CH689512A5 - New fatty acid esters of baccatin III compounds - Google Patents

Abstract

Baccatin III esters of formula (I) are new. R1 = H or R; R2 = H or acetyl; R3 = H or R; R4 = H, OH or OR; and R = 6-32C alkanoyl, alkenoyl or alkapolyenoyl or retinoyl, provided that at least one R group is present. Also claimed are spontaneously dispersible concentrates that can be diluted with water, and 5 % glucose solution of Ringer's solution to form thermodynamically stable ultramicroemulsions with a hydrodynamic micelle radius of 2.2-3.0 nm, comprising: (i) 0.1-10 wt.% of one or more (I) as above; (ii) 0-10 wt.% of one or more other drugs; (iii) 1-40 wt.% hydrotropic or coemulsifying solvent; (iv) 0.1-90 wt.% surfactant; (v) 0-10 wt.% vitamin or provitamin; (vi) 0-10 wt.% stabiliser, radical scavenger or penetration enhancer, and (vii) carriers and/or diluents.

Description

       

  
 


 introduction
 



  The present invention relates to esters of baccatin III, 10-deacetylbaccatin-III and 14-OH-10-deacetylbaccatin-III, production processes, their processing into spontaneously dispersible concentrates and aqueous ultramicroemulsions and their use for producing well-tolerated medicines with antitumoral and / or antiviral or virucidal 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, modulators and regulators.



  The esters of baccatin III, 10-deacetylbaccatin-III and 14-OH-10-deacetylbaccatin-III according to the invention have very good antitumor and antiviral or virucidal activity, and they are also suitable for the treatment of eczema and psoriasis. The therapeutic properties of these esters, however, only increase significantly if they are incorporated into spontaneously dispersible concentrates and then diluted with distilled water or 5% glucose solution or physiological saline (Ringer's solution), thermodynamically stable ultramicroemulsions with globular micelles forming, which have a hydrodynamic radius of 2.2 to 3.0 nm.


 Description of the invention
 



  The esters of baccatin III, 10-deacetylbaccatin-III and 14-hydroxy-10-deacetylbaccatin-III according to the invention have the general formulas (I) to (III):
EMI1.1
 
 
 



  (I) = baccatin III; R <1> = R <3> = H, R <2> = COCH3, R <4> = H
 (II) = 10-deacetylbaccatin-IIl; R <1> = R <2> = R <3> = R <4> = H
 (III) = 14-hydroxy-10-deacetylbaccatin-III; R <1> = R <2> = R <3> = H, R <4> = OH
 
 wherein in the formulas (I) and (II) mono- or diesters at C (7) and / or C (13) with C6-32-alkylcarbonyl, C6-32-alkenylcarbonyl or C6-32-alkapolyencarbonyl or retinoyl as ester components and in the formula (III) esters at C (7), C (13) and / or C (14) with these ester components are claimed.



  The compounds of the formula (I) in which R <1> and R <3> represent a C12-22-alkylcarbonyl, a C11-22-alkenylcarbonyl or a C11-22-alkapolyencarbonyl group.


 Examples of compounds of formula (I) include:
 
 



  7,13-diundecenoylbaccatin III
 7,13-dilauroylbaccatin III
 7,13-Dipalmitoylbaccatin-III
 7,13-distearoylbaccatin-III
 7,13-baccatin III di-oleic acid ester
 7,13-Baccatin III di-linoleic acid ester
 7,13-Baccatin-III-di-linolenic acid ester
 7,13-baccatin III di-arachidate
 7,13-Baccatin III di-behenate
 7-lauroylbaccatin III
 7-palmitoylbaccatin III


 Examples of compounds according to formulas (II) and (II) include:
 
 



  10-deacetyl-7,10-dilauroylbaccatin-III
 10-Deacety1-14 beta-hydroxybaccatin-7,10,14-trilaurate
 10-Deacety1-14 beta-hydroxybaccatin-7,10-dilaurate
 10-Deacety1-14 beta-hydroxybaccatin-10,14-dilaurate


 The fat tail principle and the formation of ultramicroemulsions in taxane esters
 



  More recently, several diterpenes with the unusual taxane structure have been isolated from representatives of the genera taxus, cephalotaxus and austrotaxus; see e.g. S. Blechert and D. Guénard in "The Alkaloids" (Ed. A. Brossi), Vol. 39, pp. 195-238, Academic Press, N.Y., 1990, and M. Suffness and G.A. Cordell, ibid., Vol. 25, 1985.



  Because of their sometimes excellent cancerostatic effects, taxane derivatives have attracted great attention. This patent relates to results from studies on the taxane derivatives baccatin-III and its hydroxylated modifications 10-deacetylbaccatin-III (10-DAS) and 10-deacetyl-14 beta-hydroxybaccatin-III (14-OH-10-DAB) .



