EP0902685A1

EP0902685A1 - Ultramicro-emulsions of spontaneously dispersible concentrates containing antitumorally, antivirally and antiparasitically active esters of pentacyclic triterpenes

Info

Publication number: EP0902685A1
Application number: EP97900535A
Authority: EP
Inventors: Carl Eugster; Conrad Hans Eugster
Original assignee: Marigen SA
Current assignee: Marigen SA
Priority date: 1997-01-24
Filing date: 1997-01-24
Publication date: 1999-03-24
Also published as: WO1998032443A1

Abstract

The invention relates to spontaneously dispersible concentrates containing selected esters of pentacyclic triterpene compounds with antitumoral, antiviral and antiparasitic action, aqueous ultramicro-emulsions of such concentrates, methods for producing and processing said esters, and their use as agents for producing a pharmaceutical systemic product active against tumours, eczema, psoriasis, and viral and parasitic infections, for lasting tumour prophylaxis and enhanced absorption of exogenous activators, modulators and regulators.

Description

ULTRAMICROEMULSIONS FROM SPONTANEOUSLY DISPERSIBLE CONCENTRATES CONTAINING ANTITUMORAL, ANTIVIRAL AND ANTIPARASITAR EFFECTIVE ESTERS OF PENTACYCLIC TRITERPE-

NEN

INTRODUCTION

The present invention relates to ultramicroemulsions from spontaneously dispersible concentrates with esters of pentacyclic triterpene compounds, new triterpene esters, processes for their preparation and processing into suitable pharmaceutical dosage forms, and the use of the ester-containing, spontaneously dispersible concentrates and microemulsions as medicaments with effectiveness against tumors, eczema, psoriasis, viral and parasitic infections, as well as for the prevention of tumor formation by increasing the absorption of exogenous activators, modulators and regulators.

The esters of the selected plant-based raw materials and their transformation products surprisingly have a very good antitumor, antiviral and / or virucidal and antiparasitic activity, and they are also suitable for the treatment of eczema and psoriasis. In preventive and therapy-supporting respects, they are important because they increase the uptake of exogenous activators, modulators and regulators and thereby increase the metabolic as well as the immune performance.

The therapeutic and prophylactic properties of such esters are more pronounced when they are incorporated into spontaneously dispersible MARIGENOL® concentrates and then diluted with distilled water or 5% glucose solution or physiological saline (Ringer's solution), resulting in thermodynamically stable ultramicroemulsions, which contain very small, globular micelles with a hydrodynamic radius of 2.2 to 3.0 nm. DESCRIPTION OF THE INVENTION

The esters according to the invention of selected, vegetable raw materials with a pentacyclic triterpene structure have the general formulas (I) to (XI) =

BETULIN [Lup-20 (29) -en-3,28-diol], iup-20 (30) -en-3ß, 28-diol; Betulinol. Merck

Index 11.1212, Sigma B 9757, Aldrich 12.376-5.

L. Ruzicka et al., Helv.Chim.Acta ü, 506 (1936)

ALLOBETULIN

Betulinic acid; Hatchet: 10 (3), 1059; Aldrich 85.505-7

BETULINE ACID METHYLESTER

ENOXOLON (3-hydroxy-11-oxoolean-12-en-29-oic acid); 18β-glycyrrhetic acid; Uralenic acid; Merck Index 11.3543 L. Ruzicka et al., Helv.Chim.Acta 26., 2143, 2278 (1943)

ENOXOLON METHYLESTER OLEA ryophylline.

Merck Index 11.6787; Aldrich 30, 170-1

L. Ruzicka et al., Helv.Chim.Acta ü, 114 (1936)

L. Ruzicka et al., Helv.Chim.Acta 29_, 210 (1946)

OLEANOLIC ACID METHYLESTER

LUPEOL (fagasterol); Merck index 11.5478

URSOLIC ACID (3ß-Hydroxyurs-12-en-28 oic acid)

L. Ruzicka et al., Helv.Chim.Acta, 28, 199 (1945), Beil. 10 (2), 202; Merck index 11.9802

URSOLIC ACID METHYLESTER where in the formulas (I) to (XI) R1 a C ₃ . ₃ ι-alkyl, a C _3-3 ι-alkenyl, a C-ι - ₂₃ -Alkapolyen or a retinoyl group.

The compounds of the formulas (I) to (XI) can generally be prepared by the following processes which are known per se: a) Reaction of a compound of the formula (XII):

R — COOH (XII) in which R2 is a C _3-3 ι-alkyl, a C ₃ . ₃ -alkenyl, a _-23 -Alkapolyen- or a retinoyl group, with 1,1 '-carbonyldiimidazole or with N, N'-dicyclohexylcarbodiimide in an indifferent solvent, such as toluene or tetrahydrofuran, at 0 to 70 ° C, with the supply of protective gas and the addition of a catalytic amount of an alcoholate, and subsequent alcoholysis of the imidazoles formed with a compound of the formulas (I) to (XI).

b) formation of the chloride of a compound of the formula (XII): R - COOH (XII) in which R ^{2 is} a C ₃ . ₃ ι-alkyl, a C ₃ . ₃₁ alkenyl or a C ₁₇ . ₂ 3-Alka-polyengruppe stands, with a chlorinating agent such as thionyl chloride or oxalyl chloride, and then reacting with a compound of the formulas (I) to (X) at a temperature from 0 ° C. to 60 ° C., with the supply of protective gas, in an inert solvent, such as toluene or tetrahydrofuran, and in the presence of a catalyst, such as dimethylformamide and / or p-dimethylaminopyridine.

c) Formation of the allobetulin by partial synthesis from betulin:

and then acylation at position 0-C (3) with an activated, saturated C5. ₃₁ - or an unsaturated C ₈ - ₃ i-carboxylic acid:

and subsequent workup as described in detail below.

) Formation of the betulinic acid methyl ester according to formula (IV):

and then reaction at 0-C (3) by reaction with the chloride of a higher carboxylic acid, including subsequent work-up as described in detail below. In a corresponding manner, the methyl ester of 18β-glycyrrhetinic acid can:

or the oleanolic acid:

(VIII) or ursolic acid: be acylated at 0-C (3).

The esters of the selected pentacyclic triterpene compounds of the formulas (I) to (XI) surprisingly have an excellent antitumor, antiviral, virucidal and antiparasitic activity, and they are also suitable for combating eczema, psoriasis, as well as metabolic and immune Disruptions, especially when they have been incorporated into spontaneously dispersible MARIGENOL® concentrates according to the present invention, which result in thermodynamically stable oil-in-water ultramicroemulsions diluted with distilled water, with 5% glucose solution or with Ringer's solution.

Such ultramicroemulsions have spherical micelles with a hydrodynamic radius of 2.2 to 3.0 nm. [Measurements with QLS = quasi-elastic, dynamic light scattering were carried out at E.T.H., Zurich, Institute for Polymers (Prof. Dr. Pier-Luigi LUISI and Prof. Dr. Peter SCHURTENBERGER). For the methodology used, see:

P. Schurtenberger, R. Scartazzini, P.-L. Luisi, Rheol. Acta 28, 372 (1989). P. Schurtenberger, R. Scartazzini, L.J. Magid, M.E. Reader, P.-L. Luisi, J. Phys. Chem. 94, 3695 (1990).

P.-L. Luisi, R. Scartazzini, G. Haering, P. Schurtenberger, Colloid Polym. Sci. 268, 356 (1990): Organogels from water-in-oil microemulsions. T. Gisler et al .: "Mode-selective dynamic light scattering: theory versus experimental realization". Applied Optics / Vol.34, No. June 18, 1995.

The present invention accordingly relates to spontaneously dispersible concentrates in advance with the antitumoral, antiviral, virucidal or antiparasitic esters of the compounds (I) to (XI), which can also induce the apoptosis of tumor or host cells. The designated esters are almost water-insoluble and highly agglomerated compounds. So that such connections pass through the membrane barrier of the tumor or host cells, or the protein sheath of veins or parasites diffuse and inside the

Cell, or the capsid can be effective, they must first be suitably solubilized in the aqueous medium. Through the formation of thermodynamically stable oil-in-water ultramicroemulsions, with the help of specially selected and combined as a system surfactants or solubilizers on the one hand and suitable surfactants on the other hand, it is possible to achieve an optimal degree of solubilization of the therapeutically and prophylactically active esters.

All experimental observations on ultramicroemulsions designed in this way can be uniformly interpreted by the assumption that the selected surfactants and cosurfactants, taken as a balanced formulation, aggregates organized in the aqueous phase, so-called Form micelles. These have more or less spherical shape, with a hydrodynamic radius of 2.2 to 3.0 nm. The surfactants and hydrotropes (cosurfactants) allow between the outer, aqueous phase and the inner, oily phase of the microemulsion [containing the esters of Formulas (I) to (XI), dissolved in the biosurfactant solubilizer], a boundary layer is formed, as a result of which the mixing of these two phases does not occur, that is to say no Marangoni effects occur. The molecules of the designated ester compounds are dissolved in the oily inner phase and are present in the micellar core in monomeric or in oligomeric agglomerated form, which makes a very decisive difference to mere macro-emulsions (e.g. liposomes). The micelles of the ester-containing inner phase of the ultramicroemulsions according to the invention are accordingly on their surface, i.e. protected at its boundary layer with a surfactant coat, which it repairs, easily diffusing through the cell membrane or the protein envelope into the interior of the tumor or host cell. The diffusion through the plasma membrane of abnormal cells occurs exclusively due to thermal molecular movements; the fractal conditions on the cell membrane accommodate her. It can be shown that the speed of the diffusion process or the strength of the active substance transport is determined by the membrane of the target cell:

1 . the difference in concentration between the intracellular and the intercellular area

2. of the particle radius of the diffusing Wi rmstoffkoleks or

Active ingredient systems

3. the viscosity of the diffusing aqueous solution

(Emulsion)

4. on the temperature. As can be seen from the table below, a globular "micelle" with a hydrodynamic radius of one centimeter has a volume of 4.189 cm ³ and a phase surface area of 1 2.564 cm2. In contrast, 1Q18 micelles with a hydrodynamic radius of only 10-6 cm (10 nm) each, which together make up the same volume of 4.1 87 cm ³ , already have a total phase surface of 1 '256.4 m2 .

MICELLES: RELATIONSHIP VOLUME TO TOTAL SURFACE

Spherical volume = - π r3 3

Spherical surface = 4 π r2

Conclusion: Due to the enormously large phase surface, which the micelles form with a hydrodynamic radius of 1 to 5 nm in ultramicroemulsions, in addition to their increased diffusion capacity, the rheological distribution (spreading) and thus the bioavailability and bioreactivity of the active substances, which are present in the inner phase of the micelles in monomeric or oligomeric agglomerated form, also greatly improved because there are no Marangoni effects. This can allow a significant reduction in the critical dosage and thus completely avoid or at least reduce unwanted side effects.

The "packing density" of a spontaneously dispersible, stable MARIGENOL® concentrate increases exponentially with the smaller particle size of the micelles. The right training is crucial the inner phase, its balanced ratio to the total concentrate and the selection of the appropriate surfactants.

COMPOSITION OF MARIGENOL® CONCENTRATES The concentrates which are spontaneously dispersible according to the invention contain: 0.1 to 10% by weight of individual esters of the formulas (I) to (XI), or combinations of such esters, and

0 to 5% by weight of a synergistic, pharmaceutical or cosmetic active substance, as enumerated in detail in patent specification CH 683 ^* 426 (US. 08 / 179,729, issued September 3, 1996),

1 to 25% by weight of a pharmaceutically acceptable solvent or solvent mixture serving as a hydrotrope or co-emulsifier,

5 to 90% by weight of a pharmaceutically compatible surfactant or surfactant mixture, up to 10% by weight of a vitamin or provitamin, up to 10% by weight of a stabilizer, scavenger, biological vector or penetration enhancer and carriers and the like / or diluent.

Because of their pronounced protective action on the host cell and their direct activity against viruses and parasites, the synergistically active ester compounds described below, mentioned particularly in patent specification CH 2239-95, can be used for therapeutic support in the spontaneously dispersible concentrates according to the invention Incorporate it: Trän s-2-B ute nsäu re-3ß-hydroxy-5-cholestanylester

(Cholestanyl crotonate) isovaleric acid 3β-hydroxy-5α-cholestanyl ester

(Cholestanyl iso-valerate)

1 0-undecenoic acid 3ß-hydroxy-5-cholestanyl ester

(Cholestanyl-1 0-undecenoate)

Lauryl acid 3β-hydroxy-5α-cholestanyl ester

(Cholestanyl Laurat)

Palmitic acid-3β-hydroxy-5α-cholestanyl -Este r

(Cholestanyl-Pa imitation)

3ß-stigmast-5-en-3-laurate (ß-sitosteryl laurate)

3ß-stigmast-5-en-3-palmitate (ß-sitosteryl-pa im itat)

3ß-stigmast-5-en-3-stearate (ß-sitosteryl stearate)

3ß-stigmast-5-en-3-arachidate (ß-sitosteryl arachidate) 3ß-stigmast-5-en-3-behenate (ß-sitosteryl-behenate)

3ß-stigmast-5-en-3-1 0-u-undecenoate (ß-S itosteryl-1 0-undecenoate)

3ß-stigmast-5-en-3-oleate (ß-sitosteryl-oleate)

3ß-Stigmast-5-en-3-all trans-retinate (ß-S itosteryl-all-trans-retinate).

(3ß, 22E) -Ergosta-5,7,22-trien-3-ol-1 0-undecenoate

[Ergosteryl-10-u undecenoate, provitamin D2-undecenoate]

(3ß, 22E) -Ergosta-5,7,22-trien-3-ol-laurate

[Ergosteryl-Iau rat, Provitami n D2-Iaurat]

(3ß, 22E) -Ergosta-5,7,22-trien-3-ol-palmitate

[Ergosteryl palmitate, provitamin D2 palmitate]

(3ß, 22E) -Ergosta-5,7,22-trien-3-ol-arachidate

[Ergosteryl laurate, provitamin D2 arachidate]

(3ß, 22E) -Ergosta-5,7,22-trien-3-ol-al l-trans-retinate

[Ergosteryl retinate, provitami n D2 retinate]

10-undecenoic acid-9,10-secocholesta-5,7,10 (19) -trien-3-ol

[Vitamin D3 undecenoate, cholecalciferyl undecenoate,

Calciol undecenoate]

Lauric acid-9, 10-secocholesta-5,7,10 (1 9) -trien-3-ol

[Vitamin D3-Iaurate, Cholecalciferyl-Iaurate, Calciol-Iaurate]

Palm itic acid-9,10-secocholesta-5,7,10 (1 9) -trien-3-ol

[Vitamin D3 palmitate, cholecalciferyl palmitate, calciol palmitate]

Oleic acid-9,1-secocholesta-5,7,10 (19) -trien-3-ol

[Vitamin Dß-oleate, cholecalciferyl-oleate, calciol-oleate]

Retinoic acid-9,1-secocholesta-5,7,10 (19) -trien-3-ol

[Vitamin Dß-all trans-retinate, cholecalciferyl-all trans-retinate,

Calciol-al l trans-retinate]

Z-Gly-Phe-Vitamin D3

[CBO-Glycyl-L-phenylalanine dipeptide calciol ester].

The surfactants or surfactant mixtures to be used according to the invention can be anion-active, cation-active, amphoteric or non-ionic. They are preferably amphoteric or non-ionic and have an HLB ratio (ie a “hydrophilic-lipophilic balance”) between 2 and 1 8; for mixtures it is preferably between 2 to 6 on the one hand and 1 0 to 15 on the other. Highly preferred for the production of spontaneously dispersible concentrates according to the invention are, on the one hand, phosphoric acid ester surfactants, such as, for example: the practically anhydrous tristyryl phenol polyoxyethylene-18-phosphoric acid ester TEA salt surfactant:

(Soprophor® FL, RHONE-POULENC);

Further.:

Diphasol® 3873 (CIBA-GEIGY), or the identical Sermul® EA 188 (SERVO), a mixed emulsifier, each consisting of 50% of the two compounds with the

Formulas:

O

^C 9 ^H 1 (-CH ₂ -CH ₂ -θ7π P- ° ^CH 3 OH

O

^C 9 ^H 1! CH ₃

Diphasol® 3873 (CIBA-GEIGY), an alkylphenol polyglycol ether phosphate surfactant

Tensi

(Surfactant 508, CIBA-GEIGY); Tinovetin® JU (CIBA-GEIGY), a hydroxybiphenyl-10-ethoxy-phosphoric acid ester

Butyl mono-4-ethoxy phosphoric acid ester (Zerostat® AT, CIBA-GEIGY), or CH ₃ - (CH ₂ ) —CH-CH ₂ -O (-CH ₂ - CH ₂ -O) ₅ -OCH,

C ₂ H ₅

OCH,

(Zerostat ® AN, CI BA-G EIGY) and on the other hand betai n associations, i.e. amphoteric, salt and water-free imidazole derivatives, e.g. :

where Me [ ⁺ ] stands for hydrogen, alkali and alkaline earth atoms and R _x for C _{1 -32} -alkyl or C _2-32 -alkenyl groups.

So-called "multi-functional glucose derivatives" such as e.g.

Glucate® SS (methyl glucose sesq uistearate) and Glucate® SSE-20 (PEG-20

Methyl Glucose Sesquistearate) from Amerchol, Edison, N.J. , UNITED STATES.

In some cases, good results can also be achieved by using non-ionogenic compounds from the "TWEEN®" series (Atlas Chem. Ind.,

Inc.; or ICI Specialty Chemicals), so-called polyoxyethylene sorbitan monoesters or "polysorbates" 20 to 85 compounds in the CTFA Classification

[Fluka 93'773 to 93'783].

Pharmaceutically acceptable solvents which can be used as hydrotropes or as co-emulsifiers can be used, e.g. :

Ester of an aliphatic alcohol (C _3-18 ) with an aliphatic carboxylic acid (C ₁₀ - ₂₂ ). such as isopropyl laurate, hexyl laurate, decyl laurate, isopropyl myristate, isopropyl palmitate and lauryl myristate; Hydrocarbons with a straight carbon chain (C _{12 -32} ) which is substituted with 6 to 16 methyl groups and can have 1 to 6 double bonds, of which terpenes such as polymethylbutanes and polymethylbutenes may serve as examples. Mono-esters from ethylene glycol or propylene glycol with an aliphatic carboxylic acid (C _6-22 ). such as propylene glycol monolaurate and propylene glycol monomyristate.

Esters of an al iphatischen alcohol (. C ₁₂ ₂₂₎ with lchsäure Mi, such as myristyl or preferably Lau ryl-lactate; Mono-, di- or triesters of Gly cerins (neutral oleum) with an ali phatic carboxylic acid (C _6. _22), such as G lyceryl- capryolat, or Miglyol® 812 neutral oil. Esters of a poly (2-7) ethylene glycol glycerol ether with at least one free hydroxyl group with an aliphatic carboxylic acid (C _6. ₂₂ ), such as, for example, aliphatic alcohols (Cι _2-22 ), thus, inter alia, dodecanol, tetradecanol, oleyl alcohol, 2-hexyldecanol and 2-octyldecanol.

Ester with at least one free hydroxyl group, from poly (2-1 0) glycol with an aliphatic carboxylic acid (Cβ- ₂₂ ). Monoether from a polyethylene glycol with an aliphatic alcohol (C ₁₂ - ₁₈ ). such as polyoxyethylene (Cι o) octyl ether.

Heterocyclic compounds, e.g. 1-methyl-2-pyrrolidone. Biosurfactant esters of the general formula (XII):

R ^ COO-R ⁴ (XII), where R ^{3 is a} C. ₃ i -alkyl-, a C ₃ . ₃ ι alkenyl or a C ₃ . ₃₁ -Alkapolyengruppe and R4 Citronellyl-, Farnesyl-, Geranyl-, Isophytyl- or Phytyl- mean.

Before being added to the spontaneously dispersible concentrates, all technical surfactants were filtered or chromatographed over neutral aluminum oxide with an inert solvent such as e.g. Tetrahydrofuran, ethyl alcohol or dichloromethane cleaned.

Vitamins and provitamins (such as vitamins A acid, retinol, tocopherols), as well as selected penetration enhancers ("flux enhancers") and radical scavengers are suitable as additives in the spontaneously dispersible concentrates according to the invention.

PRODUCTION a) Production of 3-O-AII-trans-retinoyl oleanolic acid

To 600 mg of AII-trans-retinoic acid in 50 ml of dioxane or chloroform are added 650 mg of N, N'-carbonyldiim idazole at 15 ° C. with exclusion of air. The reaction solution is left to stand at 15 ° C. for 24 hours. The solution with the resulting AII-trans-retinoic acid imidazolide is then added dropwise at 20 ° C. to a solution of 900 mg of oleanolic acid, 60 mg of dimethylformamide and 50 mg of p-dimethylamino nopyridine in 50 ml of dioxane or chloroform. After heating for 3 hours at 40 ° C, the solvent is removed by vacuum distillation. The residue is taken up in ethyl acetate or dichloromethane, neutralized, dried and then on an aluminum oxide column (neutral, Brockman activity I) with cyclohexane / ethyl acetate or methylcyclohexane / dichloromethane (90:10 or 98: 2). chromatographed as eluent. The product 3-all-trans-retinoyl-oleanolic acid is obtained. M.p. 147, 5-149, 5 ° C; Rf. value on DC hexane / ethyl acetate (90:10): 0.75.

The following connections can also be made in a comparable manner:

3-O-all-trans-retinoyl glycyrrhetinic acid,

M.p. 159-160.5 ° C; Rf. value on DC hexane / ethyl acetate (90: 1 0): 0.70.

3-O-linoleoyl glycyrrhetinic acid

3-O-linolenoyl glycyrrhetinic acid

3-O-Li noleoyl oleanolic acid

3-O-linolenoyl oleanolic acid

b) Preparation of 3-O- (10-undecenoyl) oleanolic acid

600 mg of 10-undecenoyl chloride in 30 ml of toluene are added dropwise to 900 mg of oleanolic acid and 75 mg of dimethylformamide in 50 ml of toluene at 20 ° C. The reaction solution is heated to 60 ° C. under reflux for three hours. The solvent is then removed by vacuum distillation. The residue is chromatographed on a silica gel column using cyclohexane / ethyl acetate (95: 5) as the eluent. The product is obtained:

3-O- (10-undecenoyl) oleanolic acid, with a refractive index (B1) of 1.44455. The following can also be produced in a comparable manner:

3-isovaleroyl oleanolic acid, Bl 1.43150

3-O-lauroyl ester oleanolic acid, Bl 1.43150

3-oleyl oleanolic acid

3-lauroyl glycyrrhetinic acid

3-oleyl G lycyrrhetinic acid

3,28-diundecenoyl betulin

3,28-dilauroyl betulin

3,28-dipalmitoyl betulin

c) Preparation of 3-0, 28-O-dilauroylbetulin

In a dry three-necked flask equipped with a magnetic stirrer, inlet for protective gas, cooler and septum, the solution of 347 mg betulin in 4 ml abs. Some crystallines 4-dimethylaminopyridine were added to pyridine and then cooled to 0.degree. Then 0.3 ml of lauroyl chloride was added dropwise through the septum using a syringe. After removing the cooling bath, the solution was brought to room temperature with constant stirring. After 12 h it was diluted with benzene, then the suspension sion filtered and the filtrate washed with H ₂ 0, 2N H ₂ S0 ₄ and brine and the benzene solution dried over Na ₂ S0 ₄ .

TLC on silica gel with ethyl acetate / toluene / acetone 11: 9: 1 and spraying with the cerium ammonium molybdate reagent gave an Rf value of 0.03 for betulin and 0.93 for the dilaurate.

The further purification was carried out by column chromatography on basic Alox, activity II. Yield 83% of pale yellow, very viscous oil after drying at 50 ° C. and 0.01 Torr. IR (CH ₂ CI ₂ ): 1722 cm-ss

CIMS (NH ₃ ): m / z 624.2 (6.2%; M + +1 of allobetulin laurate); 608.2 (46.4, M + +1 - lauric acid); 607.2 (100%, M + - lauric acid); 407.2 (31.65% M ⁺ - 2 lauric acid); 409.2 (5.32%, M ⁺ of allobetulin laurate - lauric acid). {In the case of HV distillation, lauric acid is partially split off and rearranged in allobetulin}.

1H NMR (CDCI3, 300 MHz): 4.69 (d, J = 2.1, 1H of C = CH.2); 4.60 (q, J = 1.3, 1H of C = CH.2); 4.48 (d, d, J = 9.5 and 10.6 (HC (3)); 4.27 (d, broad, J = 10.3, 1H of H2-C (28); 3.84 (d, J = 11.0, 1H of CH2; 1.69 (s, CH3 (29); 1.26 (s, balance of the (CH2) protons.

13C NMR; 174.3 and 173.7 (each a lauroyl CO), 150.2 (C (22)); 109.8 (C (30)); 80.6 (C (3)); 62.5 (C (28)).

d) Preparation of 3-O-lauroyl betulin

The preparation is made from 30,280-dilauroyl betulin analogous to a rule by L. Ruzicka et al. [Helv.Chim.Acta 1938, 21_, 1708]: the solution of 495 mg of 3,28-di-O-lauroylbetulin in 3 ml of benzene was mixed with 6.25 ml of 0.1N KOH in ethanol and while stirring Maintained at 50 ° C for 12 hours. We poured the clear solution on a silica gel column (silica gel Merck, 0.04 - 0.063 mm / 2, 6 x 43 cm, wet filled with ethyl acetate / benzene / acetone 11: 9: 1). Detection of the desired substance with the cerium ammonium molybdate reagent. The monolaurate has an Rf value of 0.84. Yield 280 mg of colorless, very viscous oil which slowly solidifies to a semi-solid mass when dried at 50 ° C./0.01 Torr.

Rf (silica gel Merck, AcOEt / Toluon / Acetone 11: 9: 1): 0.84. For comparison: Betulin 0.63; 3,28-di-O-lauroyl betulin: 0.94. IR (CH2CI2): 3615 (OH free), 2930, 2858, 1720; (CHCI3): 3627, 3492, 2954, 2856, 1717.

CIMS / NH3): m / z 425.3 (100%, M + +1 - lauric acid);

1H-NMR: largely analogous to that of c), the signals of H2-C (28) are characteristic at 3.80 (d, d, J = 1.5 and 10.8), and at 3.34 (d , J = 10.8) e) Preparation of 3-O-Lauroylallobetuli n

Allobetulin was prepared according to the instructions of H. Schulze and K. Pieroh (Ber. Deutsch. Chem. Ges., 1922, 55_, 2332). 0.31 and 0.47 g each of pure Allobetu lin were in 2 ml abs. Dissolved pyridine and 10 ml of 1, 2-dichloroethane, then mixed with a few crystals of 4-dimethylaminopyridine and added dropwise at room temperature via syringe with 1, 2 equivalents of acid chloride. After 2 h at RT, the mixture was heated to 50 ° C. for 30 m, then cooled, sufficient Et2θ was added and the mixture was shaken out with water, 2N HCl and brine. After drying over Na2SO4, filtering and evaporation, the mixture was taken up in benzene and the solution was filtered through a short column of activated aluminum oxide and the filtrate was evaporated.

3-O-lauroylallobetulin (n = 10): colorless, viscous oil that gradually solidifies in a vacuum. Rf 0.85

IR (CH2CI2): no OH bands, 2925, 2855, 1720 (ester carbonyl) CIMS (NH3): 425.3 (100%, M + +1 - lauric acid); NMR: analogous to 3-O-palmitoyl betulin (n = 14)

3-O-palmitoylallobetul in (n = 14): colorless crystals of tetrahydrofuran / pentane / methanol, mp (vac.) 185-189 ° C; Yield 82%. Rf laurate: 0.92, palmitate 0.93 (allobetulin 0.72, betulin 0.65) IR (CHCI3): no OH bands, 2959, 2927, 2855, 1717; (CH2CI2): no OH bands, 2950, 2931, 2855, 1767, 1720.

1 H-NMR (CDCI3), 300 MHz, selection of structure-evidencing signals): 4.487 (d, d, H-C3)); 3,775 and 3,444 (each d, J = 8.2, H2- (28)); 3.530 (broad s, H-C (19)); 2.294 (t, J = 7.3, α-CH2).

CIMS (NH3): 425.4 m / z (100%, M + 1 - palmitic acid); 13 C NMR: 171, 4 palmitoyl-CO); 87.8 (C (3)); 71, 2 (C (28)); 80.4 (C (19).

f) Preparation of 3-O-palmitoyl-betulinic acid methyl ester

The methyl ester was prepared from pure betulinic acid by esterifying its solution in THF / MeOh with an excess of freshly prepared ethereal diazomethane solution (cf. V. Bruckner, J. Koväcs, J. Kozka, J. Chem. Soc. 1 948 . 948). The solution of 0.62 g of pure methyl ester in 2 ml of abs. A little 4-dimethylaminopyridine was added to pyridine and 10 ml of 1,2-dichloroethane and then acylated and worked up as above under e). 0.75 g of colorless, very viscous oil was obtained which did not crystallize at RT. Rf: 0.91 (betulinic acid methyl ester 0.74) IR (CHCI3): 2928, 2855.171 9, 1641, 1465. In a corresponding manner, the methyl ester of oleanolic acid, 18β-glycyrrhetic acid (enoxolone) or ursolic acid can also be prepared and acylated on O-C (3) with an activated, saturated or unsaturated carboxylic acid. See also A. Winterstein, W. Hämmerle, Z. Physiol.Chem., 1931, 199.71.

g) 3-O-palmitoyloleanolic acid methyl ester (n = 14)

Colorless crystals of diisopropyl ester / pentane, mp 62-63 ° C, Rf 0.88 (methyl oleanol acid 0.69; oleanolic acid 0.60). IR (CHCI3): 2928, 2855, 1717, 1464.

h) 3-O-palmitoylglycyrrhetinic acid methyl ester (n = 14)

Colorless crystals of diisopropyl ether / pentane; Mp 130 ° C.

UV (CH2CI2): λmax 247 nm (ε 14,500)

IR (CHCI3): no OH bands, 2928, 2855, 1722, 1653, 1465 cm -

CIMS (NH3): 724.6 m / z (50%, M + +1), 723.5 (100%, M +), 467.4 (14%, M + -

Palmitic acid).

1H-NMR (600 MHz, CDCI3), selection of bands): 5.622 (s, H-C (12); 4.48 (dd,

J = 11.3 and 5.0, H-C (3); 3.68 (s, OCH3); 2.80 (m, H-C (18); 1.254 (s, CH2).

13C NMR: 200.0 (C-11); 176.9 (C-30), 173.6 (palmitoyl-CO); 169.1 (C-13); 128.5

(C-12); 80.2 (C-3); 51.7 (OCH3).

COMPOSITION EXAMPLES of spontaneously dispersible CONCENTRATES according to the invention which contain the active compounds of the formulas (I) to (VI) and which, if diluted with water or 5% glucose solution or physiological saline (Ringer's solution), thermodynamically stable ULTRAMICROEMULS IONS with a hyd rododynamic radius of 2.2 to 3.0 nm.

g) 0.1 to 10% by weight of one or more esters of selected pentacyclic triterpene compounds of the formulas (I) to (XI),

0 to 5% by weight of a known pharmaceutical active substance,

1 to 25% by weight isopropyl myristate, isopropyl palmitate or neutral oil, e.g. Mig lyol® 81 2 (DYNAMIT NOBEL or HÜLS),

0 to 45% by weight of a phosphoric acid ester surfactant, e.g. Diphasol® 3873 (CI BA-GEIGY), tenside 508 (CIBA-G EIGY), Zerostat® AN or AT (CIBA-GIGY), Tinovetin® JU (CIBA-G EIGY), Soprophor® FL (RHÖNE-POULENC), 5 up to 90% by weight Invadin JFC 800% (CIBA-GEIGY) and / or Tween®-20 (ICI S specialty Chemicals), up to 10% by weight of a vitamin or provitamin. up to 10% by weight of a stabilizer, radical scavenger, biological vector or penetration enhancer and carriers and / or diluents.

h) 0.5 to 5% by weight of one or more active compounds of the formulas (I) to

(XI),

5 to 25% by weight of one or more bio-surfactant esters of the general

Formula (XIII):

R - COO - R ⁵ _{(X | | |)} where R4 is a C ₂ - ₃ i alkyl, a C ₃ . ₃ i -Alkenyl or a C _3-3 ι -Alkapolyen- group and R5 citronellyl, farnesyl, geranyl, isophytyl or phytyl mean.

30 to 45% by weight Invadin® JFC 800% and / or Tween®-20, 30 to 45% by weight Soprophor® FL or Diphasol® 3873.

NB: INVADIN® J FC 800% (CI BA-GEIGY) is an anhydrous tert. Octylphenyl polyoxyethylene ether surfactant with 9 to 10 oxyethylene groups. TWEEN®-20 is a polyoxyethylene (20) sorbitan monolaurate, or the polysorbate-20 in the CTFA classification. EXAMPLE for the pharmaceutical production of a system preparation with concentrates according to the invention in the form of "multiple units". i) granulation

Metolose® 90 SH-4000 (SHIN-ETSU CHEMICAL) 90.0 g

Avicel® PH-101 80.3 g

MARIGENOL® CONCENTRATE according to the invention 139.4 g

Aerosil® 200 80.3 g

Σ 390.0 g

Granulate / shape in a high-speed mixer or in a rotary bed with the addition of 110 g ethanol, break, seven 18 to 42 mesh, dry for 24 h at 40 ° C.

k) MSR and RETARD equipment in a rotary bed with AQOAT® AS-HG (SHIN-ETSU CHEMICAL) and talc

I) composition of finished granules / or. Micropellets

Core material 44%

MARIGENOL® CONCENTRATE according to the invention 25%

MSR coating 31%

Σ 100%

NB: MSR = gastric juice resistance. The pellets / granules according to a) can also be filled directly into capsules, which are made from AQOAT® (HPMC-AS-M or HPMC-AS-N), and sealed with acetone / ethanol 1: 1, and so the functions adequately control the MSR and the delayed release (retard).

BIOLOGICAL TESTS

The antitumor / antiviral / antiparasitic effect of spontaneously dispersible concentrates with active substances according to the preparation examples a) to e) and the workup examples f) and g) is confirmed on the basis of the following test results:

1. In vitro tests with suitable tumor cell lines

A biological assay system has been developed that works with microtiter plates and dilution series. In each case, 1 θ4 / ml tumor cells are inactivated in RPMI 1640 culture medium with 10% fetal calf serum (GIBCO); they are sown so densely that they can grow in non-confluent monolayers during the assay. The sample is added after 6 to 24 hours, with 100 μl per row, which is then in the 1st Add 100 μl of medium to the hole. Half of this is removed and placed in the following hole, again mixed with 100 μl medium, etc. A geometric series of dilutions n ¹ / .. is formed.

The samples are incubated in the plaque assay for 3 to 5 days at 37 ° C with 3 ¹ Λ% CO2. Then dye / fix with 0.1% crystal violet (Fluka, Buchs) in a solution of 70% methanol, 1% formaldehyde, 29% water. The evaluation is carried out on a microscope, magnification 300 times. The largest cytotoxic dilution is determined. The quantitative evaluation can also be carried out by means of scanning and absorption measurement on a spectrophotometer.

2.0 Test for cytotoxicity

2.1 Cytotoxicity of MARIGENOL® CONCENTRATES tested on Py6 cells (virus-infected 3T3 mouse fibroblasts)

CONTINUATION

') 72 h dilutions: First line calculated for concentrate

Second line calculated on active substance content

REPEAT in vitro test for Py6 cells

N.B. Dilutions: Upper number calculated on concentrate

Lower number calculated on active substance content 3.0 In vitro testing with human tumor cell lines

VITALITY TEST with human tumor cell lines 1% concentrate with 3-O-lauroyl-oleanolic acid proliferation test (tritium: 1 μCi / per well H +)

Dilutions LC 89 U 937 T O M cpm% cpm% cpm%

10-2 474 16 583 0.3 788 0.4

10-3 775 26 1 * 370 0.7 8 * 328 4.4

10-4 7 * 265 (?) 250 88 ^* 61 45.5 17'766 9.5

10-5 2 * 782 95 175 * 127 89.7 22 * 895 12.2

10-6 2,926 100 191 * 432 98.0 46 * 602 24.8

10-7 3 * 800 131 198 * 796 101.0 159,207 84.9

cpm controls 2,906 195 * 125 187 * 601 Zeil lines: LC 89: pulmonary adenocarcinoma

U 938: Leucemia mieloid acuta

TOM: Melanoma umano

ID:% vitality after 48h with 1% MARIGENOL® CONCENTRATES, added once to the medium as an aqueous ultra-microemulsion. When assessing, consider the relatively short exposure time. Tests conducted by Dottoressa Anna-Rita Guarini, Università degli Studi di Torino, Clinica medica, Torino, March-April 1995.

3.1 Other findings

For the pharmacological effects found for the unesterified body of Lupeol, Betulin, Betulinic acid and Ursolic acid cf. including:

Bhattacharyya J. et al., Phytochemistry, 1976, 15, 431.

Aszalos Adorjan: "Antitumor Compounds of Natural Origin", CRC Press, Boca

Raton, Florida, 1980, N ° 220, 221, 222, 224.

Recio Maria delCarmen et al., Planta Medica, 57 (1991)

Yasukawa K. et al., Oncology 48, 72-76

Macias FA et al., Phytochemistry, 1994, 36_, 1369 Recio Maria delCarmen et al. : "Investigations on the Steroidal anti-inflammatory Activity of Triterpenoids from Diospyros leucomelas", Planta Medica, 61 (1995) pp. 9-12

Pisha Emily et al. : "Discovery of betulinic acid as a selective inhibitor of h uman melanoma that functions by induction of apoptosis", Nature Medicine 1, (1995), 1046-51.

See also: O. Gessner: "The poison and medicinal plants of Central Europe", Verlag Winter, Heidelberg, 1953.

Betulin accounts for up to 35% of the dry weight in the white bark of Betula platyphyl la. [K. Hi rota et al., Chem. Abbstr. 1948, 42., 6090], which suggests that it assumes a protective function for the plant there. The extract from the dry distillation served as a remedy for various applications, and as a "bark tar" for the manufacture of the famous Russian leather.

Among the pentacyclic triterpene compounds of the Lupan series, the effects of the allobetulin monoesters, whose backbone structure with its five fused, trans-configured cyclohexane rings and with the polar hydroxyl group on one side and the diagonal tetrahydrofuran appear to be of particular therapeutic importance ri ng on the other hand has an extremely flat molecular structure. Its central part ("core") is reinforced in its lipophilicity by the vertically standing methyl groups, while at the same time the hydrophilic (polar) groups of the molecule come to lie on opposite sides of the "core" part. This has made it possible to bind to relatively distant polar groups of the receptor. The central part therefore serves as a kind of "spacer"; he is responsible for extensive lipophilic areas.

3.2 APOPTOSE TEST with mouse fibroblasts transformed with Py6 virus

% Cell vitality

Cytofluor 2300 system; Incubation 72h, Alama blue; Plate CoStar 96 treatment: 46 h; Measurement EX filter 530/25; EM filter 590/35, sensitivity 2

Concentrate dilutions: 1:10 * 000 (100 ppm) to 1: 160 * 000 (6.6 ppm) active substance content: 1 ppm to 67 ppb

Test carried out at STI, Basel, December 15-20, 1996 (File 201296-001 / 03).

4.0 Test for stimulation of the immune response

With human tumor cells (lung carcinoma LC 89)

cpm 27 * 360, exposure 48 h Tests performed by Dottoressa Anna-Rita Guarini, Università degli Studi di Torino, Clinica medica, Torino, March 21-24, 1995

5.0 Antiviral test.

5.1 Testing for protection and antiviral activity against sensitive MT4 cells infected with the AIDS VIRUS HIV ("Human Immunodeficiency Virus"). The tests were carried out at the Institute of Infectious Diseases at the University of Turin (Director: Prof. Dott. P. GIOANNINI), and by Prof. Dott. Alberto BIGLINO, Head of the Infectious Diseases Department at Ospedale di Asti, U.S.L. 19, with the collaboration of Dotta. Brunella FORNO and Dotta. Annamaria POLLONO performed. June / July 1994 and March / April 1995.

5.2 Protection effect on the host cells (MT4 lymphocytes)

MT4 cells (an eternalized T cell line, which is very sensitive to the HIV cytopathogenic effect "CEP") from a 24-hour culture were suspended at a concentration of 2x1θ5 / ml, divided into 1.2 ml aliquots in polypropylene tubes and pelleted by centrifugation. The pellets were then either infected with 200 μl of a stock solution of HIV III B cells (titer: 600 CCIDso / ml) or sham-infected with pure medium, incubated for 90 min. At 37 ° C. and then mixed with 1 ml of pure medium to make up the adjust the initial cell concentration again and bring the HIV concentration to 100 CCIDδo / ml. [CCIDso / ml = 50% cell-culture mfective d.ose].

100 μl volumes of the MARIGENOL® concentrates used in the test, diluted to aqueous ultramicroemulsions 10-3 and then additionally diluted from 10- ³ to 10-5 with RPMI 1640 medium, were introduced in triplicate into flat-bottomed microtiter plates with 96 holes. 100 μl of the HIV-infected or sham-infected MT4 culture were introduced into each well, in order to set up 4 test series:

- simple culture (controls; testing the viability of the MT4

Cells: 1θ5 cells / ml or 2x1θ4 / well)

- Culture + HIV (virus CPE control; concentration 50 CCIDso / ml or

10 CCIDso / hole)

- Culture + active substance in microemulsion

- Culture + HIV + active substance microemulsion in the

Dilutions 10- ³ to 10-5 (= 1,000 ppm, 100 ppm and 10 ppm, or 10 ppm, 1 ppm and 0.1 ppm water content) Incubate the cultures at 37 ° C in an atmosphere of 5% CO2 + 95% humidity. 5 days after the infection, 100 μl of the supernatant are removed in each well and the viability of the cells is checked using a methyl tetraazole salt (MTT, Sigma, 25 μl / well in 5 mg / ml solution) in a 2-hour incubation test This is followed by a solubilization with 100 μl DMSO / hole and the photometric reading on optical density at 550 nm. Residual values of the Zeil vitality (survival test) are expressed as a% difference compared to the HIV CPE controls, the mean as zero. The measured titer depends on the dose. It drops from the initial value of 300% CCID50 to 120% CCID50 and down to 2% CCID50.

The best protective effect on the host cells has so far been achieved with a 1% concentrate of β-sitosteryl palmitate at a concentration of 10-4 (= 100 ppm concentrate in aqueous ultramicroemulsion); The results with 1% concentrates containing ß-sitosteryl caproylate, ß-sitosteryl laurate, ß-sitosteryl arachidate or ß-sitosteryl behenate are also very noteworthy.

5.3 Effect on HIV infection. (Virulence, direct antiviral or virucidal activity against the acquired immunodeficiency, the human "Aquired Immuno-Deficiency Syndrome: AIDS" or against pathologically active prions).

Aliquots of 2 clinically prepared preparations from AIDS patients ("isolates" with the strains 21/4 and 4/5) were resuspended in complete RPMI medium at a titer of 300 CCIDso / ml and incubated for 3 hours at + 4 ° C with a MARIGENOL® concentrate, diluted as microemulsions in complete medium to the concentrations 10-2 to 10-5, and then tested for their effect together with an "empty" carrier concentrate and with pure medium alone.

After the pre-incubation, the HIV titer was recorded using the CCIDso method (CCID50 = cell c_ulture mfective d_ose 50%). Two serial dilutions of each of the 6 virus suspensions were made briefly, of which 200 μl each were incubated with MT4 cell pellets for 90 minutes, as indicated above (see 5.1). At the end of the incubation, the pellets are brought to the original cell concentration by adding the required amount of medium to each sample and then introducing them into 8 holes of a microtiter plate with 200 μl each. After 5 days of incubation, the vitality of the cells is measured using the MTT test as shown above. The HIV titer is given as the reciprocal of the dilution which 4 of 8 samples from a series (50%) infected. The content of a hole is considered infected if the reading of the OD at 550 nm is lower than the mean of the 8 control holes minus 2.8 times the standard deviation (lower 95% confidence limit). With the active substances ergosteryl-1 0-u-decenoate and ß-sitosteryl-palm itate, a very clear inhibition of replication and actual elimination can be determined in vitro as a result of the pretreatment of the strains. Your defense against infection is dose-dependent. There was no inhibition at all after treatment with pure carrier concentrate alone.

Evaluation of the test results:

TEST SUBSTRATE RESIDUAL in

HIV TITER (CCID50)

(Mean of

3 cultures)

HIV strain 21/4 (clinical isolate) + 300

medium

HIV strain 4/5 ("clinical i solate") + 200

medium

HIV (2 strains) + 200

CARRIER CONCENTRATE (medium titer from 2 strains)

HIV (2 strains) + 25

ERGOSTERYL-1 0-U NDECENOAT (medium titer off

CONCENTRATE 1 * 000 ppm 2 strains)

ACTIVE SUBSTANCE 10 ppm

HIV (2 strains) + 2 ß-SITOSTERYL-PALM ITAT (mean titer from

CONCENTRATE 1 * 000 ppm 2 strains)

ACTIVE SUBSTANCE 10 ppm

HIV (2 strains) + 50 ß-SITOSTERYL PALMITAT (medium titer from

CONCENTRATE 1 00 ppm 2 strains)

ACTIVE SUBSTANCE 1 ppm

HIV (2 strains) + 76 ß-SITOSTERYL-PALMITAT (mean titer from

CONCENTRATE 50 ppm 2 strains)

ACTIVE SUBSTANCE 0.5 ppm

HIV (2 strains) + 120 ß-SITOSTERYL PALMITAT (mean titer from

CONCENTRATE 10 ppm 2 strains)

ACTIVE SUBSTANCE 0.1 ppm

Residual values of the H IV-1 titer after preincubation for 3 h at + 4 ° C For the test methodology used, cf. et al .: Rudi Pauwels, Erik De Clercq et al .: "Sensitive and rapid assay on MT-4 cells for detection of antiviral compounds against the AIDS virus". Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium, Elsevier Science Publishers

B.V. (Biomedical Division), 1987 (0166-0934 / 87 / 503.50); J.Virol. Methods 1987;

16, 171-85.

A. Bergamini, C.F. Perna, et al .: A tetrazolium-based colorimetric assay for quantification of HIV-induced cytopathogenicity in monocyte-macrophages exposed to macrophage colony-stimulating factor. J.Virol. Methods, 1992:

40 (3), 275-86.

Jay A. Levy: "HIV research: a need to focus on the right target," The Lancet, Vol. 345, June 24, 1995, pp. 1619-21.

Molecular Cell Biology, second edition, by J. Darneil, H. Lodish, D. Baltimore; Scientific American Books, Chapter 5: Viruses, Structure and Functions, pp.177-188. New York, 1990 (W.H. Freeman & Co.)

6.0 Other viruses

6.1 Cytomegalovirus (CMV)

The test was carried out with human, embryonic lung fibroblasts as host cells, which were then infected with the CMV strain AD 169. The strain "CVM umano AD169" was formulated as a 1% concentrate and then diluted to an aqueous microemulsion 10-3, 10 for 4 h at + 4 ° C. with different concentrations of the test substance C _6: o-ß-sitosterylester -4 and 10-5, incubated and then inoculated as pretreated virus suspensions on cultures of human, embryonic lung fibroblasts. Approach as a confluent culture with 120 * 000 cells per shell vial. The infection was carried out by centrifugation for 45 minutes at 1500 rpm and at RT, whereupon the injection suspension was removed and 1 ml of culture medium MEM with 2% fetal calf serum (as in the case of cultivation) was added; the infected cells were then kept at 37 ° C. and under a 5% CO 2 atmosphere for 20 hours.

At the end of the incubation, the medium was removed, the cells were collected, fixed with 2% acetone-methanol (2: 1) and washed 3 times with PBS. The quantification was carried out by means of immunofluorescence measurement. The vials were mixed with the monoclonal antibody "Anti-P-72 CMV" (an immediate precursor protein of the CMV, which can be detected in the infected cells after 6 hours) and incubated for 30 minutes at 37 ° C. in a moist atmosphere. This was followed by 3 washes in PBS, a second incubation with the fluorescence-labeled antibody IgG goat anti-IgG mouse. This is followed by another incubation for 30 minutes at 37 ° C., as indicated above, followed by a 3-wash with PBS. The samples are then mounted on glass slides with 50% glycerol in PBS.

At the same time, controls were prepared with infected cells, which were prepared under the same conditions but without pretreatment with the test substance.

The nuclei positive for the CMV-specific antigen are counted using a fluorescence microscope at 25x magnification in the aqueous phase.

A dose-dependent, direct virucidal effect can be clearly determined. The virus is no longer virulent enough to infect the sensitive host cells.

RESULT :

VIRUZIDE EFFECT OF ß-SITOSTERYL-PALMITA TES

(formulated as MARIGENOL® CONCENTRATE)

Number of "Nuclei positivi per P-72" (the infected fibroblasts)

Examinations carried out by Dottoressa Rossäna CAVALLO, Istituto di Microbiologia, Universita di Torino, J uli-November 1 995.

6.2 Herpes virus (herpes simplex, HSV)

The antiviral / virucidal effect of an aqueous microemulsion of suitable MARIGENOLΘ concentrates is determined using a confluent monolayer culture from VERO cells (ie "African green mon key kidney cells"). The host cells are pretreated for 3 hours at 4 ° C. with the microemulsion or with the cooling medium alone and then infected with a constant dose of 104 herpes simplex viruses, so-called "plaque forming units" = PFU. Duration of virus absorption 1 h. Then addition of 2% methyl cellulose to prevent the spread of the virus; an elastic fleece is created in the culture medium MEM + 2% calf serum. Cultivation for 48 hours, then fixing and coloring the monolayers with 1% crystal violet in methanol. The antiviral effect correlates with the number of "pIaques" from lysed cells which still form in the culture medium MEM + 2% FCS. The reduction in viral titer is given as a percentage ratio to the controls (21 PIaques per cell).

The following concentrates were used as test substances in an aqueous dilution (based on a microemulsion 1: 100):

1% C ₂ o: _θ -ß-SITOSTERYL-ESTER

1% QUERCETIN-PENTA-10-U NDECENOAT

1% C _16: o-ß-SITOSTERYL-ESTER

1% C _2: o-ERGOSTERYL ESTER

Evaluation:

The results show a clear direct effect on the VERO Wirtz cells infected with HS virus. At a concentration of 10-4 ß-sitosteryl arachidate, the number of PIaques appears in comparison to the controls

76% reduced. The HSV titer is significantly reduced.

With this concentration of the tested substances, none is found

Lysis of eternalized lymphocytes of the MT-4 type, still the VERO-

Cells themselves instead. (See above under 5.0)

Tests carried out by Dottoressa Rossana CAVALLO, Istituto di Microbiologia, Universita deg li Studi d i Torino, July 1995.

6.3 Herpes virus (Herpes Sim plex, HSV)

Repetition of the test with fluorescence labeling as in the CMV test. See. above. Result: VIRUCID EFFECT OF ß-SITOSTERYL ARACHIDATE

(formulated as MARIGENOL® CONCENTRATE)

6.4 Hepatitis B Virus (HBV)

The tests were carried out with immortalized liver hematoma cells which, after infection with the hepatitis B virus, the two antigens Hbs Ag (a "surface antigen" from the outer shell of the virus) and Hbe Ag (a "core antigen" from the core of the DNA) Virus) secrete. Orientative tests in vitro were carried out with a 1% concentrate of the three test substances ß-sitosteryl palmitate, ß-sitosteryl arachidate and ergosteryl isovalerate by Prof. Dott. Antonio Ponzetto and performed by Dotta Rossana Cavallo, Università degli Studi di Torino.

With a dilution of 10-5 and incubation for 72 hours, the ß-sitosteryl palmitate concentrate has so far shown the greatest effectiveness. The results are clearly dose-dependent and also vary with the incubation period. The effect of the surface antigen Hbs Ag is particularly pronounced.

HEPATITIS B virus

Secreted antigens

7.0 Antiparasitic effects

Orientative examinations were carried out at the Swiss Tropical Institute Basel (PD. Dr. Ronald Kaminsky and Ms. Yvonne Grether). In the first series of tests, it was examined whether significant results could be reported in vitro against Trypanosoma rhodesiense (the causative agent of sleeping sickness), against Trypanosoma cruzi (the causative agent of Chagas disease) and against Leishmania donovani (the causative agent of leishmaniasis). . Significant antiparasitic activity Hess has been demonstrated in advance for selected cholestanyl and bioflavonoid ester concentrates, as well as for the Z-Gly-Phe-D ₃ ester concentrate, at concentrations that do not have a basic cytotoxic effect on murine muscle cells or generate macrophages. It is specific against Leishmania donovani.

Preliminary results :

SWISS TROPICAL I NSTITUTE, BASEL In-vitro assays, WHO screening as SOP

All values as: μg / ml

Legend * r = active on T.b.rhodesinense d = active on L. donovani t = cytotoxic on mammalian cells c = active on T. cruzi - = i nactive or low activity

MIC = minimum inhibitory concentration IC50 = 50% killi ng rate concentration

EP 1 1% concentrate CHOLESTANYL-ISOVALERAT

EP 2 1% concentrate CHOLESTANYL-10-UNDECENOAT

EP 3 1% concentrate CHOLESTANYL-PALMITAT

EP 4 1% concentrate CHOLESTERYL-n-VALERAT

EP 5 1% concentrate CHOLESTERYL-LAURAT

EP 6 1% concentrate CHOLESTERYL-PALMITAT

EP 7 1% concentrate SITOSTERYL-LAURAT

EP 8 1% concentrate ß-SITOSTERYL-PALMITAT

EP 9 1% concentrate ß-SITOSTERYL ARACHIDATE

EP 10 1% concentrate ß-SITOSTERYL-BEHENAT

EP 11 1% concentrate STIGMASTERYL-LAURAT EP 12 1% concentrate STIGMASTERYL PALMITAT

EP 13 1% concentrate ERGOSTERYL-n-VALERAT

EP 14 1% concentrate ERGOSTERYL-LAURAT

EP 15 1% concentrate ERGOSTERYL PALMITAT

EP 16 1% concentrate ERGOSTERYL BEHENATE

1 N 1% concentrate ß-SITOSTERYL-PALMITAT

2N 1% concentrate ß-SITOSTERYL-PALMITAT

TO 100% Cι ι _: -CITRONELLYL-ESTER (COEMULGATOR)

T1 1 00% COEMULGATOR + TENSIDE

T10 1% concentrate ß-S ITOSTERYL-PALMITAT

T1 1 1% concentrate ß-SITOSTERYL ARACHIDATE

T12 1% concentrate ERGOSTERYL-LAURAT

Are the measured values of the concentrates on W.S. - In terms of content, they are 100 times smaller.

8.0 General compatibility of MARIGENOL® preparations

8.1 Toxicity in vivo

Test with normal 8-week female BALB / c nAncr (H-2d) mice supplied by Charles River Laboratories, Calco (Italy). IV injection twice daily for 4 weeks. of 0.250 ml each of aqueous microemulsion, formed from the specified concentrates, or with physiological buffer for the controls. Coloring with May Grünwald-G iemsa. Treatment time: 28 days. Blood analysis: after the last injection

Number of animals: 1 3 groups of 5 per animal

RESULT: There are no significant differences from the controls. In addition, no toxicity of the concentrates to the leukocyte population was found. The erythrocyte population also showed normal values. All animals were and remained healthy until the end of the experiments.

Carrying out the samples: Prof. Dott. Guido FORNI, Dotta stefania VAI, University of Torino, Dipartimento di Scienze Cli niche e Biologiche, Ospedale San Luigi Gonzaga, I-10O43 ORBASSANO (TO). 8.2 Examination of the blood picture

Effect on normal human LYMPHOMONOCYTEN

Cells: normal human phagocytic white blood cells

(Macrophages); 2 x 10 ⁵ cells per well

Control stimulation: 1% PHA (Phytohemaggl utinin)

Test 1: 2% concentrate containing Cι _2: o-cholesteryl ester

Test 2: 2% concentrate containing Ci ₆ : o-ergosteryl ester

Test 3: 2% concentrate containing C _{5: 0} cholecalciferyl ester

(Vitamin D3-iso-valerate) Tests carried out by Dottoressa Anna-Rita G UARINI, Universitadegli Studi di Torino, Clin ica medica, 1-10'1 26 TORINO, 21. until March 24, 1994.

Claims

PAT EN TAN SPEECH

1. Spontaneously dispersible concentrate, which dilutes with water, with 5% glucose solution or with Ringer's solution, results in thermodynamically stable ultramicroemulsions which have micelles with a hydrodynamic radius of 2.2 to 3.0 nm, characterized in that the following constituents form a pharmaceutically usable system are assembled:

0.1 to 10% by weight of one or more esters of pentacyclic triterpene compounds according to formulas (I) to (XI), or a combination of such active ingredients:

(III)

(VII) where in formulas (I) to (XI) R1 is a C ₃ . ₃ ι-alkyl, a C ₃ . ₃ ι-alkenyl, a Ci7-23 alkapolyen or a retinoyl group, 0 to 5% by weight of a synergistic, active pharmaceutical substance, 1 to 25% by weight of a pharmaceutically compatible solvent or solvent mixture serving as a hydrotrope or co-emulsifier, 0.1 to 90% by weight of a pharmaceutically compatible surfactant or surfactant mixture, up to 10% by weight of a vitamin or provitamin, up to 1 0% by weight of a stabilizer, radical scavenger, biological vector or penetration enhancer and carriers and / or diluents.

2. Spontaneously dispersible concentrate containing compounds of the formulas (I) to (XI) according to claim 1, as an agent for the preparation of pharmaceutical preparations with activity against tumors, eczema and psoriasis, and against viral and parasitic infections.

3. Spontaneously dispersible concentrate, containing compounds of the formulas (I) to (XI) according to claim 1, as a means for the production of pharmaceutical and paramedical preparations with prolonged prophylactic activity against tumors, viral and parasitic infections, and for supporting the Inclusion of exogenous activators, modulators and / or regulators.

4. Spontaneously dispersible concentrate according to claim 1, characterized in that the following components are combined:

0.5 to 2% by weight of one or more esters of pentacyclic triterpene compounds according to formulas (I) to (XI),

5 to 25% by weight of isopropyl myristate, isopropyl palmitate or neutral oil (oleum neutral), or one or more bio-surfactant esters of the general formula (XIII):

R- ^ COO-R ⁴ _{(χ | | l)} where R ^{3 is} a C ₂ . ₃ ι alkyl, a C ₃ . ₃₁ -Al kenyl- or a C ₃ . ₃₁ -Alcapolyen group and R4 mean citronellyl, farnesyl, geranyl, isophytyl or phytyl,

0 to 45% by weight of a pharmaceutically compatible phosphoric acid ester surfactant,

5 to 90% by weight of the anhydrous tert. Octylphenyl polyoxyethylene ether surfactant with 9 to 10 oxyethylene groups and / or polysorbates 20.

5. Spontaneously dispersible concentrate according to claim 1, characterized in that the following components are combined:

5 to 25% by weight of one or more biosensitive esters of the general formula (XIII):

R-COO-R ⁴ _(XIII) wherein R ^{3 is} a C ₂ . ₃₁ alkyl, a C ₃ . ₃₁ alkenyl or a C ₃ . ₃ -Al kapolyen group and R4 Citronellyl-, Farnesyl-, Geranyl-, Isophytyl- or Phytyl- mean.

30 to 45% by weight of the anhydrous tert. Octylphenyl polyoxyethylene ether surfactant with 9 to 10 oxyethylene groups and / or polysorbates 20, 30 to 45% by weight of an alkylphenol polyglycol ether phosphate surfactant or the tristyrylphenol polyoxyethylene 18-phosphoric acid ester, TEA salt surfactant.

6. Therapeutic system preparation which contains 1 to 95% by weight of the spontaneously dispersible concentrate according to one of claims 1, 4 or 5 and up to 10% by weight of a pharmaceutically acceptable additive, solvent or stabilizer or a mixture thereof and which in unit dosage form as micropellets, granules, dragees, suppositories, ampoules or as capsules.

7. A process for the preparation of esters of pentacyclic triterpene compounds according to claim 1, characterized in that a compound of the formula (XII):

R- COOH _(XM) wherein R2 is a C. ₃ ₃ alkyl, a C ₃ . ₃ i -Alkenyl-, a Cι _7-23 -Alkapolyengruppe or a retinoyl group called, with 1, 1 '-carbonyldiimidazole or with N, N'-dicyclohexylcarbodiimide in an indifferent solvent and then the resulting imidazolide with a compound of the formulas (I ) to (X) can react.

8. A process for the preparation of esters of pentacyclic triterpene compounds according to claim 1, based on the fact that a compound of formula (XII):

R— COOH _(XM) wherein R ^{2 is} a C ₃ . ₃ ι alkyl, a C ₃ . ₃ ι alkenyl or a Ci ₇ . ₂₃ -Alcapolyen group, is reacted with a chlorinating agent and then the resulting product is allowed to react with a compound of the formulas (I) to (X).

9. A process for the preparation of allobetuli n-carboxylic acid esters, consisting in that the transformation product Al lobetuli n is obtained by refluxing with 85% formic acid and then with the acid chloride of a carboxylic acid C ₅ . ₃ -ι in abs. Pyridi n acylated with the addition of 4-dimethylaminopyridine.

1 0. A process for the esterification of the betulinic acid methyl ester, the 18ß-glycyrrhetinic acid methyl ester and the oleanolic acid methyl ester at position 0-C (3), consisting in that these products with an activated carboxylic acid in abs. Pyridine acylated with the addition of 4-dimethylaminopyridine.

1 1. The use of a spontaneously dispersible concentrate according to claim 1 or an aqueous microemulsion prepared therefrom as a remedy with efficacy against tumors, psoriasis, eczema, viral and parasitic infections and for the increased absorption of exogenous activators, modulators and regulators.