as with immobilized loctins or protectins, (e.g. ConA / Sepharose) find a prewonding.

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6. Tumor-selective chemotherapy by encapsulation

of cytotoxic substances in liposonies and Erythrocytes on the surface with tumor specifics Immunolobulins (in covalent no Binding) are loaded.

In addition to stimulating the body's own Immune defense through certain substances encapsulated in liposomes also arises the possibility of using the liposomes to transport cytotoxic substances (to the tumor target cell), alone or in combination with immunostimulants, use to selectively kill the transformed cells while largely sparing them from healthy tissue cells, with the liposomes on the surface with tumor-specific Immunoglobulins must be loaded. • By binding strong toxins such as ricin, abrin, Modeccin, gelonin, diphtheria toxin etc to the F (ab) ,, - fragments e of immunoglobulins, it is possible To synthesize chimeras, so-called "immunotoxins", which "seek out" the target cells, e.g., tumor cells, and kill them selectively. The following of these immunotoxins has already been successfully tested in animal experiments or in tissue culture:

Monoclonal IgM fragment against the Thy 1,2 antigen, combined with ricin A; monoclonal antibody fragments against a colorectal tumor antigen, combined with ricin A; anti-idiotypic antibodies against immunoglobulins of a B-ZeIL lymphoma, combined with ricin A; IgG (F) ab) _O antiserum against L 121o Leukemieze11 en, combined with diphtheria A fragment and F (ab) _O antilymphocyte globulin, combined with diphtoria toxin A. With abrin, conjugated with anti-Thy1.1-F (ab) _o , Lymphoma in the AKR mice was successfully treated.

Also Vinbln.stin, Daunomycin, ADR, Act i tioinyc in 0, Methothrexate, 1-ß-D-Arab ino furanosy1cytos in i.a. were tested as liposome encapsulates and brought advantages over the classic Administration form.

6.1. Encapsulation of cytotoxics / cytostatics. According to the invention, the toxins listed under "Ximmunotoxins" and the cytostatics of the general classes of alkylants not previously described in connection with liposomes (BusuIfnn = Myleran, Chlorambucil-Leukeran, Cyclophosphamide ^ Endoxan = Cytoxan, DBM = Myelobromal, HN2 = Mustargen, Phenardylalanin-Mustard, ThIoTEPA = TSPA ₁ TEM), antimetabolites (azacytidine, cytosine arabinoside = Ara-C = cytosar, 5- ^ Li = 5-fluorouracil, 6-mercaptopurine = 6-MP = Purinetho1, TTG «thioguanine), mi tose inhibitors ( Vincristine sulfate ) antibiotics (Mitomycin C, Dleomycin, VP-l6) and other cytostatics (BCNU, CCNU, cis-Platinum (II), Hydroxyurea, DTIC, Mitotan-o, p-DI) D and statmokinetics (Colcemide, Anguidin) as liposome and / or erythrocyte encapsulate enable tumor-selective chemotherapy with minimal side effects.

6.2. Covalent attachment of liposomes to HPD

The HPD (= IIaematoporphyrin derivative) is according to the latest american. Literature selectively on tumor tissue bound. One can therefore use the LiDosomes (instead of the Ig) associate or bind covalently to HPD and act as a means of transport for cytostatic agents and toxins use.

6-3 Covalent binding of liposomes to hormones (for hormone-dependent tumors)

In the case of hormone-dependent tumors such as prostate and breast tumors, there is a covalent bond (Conjugation) of liposome vesicles to synthetic ones or semi-synthetic hormones are possible, since the hormone receptors made organ sciectivity possible.

-sat-

6.4. Covalent attachment of liposomes to tumor markers

A conjugation of liposome vosicles to CEA ₁ AFP and Tennessee antigen is also of interest if these tumor antigens are available through enrichment and purification, for example with the aid of HPLC, preparative electrofocusing, affinity chromatography or chromatofocusing.
6.5 · Substances influencing tumor cell proliferation

In the case of direct or indirect application via liposome transport vesicles, substances such as auxins, indole-substituted carboxylic acids (indolyl-3-butyric acid), naphthylic or naphthylic acid are of interest. phenyl substituted acetic acids (1-naphthyl acetic acid), derivatives of phenoxycarboxylic acids ^, 4-dichlorophenoxyacetic acid; 2, k , 5-trichlorophenoxyacetic acid; 2-methyl-'t-chlorophenoxy-acetic acid) and derivatives of naphthoxy-carboxylic acids (2-naphthoxy-acetic acid) because of their influence on tumor cell proliferation. 6.6. Cytochalasin

Liposomes covalently bound to anti-tumor Ig can also be carriers of cytochalasin, which causes disruption of the nuclear-cytoplasmic interaction in the tumor cell.
6-7- Specific inhibitors of collagenase III and IV

According to the very latest findings by American researchers, Collagenase HI and IV (as exoenzymes) the trait, the selective advantage, of the metastatic tumor cell lines of the non-metastatic subpopulations

differs. An encapsulation of specific collagenase inhibitors (e.g. collagen antimetabolites / structure analogs) in liposome vesicles loaded with anti-tumor Ig is therefore of interest. 6.8. Butyrate

Butyrate induced as "reverse transformation agent "growth inhibition and / or morphological and biochemical redifferentiation of transformed Cells.

In the following the invention is based on a Example explained in more detail.

example

Phosphatidylcholine (from hen's egg) and phosphatidylserine (from bovine brain) can be found in chromatographic purity can be obtained commercially.

For the production of raultilamellar vesicles you can use both phospholipids in a 1: 1 or 7: 3 molar ratio in chloroform dissolve. A Ί, 95 = ^. 95 = ο, 1-raolar is advantageous Mixture of egg phosphatidylcholine, bovine phosphatidylserine and lysolecithin, dissolved in chloroform because

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had shown in the experiment that the resulting liposomes had negative Ladui g or a zeta potential have, which has an approximately 12-fold increased Phagotytoae or membrane fusion as, for example, the electrically neutral liposomes from egg phosphatidylcholine has the consequence.

After pre-evaporation of the choroform in a rotary evaporator, water-soluble immunostimulators are added to the cell culture medium (e.g. RPMI 160 or CMEM or TC 199 or HISS) with 5-10% FCS (FBS-Fetal Calf Serum), and the mixture is emulsified with a high-speed device for 20 to 60 minutes and the resulting liposomes containing the test substance are filtered through Millipore filters that can be screwed onto injection syringes. The liposomes are adjusted to a total lipid content of approx. 1 μmol / ml cell culture medium and should not stand for longer than 2k hours. The average liposome content should be about 2 ~ 3 ul ± lo% per mole of phospholipid. ' The liposome-encapsulated material should be given to the macrophages to be stimulated in a dosage such that 100 μmoles of phospholipid are produced per ₁ 10% of macrophages. This Liposomdosierung corresponds to an absolute amount of from about o, 25jj1 enkapsulierten of material per ₁ I Io macrophages.

While the mechanical mixing of the liposomes after evaporation of the organic solvent and "emulsification" in the aqueous medium produces vesicles with an average diameter of ^ - ^ ογη \ , the mean diameter can be reduced to o, by ultrasonic treatment of the "emulsion" o5pn can be reduced. The mechanically generated liposomes are made up of concentric phospholipid bilayers multilamellar, with the

interlaraellar space is 5 ^ -75 ^. The average layer thickness of the Phospho1ipLd-Bi1ayer is 50 X. The water-soluble material such as immunostimulators or cytostatics and lectins are built into the Liposom-Bi layer the ipophilic compounds. In the case of non-molecularly disperse active substances, this regularly concentric build-up is disturbed, and there is much more of a kind of loading of the particulate effector materials with a multilamellar liposome outer layer. The lack of antigenicity of the liposome outer layer prevents immune responses of the delayed type while at the same time facilitating membrane approach and fusion. By replacing dicetyl phosphate in a 1: 2: 1 mixture of lecithin, cholesterol and dicetyl phosphate with stearylamine, the negative surface charge of the liposome can be converted to a positive one. This is important because it is known, for example, of proteins which are positively charged at pH 7-07.1, that they have a significantly higher immunogenicity when they are encapsulated in positively charged liposomes. The lecithin: cholesterol ratio is also important. The surface loading of the liposome vesicles with denatured immunoglobulins and is often favorable

on (active) C3 complement. It is favorable as elsewhere mentioned, the simultaneous application of adjuvants such as saponin, M. tuberculosis, B. pertussis and others when injecting lipoaom there are no granulomas at the injection site. Liposomes showed when applied at People no negative effects, a rapid mobilization from the injection site and a considerable retention time in the lymph nodes. Liposomes are biodegradable.

Claims (5)

ν Drugs for the treatment of tumors. Claims. 1. Drugs to be administered intravascularly for Tumor treatment, characterized by a content in liposomes and / or erythrocytes encapsulated immune stimulants or cytotoxics. 2. Medicament according to claim 1, characterized by a content of liposomes and / or Erythrocytes encapsulated membrane fragments which may have been subjected to a membrane change or membrane extracts from primary tumor or metastatic cells. 3 · Medicament according to claim 1, characterized by a content of encapsulated interferon, interferon inducers and / or other known immune stimulants km medicament according to claims 1-3, characterized by the addition of substances that enhance the antigen-antibody reaction, 5. Medicament according to claim 1, characterized by a content of peroxide formers, immunotoxins or specific cytostatic agents in liposomes and / or Erythrocytes are encapsulated, which are monoclonal or pre-absorbed on the liposome surface Carrying heteroantibodies against tumor antigens (TSTA, TATA). ORIGINAL INSPECTED