  Baccatin-III was isolated for the first time from the heartwood of the yew (Taxus baccata, L.), characterized and structurally elucidated: W.R. Chan, T.G. Halsall, G.M. Hornby, A.W. Oxford, W. Sabel, K. Bjömer, G. Ferguson, J. Monteath Robertson, J.Chem.Soc., Chem.Comm. 1966, 923; D.P. Della Casa de Marcano, T.G. Halsall, G.M. Hornby, ibid. 1970, 216 and 1975, 365. From a biogenetic point of view, baccatin-111 is the direct and specific precursor of TAXOL [P.E. Fleming, A.R. Knaggs, X.-G. He, U. Mocek, H.G. Floss, J.Amer.Chem.Soc. 1994, 116, 4137].



  Taxol (paclitaxel) is considered a potent cytostatic in therapeutic use [cf. Antitumor Compounds of Natural Origin: Chemistry and Biochemistry, Vol. II; Ed. Adorjan Aszalos, NCI, CRC Press, Boca Raton, 1980, pp. 170, 175] and as an anti-tumor agent; it is classified as significantly more effective than baccatin III. It remains to be seen whether this can be attributed to the esterification of the hydroxyl group at C (13) by the substituted cinnamic acid or to other factors. Because of its relatively pronounced polarity, its high melting point and its very low solubility in water, baccatin-III presents considerable difficulties when formulating it into a pharmaceutical preparation for intravenous use, but this also applies to Taxol-W.S. applies. See also Blechert and Guénard, I.c., p. 232.

   Baccatin-Ill, like recently also 10-De acetylbaccatin-III, were used as starting material for numerous partial syntheses. See also: Kyriacos Costa Nicolaou et al .: "Chemistry and Biology of Taxol", Angew.Chem. 1994, 106, 38-69 and 1672-1675, as well as Lutz Heide: "Pharmaceutical biology in the spotlight", pharmacy in our time / 23. Year 1994 / No. 2 (pp. 100-104).



  10-Deacetylbaccatin-III was obtained from Taxus baccata needles (isolation and structure determination: G. Chauvière, D. Guénard, F. Picod, V. Sénilh, P. Potier, CR Séance, Acad.Sci.Sér. 2, 1981, 293, 561) and for 10-deacetyl-14-beta-hydroxybaccatin III, their occurrence in Taxus wallichiana, Zucc., Was used. See u.a. EPA 93 102 633.0 / 0 559 019 of September 8, 1993 (Indena SpA, Milano), as well as: E. Bombardelli and B. Gabetta, J. Chem.S., Perkin Trans. I (1992) (21), 2925-29 ; (1993) (14), 1563-6.



  The fact that the preparation of taxol and taxol analogs to spontaneously dispersible concentrates according to the invention and the production therefrom of aqueous ultramicroemulsions bring about decisive improvements in the bioavailability and accordingly the bioreactivity and relative toxicity was demonstrated in patent specification CH 688 504 of October 31, 1997. We have now found that the application of the fat tail principle mentioned above, i.e. the esterification of baccatin III, of 10-deacetylbaccatin-III and of 14-HO-10-deacetylbaccatin-III with long-chain fatty acids, and the subsequent incorporation into concentrates according to the invention also triggers a significant improvement in their kinetics and thus their pharmacological activity.

   Since these three compounds can be obtained from the needles of the yew (Taxus baccata, L., and Taxus brevifolia, Nutt.) And, moreover, the total synthesis is very complex, the use of a biologically quicker renewable basis offers itself as an advantage; the felling of Taxus trees to obtain TAXOL can therefore be avoided. The base substances extracted from Himalayan Yew were obtained from DABUR India Ltd., 22, Site IV, Sahibabad (U.P.), India.



  Baccatin III contains three structurally very differently reactive hydroxyl groups. In agreement with the literature we found that the hydroxyl group at C (7) is attacked much faster than that at C (13), which is surprising since the OH group at C (13) is allylic in nature; steric reasons and an intramolecular hydrogen bond are responsible for this behavior. The OH group at C (1) is not esterified under any known conditions because of its tertiary nature and simultaneous neopentyl position. The situation is different again with the hydroxylated baccatins: OH-C (10) reacts faster than OH-C (7); the OHC (14) again slower than that at C (10). It is therefore possible to make bonds at different points during esterification.



  If one wants to esterify both hydroxyl groups on C (7) and C (13) on baccatin III, conditions have to be selected under which the oxetane function C (4) -C (5) -C (20) could also be attacked at the same time. In order to avoid this, we have used the acid chlorides, which are more reactive towards the anhydrides, but by adding suitable bases, we have prevented them from attacking the oxetane function and, in some cases, intercepted the chloride ions formed with silver cyanide. In this way, the reaction temperature for the complete reaction can also be gradually increased, so that yields of pure products of up to 70% can be achieved with suitable workup.



  The esters produced are practically colorless and only vaguely fluorescent in the UV. However, the esterification reaction can be easily followed with TLC on silica gel foils by spraying with a cerium sulfate / phosphomolybdic acid / sulfuric acid reagent and then heating over the mirror burner: the ester sought appears as a dark sepia-colored spot. 10-deacetylbaccatin-III (10-DAB) contains four and 14-HO-10-deacetylbaccatin-III (14-HO-10-DAB) contains five, structurally very differently reactive hydroxyl groups; for the reactivity found, see the corresponding esterification approaches.



  The esters produced can be introduced very well into spontaneously dispersible concentrates according to the invention, which, with the addition of distilled water, 5% glucose solution or physiological saline solution, produce stable \ l-in-water microemulsions with globular micelles in the lowest nm range.


 1.0 Preparation of Esters of Compounds (I) to (III)
 


 1.1 Preparation of 7,13-Baccatin III diundecenoate.
 



  600 mg (excess) of 10-undecenoyl chloride in 40 ml of dichloromethane are added dropwise to a solution of 550 mg of baccatin III, 20 mg of 4-dimethylaminopyridine (DMAP) and 20 mg of N, N-dimethylformamide (DMF) in 25 ml of dichloromethane at room temperature. After stirring for 2 h, the solvent is distilled off and the residue is purified by chromatography on silica gel with cyclohexane / ethyl acetate (90:10) as the eluent. The 7,13-baccatin-III-diundecenoate with an Rf value in hexane / ethyl acetate 90:10 of 0.85 is obtained, ester bond at 1710 cm <-> <1> nu (C = O) esters


 1.2 Preparation of 7-lauroylbaccatin III.
 



  In a well-dried three-necked flask with an N2 inlet tube, magnetic stirrer, reflux condenser and dropping funnel, 250 mg of baccatin III are dissolved in 0.6 ml abs. Under N2 atmosphere. Dissolved pyridine, then mixed with a few crystals of 4-dimethylaminopyridine and diluted with 5 ml of 1,2-dichloroethane with the addition of 150 mg of finely powdered, dry AgCN. After cooling in an ice bath to 0 ° C., 0.2 ml of lauroyl chloride are added dropwise using a syringe. The cooling bath is then removed and the mixture is heated to about 45 ° C. in the oil bath for 18 h.



  For working up, the suspension is mixed with a little Celite ™, diluted with ether and the suspension is filtered. The filtrate is washed in a separating funnel with dilute sulfuric acid, then with water and brine. The product obtained after customary work-up, namely 500 mg of a pale yellow, honey-like \ les, is purified on a silica gel column (KieseIgel Merck 0.015-0.04 mm, column 2.3 x 20 cm) with the solvent mixture of ethyl acetate / toluene / acetone 11 : 9: 1) chromatographed. The laurate you're looking for appears fairly quickly in a narrowly defined zone. After crystallization from diisopropyl ether at -14 ° C., colorless needles of mp 87-88.5 ° C. were obtained; Yield 65-70%.



  HPLC almost uniform peak with tR 3.6 and lambda max 242 nm on Spherisorb S-5 CN, 4.6 x 250 mm, flow 0.8 ml / min. Solvent 1% B in A (A = hexane with 0.1% ethyldiisopropylamine; B = CH2Cl2 with 5% methanol).



  UV (CH2Cl2): lambda max 235 nm (epsilon 11 100), 277 (1500), 285 (1300).



  [alpha] DELTA -60.9 DEG (CHCl3, c = 0.041%; for comparison baccatin III = -85.5 DEG)



  IR (CHCI3; cm <-> <1>, selection of bands): 3620 and 3570s (OH): 3020, 2925, 2855m-s (methylene and methyl groups), 1733ss, broad [carbonyl groups]



  CI-Ms (NH3): m / z 786.4 (100%, [M + NH3] *)



   <1> 1/4 <-> 7/81 / 8p (CDCl3, 600 MHz, delta in ppm, interpretation aids <1> H, <1> H-DFQ-CO-SY, <1> <3> C, <1> H-HMBC, <1> <3> C, <1> H-HSQC, DEPT techniques):
 H-2: 5.62 (d J = 6.9); H-3: 3.99 (d J = 6.9); H-5: 4.96 (d J = 9.0); H-6: 1.55 and 2.6 (each m); H-7: 5.6 (dd); H-10: 6.28 (s); H-13: 4.84 (t, J = 7.8); H-14: 2.3 and 2.6 (each m); CH3 (16): 1.13 * (s); CH3 (17): 1.07 * (s); CH3 (18): 2.10 (s); CH3 (19): 1.79 (s) CH2-20: 4.43 (AB system); CH3CO-4: 2.28 (s).



   <1> <3> D <-> 7/81 / 8p (selection of signals): C-4: 80.6; C-5: 84.0; C-7: 71.3; C-10: 75.8; C-11: 131.5; C-12: 144.8; C-13: 67.7; ArylCO: 166.9; LauroylCO-7: 173.0; CH3CO-4: 170.5; CH3CO-10: 168.8; CO-9: 202.4.



  Other fatty acid esters of baccatin-III, 10-deacetyl-baccatin-III and 14-OH-10-deacetyl-baccatin-III are also produced in a comparable manner.


 1.3 Preparation of 7-palmitoylbaccatin-III
 



  The reaction of baccatin III with palmitoyl chloride was carried out as in 1.3 above, but with the reaction time being doubled; the cleaning was carried out analogously. After recrystallization from diisopropyl ether / pentane, silky needles with double impulses are obtained. 77-80 / 90-92 DEG C.



  UV (CH2Cl2): lambda max 237 nm (epsilon 19 800)



  Circular dichroism (CH2Cl2, RT): 230.8 (DELTA epsilon 27.0), 258.6 (0), 300.2 (-12.4).



  IR (CH2Cl2: 3628w and 3570w (OH); 3049m, 2926s, 2852m (methyl and methylene groups), 1736ss, and 1725ss [carbonyls]



   <1> 1/4 <-> 7/81 / 8p (CDCl3, ARX 300): H-2: 5.63 (d J = 6.7); H-3: 4.00 (d J = 6.9); H-5: 4.97 (dd); H-6: 1.57 and 2.6 (each m); H-7: 5.61 (dd, partially covered); H-10: 6.29s; H-13: 4.89 (t J = 7.8); H-14: 2.2 and 2.3 (each m); CH3 (16): 1.14 * (s); CH3 (17): 1.12 * (s); CH3 (18): 2.11 (d, J = 1.0); CH3 (19): 1.80 (S); CH2-20: 4.23 (AB system); CH3CO-4: 2.28 (s).



   <1> <3> D <-> 7/81 / 8p C-5: 84.1; C-7: 71.3; C-11: 131.6; C-12: 144.7; C-13: 67.9; C-14: 38.6; ArylCO: 167.0; LauroylCO-7: 173.1; CH3CO-4: 170.6; CH3CO-10: 168.8; CO-9: 202.4.


 1.4 Preparation of 10-deacetyl-7,10-dilauroylbaccatin-III.
 



  500 mg of 10-deacetylbaccatin-111 were reacted with lauroyl chloride as in Example 1.2 and the crude product obtained was purified by column chromatography. Crystallization from diisopropyl ether at -14 ° C. gave 660 mg as a fine, white powder, mp. 106.5-108 ° C.



  UV (CH2Cl2): lambda max 243.5 nm (epsilon 15 400)



  IR (CH2Cl2: 3677w and 3608w (OH); 3006m, 2925s, 2352m (methyl and methylene groups), 1713s, broad [carbonyls]



  CI-MS (NH3): m / z 926.6 (47%, [M + NH3] <+>); 909.5 (15% [M <+>]; 804.5 (30%, [M + NH3] <+> - H2O; 709.3 (100% [M <+> - lauric acid], etc.



   <1> 1/4 <-> 7/81 / 8p (CDCl3, 300 MHz): H-2: 5.62 (d J = 7.11); H-3: 3.89 (d J = 7.1); H-5: 4.99 (d with fine splitting, J = 7.6); H-6: 1.7 and 2.5 (each m); H-7: 4.48 (dd, J = 10.9 / 6.9); H-10: 6.32 (s); H-13: 4.89 (t, J = 7.5); H-14: 2.3 and?, Covered (m); CH3 (16.17): 1.11 (s); CH3 (18): 2.05 (d, J = 1.0); CH3 (19): 1.67 (s); CH2-20: 4.23 (AB system).



   <1> <3> D <-> 7/81 / 8p (CDCl3): C-1: 79.1; C-4: 80.8; C-7: 72.3; C-10: 76.4; C-11: 131.9; C12: 146.3; C-13: 67.9; C-14: 38.6; ArylCO: 167.0; CH3CO-4-. 170.7; LaurylCO-7: 174.1; LaurylCO-10: 179.0.


 1.5 Esterification of 10-deacetyl-14 beta-hydroxybaccatin-III with lauroyl chloride.
 



  738 mg of the formula (III) were reacted with 1.3 ml of lauroyl chloride as indicated above. