WO1998011889A1

WO1998011889A1 - Ultramicroemulsions from spontaneously dispersible concentrates with antitumor, antiviral, virucide and antiparasitically active esters of bioflavonoid compounds

Info

Publication number: WO1998011889A1
Application number: PCT/CH1997/000168
Authority: WO
Inventors: Carl Eugster; Conrad Hans Eugster
Original assignee: Marigen S.A.
Priority date: 1996-09-18
Filing date: 1997-04-28
Publication date: 1998-03-26
Also published as: WO1998011876A1; EP0862427A1; EP0863746A1

Abstract

This invention concerns spontaneously dispersible concentrates of esters with antitumor, antiviral, virucidal and antiparasitic effect which are made from bioflavonoid compounds, aqueous ultramicroemulsions out of such concentrates, new esters of selected bioflavonoid compounds, processes for producing and preparing them, and their use as agents for producing medicaments effective against viral and parasitic illnesses, against tumors, eczema and psoriasis, for ongoing tumor prophylaxis and for enhanced absorption of exogenic activators, metabolic modulators and regulators.

Description

ULTRAMICROEMULSIONS from

SPONTANEOUSLY DISPERSIBLE CONCENTRATES with ANTITUMORAL, ANTIVIRAL, VIRUZID and ANTIPARASITARY

EFFECTIVE ESTERN from BIOFLA VONOID COMPOUNDS

INTRODUCTION

The present invention relates to ultramicroemulsions from spontaneously dispersible concentrates with lipophilic, aliphatic esters of selected, naturally occurring flavonoids (without anthocyanins and chalcones), new esters of these compounds, processes for their preparation and workup, the use of the ester-containing spontaneously dispersible concentrates for the production of medicinal products with antitumoral, antiviral, direct virucidal and / or antiparasitic activity, for combating eczema and psoriasis, as well as for tumor prophylaxis and tumor therapy, as well as for the increased absorption of exogenous activators, metabolic modulators and regulators.

The esters of these bioflavonoid compounds surprisingly have a very good antitumor, antiviral, direct virucidal, and in some cases also an antiparasitic effect, and they are also suitable for treating eczema and psoriasis and for stimulating immune responses. The therapeutic properties of such esters occur significantly more when they are incorporated into spontaneously dispersible concentrates and then diluted with distilled water or 5% glucose solution or also with physiological saline (Ringer's solution), resulting in thermodynamically stable ultramicroemulsions with micelles of a hydrodynamic radius of 2.2 to 3.0 nm.

The greatly increased, sustained biological effectiveness of the new flavonoid peresters and their presentation in the concentrate and ultra-microemulsion form according to the invention is based on the following factors:

- The esterification of the selected flavonoids with medium and long-chain fatty acids leads to an extraordinarily increased lipophilicity and thus to an inversion of the original property of the basic body. Together with the significant increase in migration and spreading Due to the ultra-fine micelles, this causes rapid permeation of hydrophobic membranes;

- The flavonoid peresters are to be regarded as "activated esters". They have the ability to transfer acyl groups to suitable nucleophilic groups, which is particularly true of the acyl group at C-5 of the flavonoid backbone. This activation can easily be seen in 1 H-NMR spectra when using solvolytic solvents: the typical hydrogen bond between OH-C-5 and the carbonyl at C-4 appears quickly at approx. 12-13 ppm. From this it can be concluded that an acylation of (R) ₃ -C-SH, R-CH ₂ OH and R-NH ₂ groups, and possibly also of secondary alcohols and secondary amines, which are present in the cell components are, should be done quickly;

- The release of the intramolecular hydrogen bond between OH-C-5 and the carbonyl at C-4 at a suitable point in the cell opens up the inherent chelation potential of the flavonoids for different cations with the coordination numbers 4 and 6. Since ancient times, this fact has been due to the strong chelation of cations used in an aqueous environment for dyeing cellulose fabrics (hemp, flax, cotton, etc.);

- The lability, especially the C-5-acyl group, prohibits the workup of the esterification batches with aqueous bases, as well as column chromatography on materials with nucleophilic traps. The esterification must therefore be carried out with a minimum of purification steps.

DESCRIPTION OF THE INVENTION

The basic form of the esters of bioflavonoid compounds has the general formulas (I) to (IV): Flavones:

R = OH, OCH ₃ , in different positions, such as chrysin with OH at (C-5,7) or apigenin with OH at (C-5, 7, 4 ') Flavonols:

with R as above, e.g. camphor oil with OH (C-5, 7, 4 '), quercetin with OH (C- 6.7 _I 3 \ 4 ^, ) _I Morin with OH (C-5, 7, 2', 4 '),

Flavanols (2,3-dihydroflavanols):

with R as above, e.g. (+) - catechin with OH (C-5, 7, 3 ', 4')

Isoflavonols:

with R as above, for example genistein with OH (C-5, 7, 4 ') where in the formein (I) to (IV) R for hydroxyl, a C ₆ - ₃₁ alkyl, a C ₉ . ₃ 1 - alkenyl or a C ₉ . ₃₁ -Alkapolyengruppe stands.

The esterification of the base bodies mentioned can be carried out by a process known per se:

Formation of the chloride of a compound of formula (V):

R ¹ COOH _(V) where R is a Cβ- ₃₁ alkyl, a Cg. ₃ -alkenyl or a C ₉ _ ₃₁ -Alkapolyen- group, with a chlorinating agent such as thionyl chloride or lylchlorid oxa, and subsequent reaction with a compound of Formein (I) to (IV) at a temperature from 0 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.

The esters of the bioflavonoid compounds according to formulas (I) to (IV) surprisingly have an excellent antitumor, antiviral, virucidal and also antiparasitic activity, and are also suitable for combating eczema and psoriasis and for compensating metabolic disorders . These effects occur primarily when they have been incorporated into spontaneously dispersible MARIGENOL® concentrates according to the invention which, when diluted with distilled water, 5% glucose solution or also Ringer's solution, result in thermodynamically stable oil-in-water ultramicroemulsions.

Such ultramicroemulsions have spherical micelles with a hydrodynamic radius of 2.2 to 3.0 n. [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)].

The present invention relates in advance to spontaneously dispersible concentrates with antitumoral, antiviral, virucidal or antitrypanosomal active esters of the formulas (I) to (IV). These esters are almost water-insoluble and highly agglomerated compounds. However, in order that such compounds can diffuse through the membrane barrier of the tumor or host cells or also through the protein envelope of viruses and become effective in the interior of the cell or the virus, they must first have been suitably solubilized in the aqueous medium. The formation of spontaneously dispersible concentrates and the thermodynamically stable oil-in-water ultramicroemulsions produced from them enables the new ester to be optimally solubilized. To this end, selected cosurfactants or solubilizers and, on the other hand, suitable surface-active compounds (surfactants) are used and combined to form a balanced system and then diluted.

All experimental observations on the aqueous ultramicroemulsions formed in this way can be interpreted uniformly by the assumption that the selected cosurfactants and surfactants are organized in the aqueous phase, so-called aggregates. Form micelles. These have a more or less spherical shape, with a hydrodynamic radius of 2.2 to 3.0 nm. The surfactants and the hydrotropes (cosurfactants) leave between the outer, aqueous phase and the inner, oily phase of the microemulsion [containing an ester of the formulas (I) to (IV), dissolved in the biosensitive solubilizer], an interface is formed, which is prevented by the mixing of these two phases. In the oily, inner phase of the micelles, the micellar nucleus, the molecules of the bioflavonoid ester are present in monomeric or in oligomeric agglomerated form.

The micelles of the inner phase of such ultramicroemulsions are accordingly protected on their surface with an elastic surfactant coat, which they repair, easily diffusing through the cell membrane into the interior of the tumor or host cell, or through the protein envelope of viruses. The diffusion through the plasma membrane of tumor or host cells or through the capsid of viruses occurs exclusively due to thermal molecular movements.

The direction that a specific diffusion process takes is determined by the difference in concentration which exists on the plasma membrane between outside and inside the tumor or host cell or on the protein envelope. The diffusion continues along the concentration gradient until it is broken down. The concentration of active substance or an active substance system is balanced between the extracellular zone and the interior of the individual tumor or host cell or a virus, whereby delayed release effects can also occur. Such diffusion processes take place independently of any energy supply. They have no relation to cellular metabolic energy.

The speed of the diffusion process or the strength of the active ingredient transport through the membrane of the tumor or host cell are determined:

1 . of the difference in concentration in the two compartments

2. from the particle radius of the diffusing drug molecule 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 12.564 cm2. In contrast, point 1018 micelles with a hydrodynamic radius of only 10-6 _C m (10 nm), which together make up the same volume of 4.189 cm ³ have a total surface area of Phase 1 '256.4 m2 on. MICELLES: RELATIONSHIP VOLUME TO TOTAL SURFACE

Sphere volume = - π r3 3

Spherical surface = 4 π r2

Conclusion: Due to the large phase surface, which the micelles form with a hydrodynamic radius of 2.2 to 3.0 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 in the The core of the micelles is in monomeric or oligomeric agglomerated form, also greatly improved. Dispensing them as very fine micelles can allow a considerable reduction in the pharmacologically critical dosage and thus avoid or at least help to reduce undesirable side effects.

The "packing density" of a spontaneously dispersible, stable MARIGENOL® concentrate increases in an exponential manner with the smaller particle size of the micelles. The decisive factors are the correct formation of the inner phase, its balanced relationship to the total concentrate and the selection of the appropriate surfactants.

The concentrates which are spontaneously dispersible according to the invention contain: 0.1 to 5% by weight of individual esters of the formulas (I) to (IV), or a combination of such esters, and 5 to 25% by weight of a pharmaceutically acceptable solubilizer or solvent mixture serving as a hydrotrope, ie serving as a co-emulsifier,

5 to 90% by weight of a pharmaceutically compatible surfactant or surfactant mixture, and optionally

0 to 10% by weight of a vitamin or provitamin,

0 to 10% by weight of a stabilizer, radical scavenger or penetration enhancer.

The oil-in-water ultramicroemulsions which can be used according to the invention contain:

0.01 to 5% by weight of a spontaneously dispersible concentrate as described above,

85 to 99.99% by weight of distilled water, 5% glucose solution or physiological saline (Ringer's solution),

0 to 10% by weight of pharmaceutical carriers or additives and / or auxiliaries.

Such aqueous ultramicroemulsions have a reduced surface tension of 28 to 32 mNm- ¹ and a low internal resistance (ie a dynamic viscosity η at 20 ^C C) of approx. 1.0 cP = 10-3 Pa.s.

The surfactants or surfactant mixtures to be used according to the invention can be anionic, cationic, amphoteric or non-ionic. They are preferably amphoteric or non-ionic and have an HLB ratio (i.e. a "hydrophilic-lipophilic balance") between 2 and 18; for mixtures it is preferably between 2 to 6 on the one hand and 10 to 15 on the other. HLB values provide information about the hydrophilic and lipophilic properties of an emulsifier. See . also "Hydrophile-Lipophile Balance: History and recent Developments" by Paul Becher in the Journal of Dispersion Science and Technology 5 (1), 81-96 (1984).

Highly preferred for the production of spontaneously dispersible concentrates according to the invention are, on the one hand, phosphoric ester surfactants, such as, for example: the practically anhydrous tristyrylphenol polyoxyethylene-18-phosphoric acid ester triethanolamine salt surfactant (TEA salt surfactant):

(Soprophor® FL, RHONE-POULENC);

Diphasol® 3873 (CIBA-GEIGY), or the identical Sermul® EA 1 88 (SERVO), a mixed emulsifier, each consisting of 50% of the two compounds with the

Formulas:

O

^C 9 1 CH,

O CH,

Diphasol® 3873 (CIBA-GEIGY), an alkylphenol polyglycol ether phosphate surfactant surfactant 508 (CIBA-GEIGY)

(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 - O) "OCH ₃

^ OCH ₃

(Zerostat ® AN, CIBA-GEIGY) and on the other hand betaine compounds, i.e. amphoteric, salt and water-free imidazole derivatives, e.g. :

wori n Me [ ⁺ ] for hydrogen, alkali and alkaline earth atoms and R _x for Cι. ₃₂ alkyl or C ₂ . ₃₂ alkenyl groups are available.

"Polysorbate" compounds are also used, e.g. Tween® 20-85 (Atlas Chem. Ind.lnc; or ICI Specialty Chemicals) and so-called "multifunctional glucose derivatives", such as e.g. Glucate® SS (methyl glucose sesquistearate) and Glucamate® SS E-20 (PEG-20 methyl glucose sesquistearate) from Amerchol, Edison, N.J., U.S.A.

As a hydrotrope, i.e. Pharmaceutical compatible solvents serving as co-emulsifiers can be used, e.g. :

Esters of an aliphatic alcohol _(C3. ₁₈₎ with an aliphatic carboxylic acid (C ₁ 0. _22). such as isopropyl laurate, hexyl laurate, decyl laurate, isopropyl myristate, isopropyl palmitate and lauryl myristate; Hydrocarbons with a straight carbon chain (C _{1 2.} 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 of 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 aliphatic alcohol with lactic acid, such as myristyl, lauryl lactate or, preferably, (Cι _2. _22); Mono-, di- or triesters of Gly cerins such as glyceryl caprylic lat with an aliphatic carboxylic acid (C _6. _22), or Miglyol® 812 neutral oil (Oleum neutral).

Ester of a poly (2-7) ethylene glycol glycerol ether with at least one free hydroxyl group with an aliphatic carboxylic acid (C6-22). such as aliphatic alcohols (C _{1 2.} ₂₂ ), including dodecanol, tetradecanol, oleyl alcohol, 2-hexyldecanol and 2-octyldecanol.

Esters with at least one free hydroxyl group, from poly (2-10) glycol with an aliphatic carboxylic acid (C ₆ - ₂ 2). Monoether from a polyethylene glycol with an aliphatic alcohol (Cι_-ιβ) _ι such as polyoxyethylene (C ₁₀ ) octyl ether.

Heterocyclic compounds, such as 1-methyl-2-pyrrolidone. Zwitterionic compounds (Good buffer) such as' zBEPES "with formula: 4- (2-hydroxyethyl) piperazin-1-ethanesulfonic acid, or" CHAPS "with formula: 3- [(3-cholamido-propyl) -dimethyl-ammonio ] propane sulfonate or biots:

wherein R ^{2 is} an aliphatic C ₂ . ₃₁ alkyl, is a C ₃ - ₃ i alkenyl or alkapolyen group and R ^{3 is} citronellyl, farnesyl, geranyl, isophytyl or phytyl, and straight-chain carboxylic acid esters of DL-α-tocopherol, especially the undecenoate and the laurat.

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

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

PROCEDURE for bioflavonoid peresters with medium and long chain carboxylic acids

1.0 Manufacturing

1.1 According to regulation A: reprocessing using water. A suitable three-necked flask dried at 150 ° C with a magnetic stirrer can be cooled to RT in a stream of N ₂ . At the same time, it is provided with a discharge pipe and gas meter, a thermometer and a septum. Then 1 g of anhydrous flavonoid is added and this is dissolved in 10 ml of abs. Pyridine, if necessary by briefly heating over the infrared burner. Then cool to RT and dilute with 10 ml abs. Benzene and dichloromethane. Then further cooling to 0 ° to -5 ° C. Now use a syringe via the septum to achieve a complete - 1 1 -

Esterification equivalents of acid chloride added dropwise and with good stirring. A white precipitate of pyridine hydrochloride quickly forms. The mixture is advantageously stirred overnight, while being allowed to come to RT. It is worked up by adding 50 ml of dilute brine (eg 5% saline) and 150 ml of solvent, including the amount required for transfer to a separating funnel. Depending on the solubility, the solvent consists of petroleum ether (30-60 °) or its mixture with benzene or pure benzene. After discarding the hypophase, wash with four further 50 ml portions. If emulsions appear during shaking, it is advisable to break them by filtration through a layer of Celite. The extract, which is usually pale yellow in color, is briefly dried over anhydrous Na ₂ S0 ₄ or MgS0, then filtered and evaporated in vacuo. The resulting oily or crystalline product is then transferred to a suitable crystallization vessel and recrystallized from 2-propanol or from benzene / acetonitrile or from dichloromethane / acetonitrile. Colorless to pale yellow colored peresters of the flavonoids used are obtained. They are dried at 40-55 ° C and 0.01 Torr.

1.2 According to regulation B: reprocessing under anhydrous conditions.

The esterification is carried out according to regulation A. However, the resulting suspension is placed directly on a dry-packed column of 100 to 150 ml of silica gel 60 (0.063-0.200 mm, Merck 7734), prewashed with benzene / acetone 99: 1, and then developed with the same solvent. The fast-running, pale lemon-yellow main zone is collected, evaporated in vacuo and the mostly crystalline residue is recrystallized as indicated under 1.1.

1.3 Comments.

- With larger batches, reprocessing according to regulation A is advisable because it usually yields higher yields and is also more cost-effective. Processing according to regulation B not only avoids annoying emulsions; it also brings pure products faster and is time-saving. The pyridine content deactivates the silica gel and thus also protects unstable esters at HO-C (5).

- The flavonoids used were dried at 130 ° C / 0.001 Torr for 20 h.

- Acyl groups on HO-C (5) are unstable, especially those with relatively short chains. Transacylation occurs rapidly, especially with primary alcohols on. It is greatly reduced with secondary alcohols. Therefore 2-

Propanol can be used for recrystallization. Evidence of a free

H-0-C (5) can be conveniently carried out by NMR using the band at approx. 12.5 ppm.

- Rapid coarse thin layer chromatography on silica gel with cyclohexane / acetone 17.5: 2.5 and detection in short- and long-wave UV is suitable for rough checking of the purity of the flavonoid esters. The Peresters show high and relatively little different Rf values; however, polar contaminants can be easily recognized.

- Quantitative UV spectra were recorded in dichloromethane, λmax, nm and ε values are given. The extinctions were calculated using the MG of the Perester.

- IR spectra were measured in CHCI ₃ . Bands in the fundamental range are given at cm ^' ; ss = very strong, m = medium, w = weak with a qualitative estimate of their intensity, based on the strongest band.

- Mass spectra mostly with ESI (electrospray with CHCI ₃ / methanol + Nal), otherwise Cl (chemical ionization with NH ₃ ); Selection of important fragment ions with m / z and intensity.

- 1 H-NMR spectra were recorded in CDCI ₃ solution at 300 MHz, selected signals and their <values are given in ppm.

- Melting points were determined in the open tube and are not corrected. In several cases, intervals are observed that are obviously due to phase changes.

1: R = (CH ₂ ) ₁₀ CH ₃

2: R = (CH ₂ ) 14CH ₃

2.1 Apiqin intrilaurate (1)

C ₅ ι H ₇₆ 0 ₈ (817.12); Yields: 80% according to regulation A, 65% according to regulation B; creamy white crystals of dichloromethane / acetonitrile; Mp 103.5-105.5 ° C.

UV: 230 (18 * 300), 275 (37 * 500), 303 (22,700), sh. 330 (14000). _ \ R: 2924 ss, 2856 s, 1398 m, 1760 s, 1657 ss, 1620 ss. MS: 657.5 (100%) [dilaurate + Na] +.

NMR: 1.05 [t, <_ -CH ₃ ); 1.44 (s, chain CH ₂ ); 1.93 // 2.75 (each m α-CH ₂ ); 6.7-8.0

(Aromatic protons, including 6.85 s, H-C (3)); 12.7 (s, HO-C (5), lane).

Spend nunqsanalvse: Ber. C 74.96 H 9.38%

Found C 75.00 H 9.49%

2.2 Apiqenintripa imitation (2.)

C ₆₃ Hi00 ₈ (985.43); Yield 70% according to regulation B; almost colorless crystals of dichloromethane / acetonitrile; M.p. 101 -102 ° C. UV: 232 (18 * 900), 276 (39 * 400), 303 (22 * 700), sh. 330 (14 * 900). 1R {CH2CI2): 2926 ss, 2853 s, 1762 ms, 1758 s, 1620 ss, 1597 ms. NMR: 0.90 (f, ω-CH ₃ ); 1-29 (s, chain CH ₂ ); 1.8 // 2.6 (each m α-CH.); 6.6-7.9 (aromatic protons, including 6.72 s, HC (3)); 12.7 (s, HO-C (5), lane).

3.0 Chrvsinperester

3: R = (CH ₂ ) ₁₀ CH ₃

4: R = (CH ₂ ) i 4CH ₃

3.1 chrysin dilaurate (3)

C ₃₉ H ₅₄ O ₆ (61 8.82); Yield 69% according to regulation A; pale yellow crystals

Benzene / CH ₃ CN; Mp 134 ° C.

UV: 230 (16OO0), 270 (48'100), sh. 295 (24 * 600), 327 (15,700)

1R: 2925 ss, 2853 s, 1763 s, 1656 ss, 1619 ss, 1593 w, 1572 w (only slight differences to quercetin pentalaurate)

MS: 1333 (69%) [3 monolaurate + Na] ⁺ ; 1,260 (19%) [2 dilaurate -1 1,8 + Na] +; 1078 (42%) [dilaurate + monolaurate + Na] ⁺ ; 896 (100%) [2 monolaurate + Na] +; 641 (44%) [dilaurate + Na] +; 460 (98%) [monolaurate + Na] +;

NMR: 0.94 (r, ω-CH ₃ ); 1.34 (s, chain CH ₂ ); 1 .8 / 2.6 (each m, -CH ₃ ); 6.6-7.9 (aromatic protons, including 6.78 (s, HC (3)); 12.8 (s, HO-C (5), trace). Combustion analysis: calc. C 75.69 H 8.80%

Found C 74.78 H 7.55%

C 74.37 H 7.32%

C 74.44 H 7.03%

C 76.29 H 7.78% Recrystallization from benzene / 2-propanol gives the monolaurate.

Combustion analysis: Ber. C 74.28 H 7.39%

Found C 74.18 H 7.1 6%

C 73.92 H 7.26%

C ₄₇ H70O ₆ (731.03); Yield 70% according to regulation A; pale brownish yellow crystals from benzene / 0 ° or from dichloromethane / acetonitrile; Mp 104-105 ° C. UV: 230 (16 ^* 800), 270 (43 * 500), sh. 296 (22 * 300), 327 (13 * 300) [R. : 2923 ss, 2856 s, 1765 s, 1648 ss, 1619 ss, sh. 1595 w, 1572 w (only slight differences to the spectrum of quercetin pentalaurate)

MS_ (CI with NH ₃ ): 493.2 (100% [M + 1 -126.6]

NMR: 0.87 (t, ω -CH ₃ ); 1.25 (s, chain CH ₂ ); 1.8 / 2.6 (each, α-CH ₂ ); 6.5-7.9 (aromatic protons, including 6.72 (s, HC (3)); 12.7 (s, HO-C (5), trace). Combustion analysis: Calc. C 77.22 H 9.65%

Found C 77.36 H 9.60%

3.2 Chrvsindipalmitate (4)

Hs C ₃₉ ₄ 0 ₆ _(618.82); Yield 69% according to regulation A; pale yellow crystals

Benzene / CH ₃ CN; Mp 131.5-132.5 ° C.

UV: 230 (16 * 000), 270 (48 * 100), sh. 295 (24 * 600), 327 (15 ^* 700)

IR_: 2925 ss, 2853 s, 1763 s, 1656 ss, 16190 ss, 1593 w, 1572 w (only slight differences to Querceti npentalaurat)

MS: 1333 (69%) [3 monolaurate + Na] +; 1260 (19%) [2 dilaurate -118 + Na] ⁺ ; 1078 (42%) [dilaurate + monolaurate + Na] ⁺ ; 896 (100%) [2 monolaurate + Na] ⁺ ; 641 (44%) [dilaurate + Na] +; 460 (98%) [monolaurate + Na] +;

NMR: 0.94 (t, ω-CHj); 1.34 (s, chain CH ₂ ); 1.8 / 2.6 (each m, α-CH ₃ ); 6.6-7.9 (aromatic protons, including 6.78 (s, HC (3)); 12.8 (s, HO-C (5), trace). Combustion analysis: calc. C 75.69 H 8.80%

Found C 74.78 H 7.55%

C 74.37 H 7.32%

C 74.44 H 7.03% 4.0 Combat ester

5: R = (CH ₂ ) ι ₀ CH ₃

6: R = (CH ₂ ) ι ₄ CH ₃

4.1 Combatant tetralaurate (5)

C ₆₃ H ₉₈ θ ₁₀ (1015.41); Yields: 90% according to regulation A, 70% according to regulation

B; light lemon yellow crystals from Et ₂ 0 / CH ₃ CN; Mp 85.5-98 ° C

UV: 230 (25 * 700), 268 (36 * 000), 307 (21 * 600), 338 (20 * 100).

IR. : 2930 ss, 2850 s, 1765 s, 1665 s, 1645 s, 1 617 s (Minor

Differences compared to quercetin pentalaurate).

MS (Cl, NH3): 833.2 (17%) [M + 1 -x] +; 651.2 (81%) [M + 1 -2x] +; 469.2 (100%)

[M + 1 -3x] +; 287.1 [M + 1 -4x] +; x = m / z 182 (from the α cleavage on the ester)

NMR: 0.81 (r, ω -CH ₃ ); 1.19 (s, chain CH ₂ ); 1.7 / 2.5 (each m α-CH ₂ ); 6.1 -7.8

(Aromatic protons); 12.1 (s, HO-C (5), lane).

Combustion analysis: Ber. C 74.51 H 9.73% Found C 74.62 H 9.59%

4.2 Fighting Eraltripalmitate (6)

C ₇₉ H _{1 30} O ₁₀ (1239.83); Yield 75% according to regulation B; light yellow crystals of benzene / acetonitrile; Mp 105-110 ° C (crystallized from 2-propanol) UV: 230 (25,700), 268 (35,900), 307 (21 * 600), 338 (20 * 100).

IR. (CH3CI2): 2922 ss, 2850 m, 1765 m, 1654 m, 1617 s (only slight differences compared to quercetin pentapalmitate).

MS: (DCI, NH ₃ ): m / z 1239.8 (15%, M +: next to it peak with M ++ 1, M + -1, M + -2, and m / z 1001 .7 (15%), 763.5 (45 %), 525.3 (100%), 287.3 (50%) This is the consecutive split off of four pamitoyl radicals.

NMR: 0.83 (f, ω -CH ₃ ); 1.20 (s, chain CH ₂ ); 1.7 // 2.5 (each α-CH ₂ ); 6.1 -7.8 (aromatic protons); 12.1 (s, HO-C (5), lane). - 1 6

3.0 Morinperester

7: R = (CH ₂ ) ι ₀ CH ₃

8: R = (CH ₂ ) ₁₄ CH ₃

5.1 Morinpentalaurate (7)

C ₅ H _{1 20} O ₂ (1213.71); brownish, viscous oil, colorless after chromatography on silica gel with benzene / acetone 17: 3; Yield according to regulation A 90% UV: 232 (30'800), 252 (30 * 400), sh.ca. 295 (16 * 500).

IR: 2923 ss, 2853 s, 1766 s, 171 2 w, 1640 m, 1622 m (only slight differences to quercetin pentalaurate).

MS: 1236 (81%, [pentalaurate + Na] +; 1054 (100%), [tetralaurate + Na] +. NMR: 0.91 (., Ω -CH ₃ ); 1.28 / 1.30 (chain CH ₂ ); 1.64 /1.78/2.6 (α-CH ₂ ); 6.60-7.6 (aromatic protons); 12.14 (s, HO-C (5), traces).

5.2 Morin pentapalmitate (8)

C sHi ₆ oOι ₂ (1494.23); almost colorless crystals of benzene / 2-propanol; Yield according to regulation A 90%; M.p. 57-57, 5 ° C.

UV: 232 (24 * 200), 248 (23 * 500), 286 (14 * 400), sh. 304 (13 ^* 300). IR: 2921 ss, 2850 s, 1767 s, 1652 s, 1622 s (only slight differences from the corresponding quercetin derivative).

MS: 1517 (100%) [pentapalmitate + Na] +; 1279 (31%) tetrapalmitate + Na] +. NMR: 0.91 (t, ω-CHj); 1.30 (d, chain CH ₂ ); 1.66 / 1.78 / 2.6 (each m α-CH ₂ ); 6.6-7.6 (aromatic protons); 12.15 (s, HO-C (5), lane). Combustion analysis: Ber. C 76.35 H 10.79%

Found C 76.32 H 1 0.39%

R = ω

10: R = ψ 0)

CSJ— icπjt - -CH = CH

1 1 R = ω

CHj— (CH.) - -CH ₃

12 R = ω

Quercetinpentacaproat (9)

C ₄ 5H _S oOι ₂ (792.93); Yield according to regulation A 60%; recrystallized from 2-

Propanol, creamy white crystals, mp. 85-86 ° C.

UV: 231 nm (ε 23 * 900), 269 (37 * 600), 298 (21 * 000), 333 (1 1 * 600)

IR: 2962 ss, 2933 s, 2870 m, 1770 ss, 1657 s, 1616 ss.

MS_: 717 (100%), [tetracaproate + Na] +

NMR: 0.89 (t, ω -CH ₃ ); 1,352 and 1,714 (chain CH ₂ ); 2.52 (m, α-CH ₂ ); 6.53 (d. J

= 1.8, H-C (6)); 6.79 (d, J = 1.7, H-C (8)); 7.25 and 7.65 (m, H-C (2 ', 5', 6 ')); 1 2.1 (s,

HO-C (5), clearly).

Combustion analysis: Ber. C 68.16 calc. H 7.63%

Found C 68.04 Found H 7, 70%

6.2 quercetin pentaundecenoate (1 0)

C ₇ oHι ooOι ₂ (1133.50), yield according to regulation A 75%; recrystallized from 2-propanol, pale crystals, mp. 63-64 ° C. UV: 230 (22 * 500), 373 (35 * 690), 292 (18'800), 339 (12 * 200). IR: 2930 ss, 2856 m, 1769 ss, 1650 s, 1639 s, 1620 s.

MS: 1156 (100%), [pentaundecenoate + Na] ⁺ ; 990 (32%) [tetraundecenoate + Na] +

NMR (400 MHz): no ω-CH ₃ ; 1.33 / 1.75 / 2.04 / 2.56 / 2.73 (per m, CH ₂ groups); 4.98 (, 5ω-CH ₂ ); 5.81 (m, ψ-CH); 6.58 (d, J = 2.2, HC (6)); 6.84 ((/, J = 2.2, H- C (8)); 7.3 / 7.7 (each m, HC (2 ', 5 *, 6 *)); 12.12 (HO-C (5), track) Combustion analysis: calc. C 74.17 calc. H 8.89%

Found C 74.09 H 8.78% 6.3 quercetin pentalaurate (11)

C ₇ SH ₂ OOi 2 (1213.71); Yield according to regulation A 75%; cream white crystals of 2-propanol; Mp 84.5-86 ° C. UV: 233 (20 * 900), 270 (29 * 200), 300 (18 * 200). UR: 2927 ss, 2855 s, 1770 SS, 1654 s, 1616 s.

MS: 1236 (25%), [pentalaurate + Na] +; 1054 (100%) [tetralaurate + Na] ⁺ NMR: 0.93 (t, ω -CH ₃ ); 1,318 (see chain CH ₂ ); 2.6 (m α-CH2); 6.62 (d, J = 2.2, HC (6)); 6.89 (d, J = 1.4 HC (8)); 7.3 and 7.8 (each m, HC {2 ', 5', 6 ')); 12.16 (HO-C / 5), clearly).

Combustion analysis: Ber. C 74.21 calc. H 9.97%

Found C 74.08 Found H 10.1 3%

6.4 Quercetin pentapalmitate (12)

C_ ₅ H ₆ o ₂ (1494.23); Yield according to regulation A 80%; almost colorless crystals from 2-propanol; Mp 90.5-91.5 ° C. UV: 233 (20,800), 262 (23 ^* 800), 299 (19 * 900). eats. : 2929 ss, 2858 s, 1771 s, 1655 s, 1622 s.

MS: 1518 (100%), [pentapalmitate + Na] +, 1278 (80%, [tetrapalmitate + Na] + NMR: 0.90 (r, ω -CH ₃ ); 1.29 (see chain CH ₂ ); 2.6 (m α-CH ₂ ); 6.60 (d, J = 2.2, H-C (6)); 6.86 (d, J = 2.1 HC (8)); 7.3 and 7.7 (each m, HC ^ '.δ'.β')). Combustion analysis: Ber. C 76.35 calc. H 10.79%

Found C 76.38 Found H 10.86%

7.0 Genistein esters

13a: R1 = R2 = CO (CH ₂ ) 10CH ₃

13b: R2 = H, R = CO (CH ₂ ) i oCH ₃

14a: R1 = R2 = CO (CH ₂ ) i 4CH ₃

14b: R2 = H, R2 = C0 (CH ₂ ) 14CH ₃

7.1 Genistein dilaurate (13)

A trilaurate (13a) can be obtained under forced acylation conditions, but this proves to be so unstable during processing that a more or less complete transition into the 7,4'-dilaurate (1 3b) occurs.

C ₃₉ H ₅₄ O ₇ (634.82; yield 70% according to regulation A and recrystallization from 2-propanol; colorless crystals; mp. 96.5-97 ° C. UV: 232 (22 * 100), 252.5 (32 * 600) , 324 (4,800).] R: 2923 ss, 2855s, 1758s, 1650ss, 1620s.

MS (ESI): (839.6 [trilaurate + Na] +; 721.7 [trilaurate-118 + Na] +; 657.8 [dilaurate + Na] +

NMR: 0.81 (f, -CH _3); 1.20 (s, chain CH ₂ ); 6.5-7.9 (aromatic protons, including 7.89 (s, HC (2)); 1 2.7 (s, HO-C (5), combustion now: Calc. C 73.78 H 8.57%

Found C 73.52 H 8.21%

7.2 Genistein dipalmitate (14b)

C ₄₇ H ₇₀ O ₇ (747.03); Yield 85% according to regulation B; colorless crystals of 2-

Propanol; Sm p. 101 -102 ° C.

UV: 230 (15,600), 256 (36 * 000), 326 (5 * 300).

IR. (CH2CI2): 2928SS, 2855s, 1760s, 1652s, 1622m-s, 1 592m.

MS: (DCI, NH _3): m / z 746.6 (M + -1); 507.4 (M + palmitoyl; 85%); 269.1 (507.4-

Palmitoyl; 70%). .

NMR: 0.91 (_, -CH ₃ ); 1.30 (s, chain CH ₂ ); 6.6-8.0 (aromatic protons, including 7.99 (s, HC (2)); 12.8 (s, HO-C (5). Combustion analysis: calc. C 76.56 H 9.4%

Found C 75.60 H 9.1 6%

8.0 COMPOSITION EXAMPLES of spontaneously dispersible CONCENTRATES according to the invention which contain the active compounds of the formulas (I) to (IV) and which, when diluted with water or 5% glucose solution or physiological saline (Ringer's solution), give thermodynamically stable ULTRAMICROEMULSIONS with micelles which have a hydrodynamic radius of 2.2 to 3.0 nm.

a) 0.1 to 5% by weight of one or more esters of selected bioflavanoid compounds of the formulas (I) to (IV),

5 to 25% by weight isopropyl myristate, isopropyl palmitate, CHAPS, HEPES or neutral oil, e.g. Miglyol® 812 (Dynamit Nobel or Hüls), 0 to 45% by weight of a phosphoric acid ester surfactant, e.g. Diphasol® 3873 (CIBA-GEIGY), surfactant 508 (CIBA-GEIGY), Zerostat® AN or AT (CIBA-GEIGY), Tinovetin® JU (CIBA-GEIGY), Soprophor® FL (RHÖNE-POULENC), 5 to 90 weights % Invadin JFC 800% (CIBA-GEIGY) and / or TWEEN®-20 to TWEEN®-85 (ICI SPECIALTIES), and optionally 0 to 10% by weight of a vitamin or provitamin,

0 to 10% by weight of a penetration enhancer, radical scavenger or stabilizer.

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

0 to 5% by weight of CHAPS or HEPES,

5 to 25% by weight of one or more biosurfactant terpinyl esters of the general formula (VI):

2 3

R - COO - R _{(from left)} where R is a C ₂ . ₃ ι alkyl, a C ₃ . ₃ ι alkenyl or a C ₃ . _{31 is an} alcapolyen group and R ^{3 is} citronellyl, farnesyl, geranyl, isophytyl or phytyl,

35 to 45% by weight Invadin® JFC 800% and / or TWEEN®-20, -80 or -85,

35 to 45% by weight of Soprophor® FL or Diphasol® 3873.

N.B .: INVADIN® JFC 800% (CIBA-GEIGY) is an anhydrous tert. Octyl phenyl polyoxyethylene ether surfactant with 9 to 10 oxyethylene groups. SOPROPHOR FL (RHÖNE-POULENC) is a practically anhydrous tristyryl-polyoxyethylene-18-phosphoric acid ester TEA salt surfactant.

CHAPS (Fluka 26 * 680; Merck Index 11.2034) is a zwitterionic compound with the formula: 3 - [(3-cholamido-propyl) -dimethylammonio] propane sulfonate. HEPES is a good buffer: 4- (2-hydroxyethyl) piperazin-1-ethanesulfonic acid. TWEEN® 20 is a polyoxyethylene (20) sorbitan monolaurate surfactant (CAS No. 9005-64-5). [Polysorbate 20 in the CTFA Classification].

TWEEN® 80 is a polyoxyethylene (20) sorbitan monooleate surfactant (CAS No. 9005-65-6). [Polysorbate 80 in the CTFA Classification].

TWEEN® 85 is a polyoxyethylene (20) sorbitan trioleate surfactant [Polysorbate 85 in the CTFA Classification].

9.0 EXAMPLE for the pharmaceutical production of a system preparation with concentrates according to the invention in the form of "multiple units". a) 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 / form in a high-speed mixer or in a rotary bed with the addition of 1 10 g ethanol, break, sieve 18 to 42 mesh, dry for 24 hours at 40 ° C.

b) MSR and RETARD equipment in a rotary bed with AQOAT® AS-HG (Shin-Etsu Chemical) and talc

c) Composition of finished granules / or. Micropellets core material 44% MARIG ENOL® CONCENTRATE 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 effect of spontaneously dispersible concentrates according to composition examples a) and b) is confirmed on the basis of the following test results:

1.0 In vitro tests with suitable tumor cell lines

A biological assay system has been developed that works with microtiter plates and dilution series. Each 10 / ml tumor cells are inactivated in culture medium RPMI 1640 with 10% fetal calf serum (GIBCO); they are sown so leaky 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 Add 100 μl medium to the hole. Half of this is removed and placed in the following hole, again mixed with 100 μl of medium, etc. A geometric dilution series nV_ is formed.

The samples are incubated in the plaque assay for 3 to 5 days at 37 ° C. with 3V_% CO ₂ . 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 using scanning and absorption measurement on a spectrophotometer.

2.0 Testing for cell toxicity

2.1 Cell toxicity of MARIGENOL® CONCENTRATES tested on Py6 cells (Polyoma Virus transformed 3T3 mouse fibroblasts)

Py6 Cytotoxicity Test April 24-28, 1995

2% concentrates 24 h 48 h 72 h with QUERCETIN exposure exposure exposure PENTA-ESTERN concentrate concentrate concentrate

W.S. W.S. W.S.

(C ₆ : θ) ₅ "64 ^* 000 128 * 000 256 * 000

QUERCETIN 3.2 million 6.4 million 2.8 million

(Cn: l) 5 - 128 * 000 512 * 000 512 * 000

QUERCETIN 6.4 million 25.6 million 25.6 million

(Cn: l) 5 -

QUERCETIN 256 * 000 512O00 1O24O00 analytical grade 12.8 million 25.6 million 51.2 million

(Ci2; θ) 5 - 128 * 000 256 * 000 1 * 024 * 000

QUERCETIN 6.4 million 12.8 million 51.2 million

(Cl6: θ) δ ^■ 256O00 512O00 1 * 024 * 000

QUERCETIN 25.6 million 51.2 million

Largest cytotoxic dilution: on concentrate or W.S. -Calculated content.

For the eternalized Py6 cells see: "Biochemistry", Coordinating Editor Geoffrey L. Zubay, Addison-Wesley Publishing Company, 1983, p.1079. See also "Molecuiar 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 (WH Freeman & Co.) 2.2 Cell toxicity of MARIGENOL® CONCENTRATES / CONTINUED tested on Py6 cells (polyoma virus transformed 3T3 mouse fibroblasts)

Py6 cytotoxicity test July 1995

1% concentrates 24 h 48 h 72 h with FLAVONOID exposure exposure exposure

ESTERN M.E. 1: 100 M.E. 1: 100 M.E. 1: 100

W.S. W.S. W.S.

APIGENIN- 32O00 128 * 000 -

TRIPALMITAT 3.2 million - 12.8 million

CHRYSIN-128O00 512 * 000 1 million

DILAURAT 12.8 million 51 .2 million 100 million

CHRYSIN- 128 * 000 512O00 512O00

DIPALMITAT 12.8 million 51 .2 million 51.2 million

GENISTEIN- 32 * 000 256 * 000 -

DIPALMITAT 3.2 million 25.6 million

KÄMPFEROL- 32 * 000 128 * 000 -

TETRAPALMITAT 3.2 million 12.8 million

MORIN- 1 28 * 000 1 million 1 million

PENTALAURATE 12.8 million 100 million 100 million

MORIN- 64 * 000 512O00 51 2 ^* 000

PENTAPALMITAT 6.4 million 51 .2 million 51 .2 million

Largest cytotoxic dilution: calculated on microemulsion or WS content.

2.3 In Vitro Testing on Human Tumor Cell Lines (Dose Dependence)

VITALITY TEST QUERCETIN-PENTA-10-UNDECENOAT 1 x 104 cells per hole

Proliferation test (tritium: 1 μCi / well H ⁺ )

CELL LINE LC 89 U 937 THINNINGS cpm% cpm%

10-2 = 1 _: 100 470 16 788 0.4

10-3 = 1 _: V000 628 21 838 0.4

10-4 = 1 _: 1 0O00 10 * 064 347 79 * 515 40.7

10-5 = 1 _: 100 * 000 3'506 120 204'835 104

10-6 = 1 _: 1 'OOOOOO 3 * 465 119 197 * 321 101

10-7 = 1 _: 1 * 000 * 000 4 * 098 141 199'231 102

Controls: 2 * 906 cpm 195'1 25 cpm

LC 89: lung adenocarcinoma

U 937: Acute mieloid leukemia

Work carried out by Dottoressa Anna Rita GUARINI, Universita degli

Stud i di Torino, Clinica medica, June 1 to June 16, 1995.

IDENTIFICATION:% vitality after 48 hours with 2% MARIG ENOL® CONCENTRATES, added once to the medium as an aqueous ultra-microemulsion. When assessing, consider the relatively short exposure time of 48 hours. Dilution 1:10 = 10,000 ppm concentrate, 200 ppm active substance; Etc.

2.4 In vitro testing on human tumor cell lines (dose-response relationship)

VITALITY TEST / 1% MARIGENOL CONCENTRATES Erythrocyte LEUKEMIA K 562 1 x 104 cells per well

Proliferation test (tritium: 1 μCi / well H ⁺ )

PREPARATION 10-3 10-4 10- ■ 5

THINNING cpm% cpm% cpm%

MORIN- 448 0.8 1'083 2.1 14'818 29.6

PENTALAURATE

MORIN- 544 1.0 899 1 .7 23 * 666 47.2

PENTAPALMITATE

CHRYSIN- 498 0.9 601 1 .2 16 * 532 33.0

DILAURATE

CHRYSIN- 484 0.9 492 0.9 3 * 928 7.8

DIPALMITAT

QUERCETIN 392 0.7 536 1 .0 2 ^* 745 5.4

PENTALAURATE

Work performed by Dottoressa Anna Rita Guarini, Universita degli Studi d i Torino, Clinica medica, 21. until 8/24/1995. Controls 50 * 037 cpm. Test facility as in 2.3

IDENTIFICATION:% vitality after 48 hours with 1% MARIGENOL® CONCENTRATES, added once to the medium as an aqueous ultra-emulsion. When assessing, consider the relatively short exposure time of 48 hours. Dilution 10 ^{~ 3} = 1 * 000 ppm concentrate, 10 ppm active substance; Etc .

2.4 In vitro testing on human tumor cell lines (dose-response relationship)

VITALITY TEST / 1% MARIGENOL CONCENTRATE LEUKEMIA DAUDI LINE 1 x 10 ⁴ cells per hole

Proliferation test (tritium: 1 μCi / well H ⁺ )

s

PREPARATION 10 ^"3 10- ⁴ 10

THINNING cpm% cpm% cpm%

MORIN- 393 1.7 463 2.0 12'508 54.1

PENTALAURATE

MORIN- 374 1.6 632 2.7 15 * 234 65.9

PENTAPALMITATE

CHRYSIN- 271 1.1 574 2.4 10'286 44.5

DILAURATE

CHRYSIN- 401 1.7 624 2.7 9 '173 39.7

DIPALMITAT

QUERCETIN353 1 .5 384 1.6 9'541 41 .3

PENTALAURATE

Work performed by Dottoressa Anna Rita GUARINI, Universita degli Studi di Torino, Clinica medica, 21. until Aug 24, 1995. Controls 23O90 cpm. Test facility as in 2.3

3.0 Testing for MT4 cells infected with the AIDS VIRUS

Tests carried out by Prof. Dott. Alberto BIGLINO, Direttore deM'Ospedale Generale di Asti, am istituto delle Malattie Infettive, Universita degli Studi di Torino (Direttore: Prof. Dr. P. Gioan nini), Assistant: Dotta. Brunella FORNO and Dotta. Annamaria POLLONO. March to April 1995.

3.1 protective effect on the host cells (MT4 lymphocytes)

The HIV III B complex was used for 4 days in a concentration of 10 CCID ₅₀ / culture and well ("CCID ₅₀ / ml = 50% cell c_ulture mfective dose"). The best protective effect was achieved with a concentration of the tested MARIGENOL® concentrate of 10 ^'4 (= 100 ppm concentrate in aqueous microemulsion) of quercetin penta-10-undecenoate. 3.2 Direct antiviral effect or therapeutic effect against the so-called acquired immune deficiency, the human "Aq uired Immuno-Deficiency Syndrome: AIDS", or against pathologically active prions.

Use of the HIV I II B complex at an average concentration of 300 CCIDso / rnl. Incubation for 3 hours at 4 ° C in the presence of the antiviral or virucidal microemulsion. Then transfer to the MT4 cells. A clear, dose-dependent inhibitory effect can be demonstrated compared to the non-infected controls.

For the test methodology used, cf. et al : Rudi Pauwels, Erik De Clercq et ai .: Sensitive and rapid assay on MT4 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.

Cf. also: A. Bergamini, CF. Perna, et al. : A tetrazolium-based colorimetric assay for quantification of HIV-induced cytopathogenicity in monocyte-macrophages exposed to macrophage colony-stimuiating 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.

Molecuiar Cell Biology, second edition, by J. Darneil, H. Lodish, D. Baltimore; Scientific American Books, Chapter 5: Viruses, Structure and Functions, pp. 177-1888. New York, 1 990 (W.H. Freeman & Co.)

4.0 Other viruses

4.1 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 ^{' 4} and for ⁴ h at + 4 ° C with different concentrations of the test substance C _{1 6} op-sitosterylester 10 ^s , incubated and then inoculated as pretreated virus suspensions on cultures of human, embryonic lung fibroblasts. Approach as a confluent culture with 1 20 ^* 000 cells per shell vial. The infection was carried out by centrifugation at 1,500 rpm and at RT for 45 minutes, 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. would give; 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 sensitive host cells.

RESULT :

VIRUZIDE EFFECT OF ß-SITOSTERYL PALMITATE

(formulated as MARIGENOL® CONCENTRATE)

CHECKS: CONCENTRATION CONCENTRATION CONCENTRATION Number of anti-MICROEMULSION MICROEMULSION MI CROEMULSION gene (anti P-72) 1: 1'000 1: 10 * 000 1: 100 * 000

1 15 * 000 0 2 82 ^* 000

Number of "Nuclei positivi" per P-2

Exams conducted by Dottoressa Rossana CAVALLO, Istituto di Microbiologia, Università degli Stud i di Torino, Jul i-November 1 995. 4.2 Herpes virus (herpes simplex, HSV)

The antiviral effect of an aqueous microemulsion of suitable MARIGENOL® concentrates is determined using a confluent culture of VERO cells (i.e. "African green monkey kidney cells"). The host cells are pretreated with the microemulsion at 4 ° C. for 3 hours and then infected with a constant dose of 10 herpes simplex viruses, so-called "plaque forming units" = PFU. Duration of virus absorption 1 h. Then add 2% methyl cellulose to prevent the spread of the virus; an elastic fleece is created in the culture medium MEM + 2% fetal calf serum. Cultivation for 48 h, then fixing and coloring of the monolayers with 1% crystal violet in methanol.

The antiviral effect correlates with the number of "plaques" of lysed cells treated with a test substance which are still formed in the culture medium MEM + 2% FCS. The reduction in viral titer is given as a percentage of the controls (21 plaques per cell).

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

1% C _20: o-ß-sitosteryl ester

1% QUERCETIN-PENTA-10-UNDECENOAT

1% C _{1 6:} o-ß-SITOSTERYL-ESTER

1% C _{i 2: 0} -ERGOSTERYL-ESTER evaluation:

C ₂ o o-ß- QUERCETIN- Cι β: 0-ß - i _2: o-ER-

THINNING SITO-ESTER: PENTA-10 SITO-ESTER GO-ESTER:

(ARACHIDAT) UNDECENOAT (PALMITAT) (LAURAT)

1: 1 0 * 000 76% 48% 24% 62%

1: 100 * 000 5% 0% 0% 0%

The results show a clear direct effect on the HS virus

• 4 infected VERO host cells. At a concentration of 10 β-itosteryl arachidate, the number of plaques appears to be reduced by 76% in comparison with the controls; for the quercetin penta-1 0-undecenoate this value is 48%. The HSV titer is significantly reduced.

At this concentration of the tested substances, there is also no lysis, neither of eternalized lymphocytes of the MT4 type, nor of the VERO cells themselves. (MT4 lymphocytes are a human, leukemia-transformed T-line line). Repeat the test with fluorescence labeling as in the CMV test. See 4.1 above

VIRUZIDE EFFECT OF ß-SITOSTERYL-ARA CHIDA TES

(formulated as MARIGENOL®-KONZENTRA T)

CONTROLS: CONCENTRATION CONCENTRATION CONCENTRATION Number of MICROEMULSION MICROEMULSION MICROEMULSION infected cells 1: 1 * 000 1: 10 * 000 1: 100 * 000

38 0 0 20

Tests carried out by Dottoressa Rossana CAVALLO, Istituto di Microbiologia, University of Studi di Torino. July and November / December 1995.

4.3 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 Secrete DNA virus). Orientative experiments in vitro were carried out with a 1% concentrate of the three test substances ß-sitosteryl palmitate, ß-sitosteryl arachidate and ergosteryl isovalerate from Prof. Dott. Antonio Ponzetto and performed by Dotta Rossana Cavallo, University of Torino.

With a dilution of 10 ^' and incubation for 72 h, 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

Secreted antigens

PREPARATION Hbs Ag Hbe Ag

CHECKS in medium 1: 4 17.97 pos. 1 8,707 pos. Titer determination ß-SITOSTERYL-PALMITAT 1 .47 neg. 1 .777 neg. Ss-SITOSTERYL-ARACHIDAT 3.76 pos. 3,935 pos.

ERGOSTERYL ISOVALERATE 5.78 pos. 6.27 pos.

5.0 Analytical evidence

5.1 Identification of flavonoid ester active substance

Capillary zone electrophoresis using a P / ACE 2100 from Beckman Instruments or a BioFocus Integrator from BIO-RAD Laboratories. Conditions: buffer pH = 9.0 Na tetra borate; 50 mM SDS

Injection: 20 psi * sec. Run 15 kV, measurement at 192 nm The active substance peak appears after approx. 5 minutes. Very high resolution

5.2 Identification of the concentrate micelles in the aqueous microemulsion / or. in the cleaned and highly centrifuged supernatant of the cell plasma of the tumor cells.

Same methodology as 5.1.

The characteristic peak appears after about 5 minutes.

5.3 Detection of membrane penetration on the tumor cell

On the light microscope (as well as on the electron microscope) it can be shown that a few hours after the incubation (example Py6 cells = polyoma virus transformed 3T3 mouse fibroblasts; thinly sown, medium dilution of the active ingredient concentrates) there is a ring of vacuoles around the Cell nucleus forms around. As a marking substance, a small amount of Caπthaxanthin can be added to the concentrate, which has a clear fluorescence. Phenosafranin, Tinopal® GS or Uvitex® CF or EBF are also suitable. The analytical proof that these vacuoles contain the flavonoid esters as active substance is also carried out using micellar capillary zone electrophoresis "MECC" (Beckman Instruments, P / ACE System 2100 or the Bio Focus integrator from Bio-Rad). See also: Koji Otsuka et al. : "Separation of iipophile compounds by micellar electrokinetic chromatography with organic modifiers", Electrophoresis, 1994, 15, 1280-83 (VCH Verlagsgesel lschaft mbH, Weinheim).

6.0 General compatibility of MARIGENOL® preparations

EFFECT ON BLOOD

TOXICITY of MARIGENOL® CONCENTRATES on the BALB / c mouse

% Of blood cells

Preparation L M N E B

G 17 10-7 70 ± 6 1 1 + 3 13 ± 4 6 + 4 0 10-5 77 ± 6 6 ± 3 1 1 ± 4 5 + 4 1 ± 1 10-3 69 + 10 7 + 5 22 ± 8 2 ± 2 0

G 41 10-7 77 ± 6 6 ± 3 1 3 ± 5 3 ± 3 0 10-5 78 ± 4 10 ± 2 10 ± 4 1 + 1 1 ± 1 10-3 80 + 6 8 ± 2 10 ± 6 12 ± 1 0

G 44 10-7 74 ± 17 10 ± 1 20 ± 9 1 ± 1 0 10-5 74 ± 6 9 + 4 14 ± 7 4 ± 3 0 10-3 76 ± 5 6 ± 4 16 ± 8 2 ± 1 0

G 55 1 0-7 78 ± 4 10 ± 4 10 + 4 2 + 1 0 10-5 69 + 10 11 + 3 18 ± 4 1 ± 1 0 10-3 77 + 5 6 ± 4 14 + 2 2 + 1 1 ± 1

CHECKS 76 ± 5 8 ± 2 15 ± 4 1 ± 1 0 (physical buffer)

G 17 2% concentrate with C _{5 0} -CHOLESTERYL-ESTER

(Cholesteryl iso-valerate)

G 41 2% concentrate with C _{1 1: 1} -ERGOSTERYL-ESTER

(Ergosteryl 10 undecenoate)

G 44 2% concentrate with C _{1 8: 2} CHOLECALCIFERYL-ESTER

(C _{1 8: 2} -D ₃ ; vitamin D ₃ linolate)

G 55 2% concentrate with C _{4: 1} CHOLECALCIFERYL-ESTER

(C _{: 1} -D ₃ ; vitamin D ₃ -cronate)

Dilutions: 10-7 = 0.1 ppm concentrate; 0.002 ppm active substance

10-5 = 10 ppm concentrate; 0.200 ppm active substance 10-3 = 10000 ppm concentrate; 20,000 ppm active substance (calculated on the aqueous microemulsion)

Legend :

L = lymphocytes

M = monocytes (macrophages)

N = neutrophil granulocytes

E = eosinophilic granulocytes

B = basophilic granulocytes

Execution of the samples: Prof. Dott. G uido FORNI, Dotta Stefania VAI, Universita degl i Stud idi Torino, Dipartimento di Scienze Cliniche e Biologiche, Ospedale San Luigi Gonzaga, 1-10 ^* 043 ORBASSANO (TO), August / September 1993.

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 aqueous microemulsion, formed from the specified concentrates or with physiological buffer for the controls. Coloring with May Grünwald-Giemsa.

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. No toxicity of the concentrates to the leukocyte population could be determined. The erythrocyte population also showed normal values. All animals were and remained healthy until the end of the experiments. 6.1 EXAMINATION OF THE BLOOD PATTERN

Effect of MARIGENOLΘ CONCENTRATES on normal human lymphomonocytes

Cells: normal human phagocytic white blood cells

(Macrophages); 2 x 105 cells per well

Control stimulation: 1% PHA (phytohemagglutinin)

PREPARATION THINNING TEST 1 TEST 2 TEST 3 cpm cpm cpm

CHECKS 2682 3386 3386

PHA 1% (stimulated) 80 ^* 981 61 ^* 966 61 * 966

KONZEN10-1 1076 3826 3936

TRATE as 10-2 1396 3726 3845 aqueous 10-3 1431 2141 2939

MICROEMUL¬

SIONS 10-4 1386 3221 2752

10-5 1656 2888 3093

10-6 2536 2105 3046

Test 1 2% concentrate containing Cι _2: o-cholesteryl ester Test 2 2% concentrate containing Cι _6: o-ergosteryl ester Test 3 2% concentrate containing C _{ε: 0} -cholecalciferyl ester (vitamin D ₃ -iso-valerate )

Tests conducted by Dottoressa Anna Rita GUARINI, Università degli Studi di Torino, Clinica medica, 1-10 ^* 126 TORINO, March 21-24, 1994.

7.0 Antiparasitic effect

Swiss Tropical Institute, Basel In-vitro experiments, WHO screening as SOP

Parasite Strain Stage Standard Test N "

T.b.rhodesiense STIB 900 trypomastigotes Melarsoprol T961 1

L.donovani MHOM-ET- amastigotes Pentostam L9604 / 05 67 / L82

T.cruzi MHOM / Br / 00 / Y trypomastigotes benznidazole C0606 / 08

All values: μg / ml Legend * r = active on Tbrhodesiense d = active on L. donovani t = cytotoxic on mammaiian cells c = active on T. cruzi - = inactive or low activity

MIC = minimum inhibitory concentration ICβo = 50% killing rate concentration

T.b. rhodesiense T. cruzi L. cytotoxicity test donovani score *

TDR code L-6 Macroph r.d.c.

MIC IC.0 MIC ICβo MIC MIC t.-,

Standards 0.011 0.0004 3.7 30> 100> 90

EP1 100. 100 20> 100 90 d

EP2 100 - 100 20> 100 90 d

EP4 100 - 100 20> 100 90 d

EP5 100-100 <30 100 90 d

EP10 100 100 <30 100 90 d

EP17 100 - 100 20> 100 90 d

EP19 100 - 100 20> 100 90 d

4 N 33 17 100 20> 100> 90 d

7N 11 6.9 33 8 100 90 d

T1> 100 18.0 100 <30 100 90 d

T5 100 17 100 6 100 90 d

T6 100 17 100 <30 100 90 d

T7 100 16 100 <30 100 90 d

T9 100 16 100 <30 100 90 d

T10 100 16 100 <30 100 90 d

T11 100 18 100 <30 100 90 d

T12 100 20 <30 90 d

EP 1 1% concentrate CHOLESTANYL ISOVALERATE

EP 2 1% concentrate CHOLESTANYL-10-UNDECENOAT

EP 4 1% concentrate CHOLESTERYL-n-VALERATE

EP 5 1% concentrate CHOLESTERYL-LAURAT

EP 10 1% concentrate p-SITOSTERYL-BEHENAT

EP 17 1% concentrate VITAMIN Dj-UNDECENOAT

EP 19 1% concentrate VITAMIN Dj-OLEAT

4N 1% concentrate QUERCETIN-OCTALAUROYL-GLUCOSIDE

7N 1% concentrate Z-Gly-Phe-VITAMIN D ₃ -ESTER

T1 1% concentrate COEMULGATOR + TENSIDE

T5 1% concentrate 14-OH-10-DEACETYLBACCATIN-III T6 1% concentrate 3.28-LAUROYL-BACCATIN-ll l

T7 1% concentrate QUERCETINE PENTALAURATE

T9 1% concentrate DECALAUROYL RUTINOSIDE

T10 1% concentrate ß -SITOSTERYL-PALMITAT

T11 1% concentrate ß-SITOSTERYL ARACHIDATE

T12 1% concentrate ERGOSTERYL-LAURAT

The experiments were conducted under the direction of Prof. Dr. Ronald Kaminsky performed by Mrs. Yvonne Grether.

Result: A remarkable anti-parasitic effect was shown with selected cholestanyl and bioflavonoid ester concentrates, as well as with the Z-Gly-Phe-D ₃ ester concentrate. The tests were carried out at a concentration which does not cause basal cytotoxicity in murine muscle cells or in macrophages. The effect is specific against Leishmania donovan i.

Claims

PA TE N TA NS PRÜ CHE

1. Spontaneously dispersible concentrate, which with water, with 5%

Glucose solution or diluted with Ringer's solution gives thermodynamically stable ultramicroemulsions which have micelles with a hydrodynamic radius of 2.2 to 3.0 nm, characterized in that it is pharmaceutically usable and consists of the following components: 0.1 to 5% by weight of one or more esters of selected bioflavonoid compounds according to formulas (I) to (IV), or a combination of such active ingredients:

R = OH, OCH ₃ ,

R = OH, OCH3,

R = OH, OCH ₃ , where in the formulas (I) to (IV) R is hydroxyl, a C ₆ - ₃₁ alkyl, a C ₉ .31 -

Alkenyl or a Cg. ₃ i -Alkapolyengruppe stands,

5 to 25% by weight of a pharmaceutically acceptable serving as a co-emulsifier

Solubilizer or solvent mixture,

0 to 10% by weight of a vitamin or provitamin,

0 to 10% by weight of a stabilizer, radical scavenger or penetration enhancer.

2. Pharmaceutically usable combination preparation which consists of 90% by weight of the spontaneously dispersible concentrate according to claim 1 and 10% by weight of pharmaceutically compatible additives or carriers and / or solvents.

3. Spontaneously dispersible concentrate, containing one or more compounds of the formulas (I) to (IV) according to claim 1, as an agent for producing a pharmaceutical preparation of high bioavailability and with activity against tumors, eczema and psoriasis, and for the increased absorption of exogenous activators , metabolic modulators and regulators.

4. Spontaneously dispersible concentrate, containing one or more compounds of the formulas (I) to (IV) according to claim 1, as an agent for producing a pharmaceutical preparation with antiviral, virucidal or antiparasitic activity.

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

0.5 to 5% by weight of one or more esters of bioflavonoid compounds of the formulas (I) to (IV), 0 to 5% by weight of a good buffer,

5 to 25% by weight of isopropyl myristate, isopropyl palmate, neutral oil (neutral oleum), and / or one or more biosurfactant terpinyl esters of the general formula (VI): 2 3

R— COO— R _{(V |)} wherein R ^{2 is} a C ₆ - ₃ i alkyl, a C _β . ₃₁ alkenyl or a C ₉ . ₃ ι -Alkapolyen- group and R ^{3 is} 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 polyoxyethylene sorbitan monolaurate.

6. Spontaneously dispersible concentrate according to claim 1, characterized in that it comprises the following components:

0.5 to 5% by weight of one or more esters of bioflavonoid compounds of the formulas (I) to (IV),

R- COO- R ³ _{(V |)} where R ² is a C ₆ - ₃ i alkyl, a Cg. ₃ i alkenyl or a C ₉ . ₃ i -Alkapoyen- group and R ^{3 means} citronellyl, farnesyl, geranyl, isophytyl or phytyl, up to 10% by weight DL- tocopheryl 10-undecenoate or DL-α-tocopheryl laurate, bis to 25% by weight polyoxyethylene sorbitan monolaurate, up to 45% by weight of the anhydrous tert. Octyl phenyl polyoxyethylene ether

Surfactants with 9 to 10 oxyethylene groups,

35 to 45% by weight of an alkylphenol polyglycol ether phosphate surfactant or the practically anhydrous tristyrylphenol polyoxyethylene-18-phosphoric acid ester triethanolamine salt surfactant.

7. Therapeutic system preparation, which is made up to 90% by weight from the spontaneously dispersible concentrate according to one of claims 1, 2, 5 or 6, and at least 10% by weight from pharmaceutically acceptable carriers, additives and / or a solubilizer and that Available in unit dose form as micropellets, granules, dragees, suppositories, ampoules or capsules.

8. Therapeutic system preparation according to claim 7, which contains 64 parts of core material for the formation of granules or pellets and 36 parts of the spontaneously dispersible concentrate according to one of claims 1, 2, 5 or 6 contains and is filled in pharmaceutically suitable capsule from hydroxylpropyl methyl cellulose acetate succinate.

9. An ester of bioflavonoid compounds of the formulas (I) to (IV):

R = OH, OCH ₃ ,

(II)

R = OH,

R = OH, OCH3,

R = OH, OCH3, where in formulas (I) to (IV) R for a C ₆ . ₃₁ alkyl, a C ₉ . ₃₁ alkenyl or a C ₉ - is ₃ i -Alkapolyengruppe.

10. A process for the preparation of esters of bioflavonoid compounds of the formulas (I) to (IV) according to claim 9, characterized in that a compound of the formula (V):

R ¹ COOH _(V) wherein R ^{1 is} a C ₆ . ₃ ι -Al kyl-, a C ₉ . ₃ ι alkenyl or a C ₉ . ₃₁ -Alkapolyen group is reacted with a chlorinating agent and then the resulting product is esterified with a compound of the formulas (I) to (IV).

11. The use of a spontaneously dispersible concentrate according to one of claims 1, 2, 5 or 6 for the manufacture of a remedy with prophylactic and therapeutic activity against viral or parasitic diseases, against tumors, psoriasis and eczema and for the increased absorption of exogenous activators, metabolic modulators and Regulators.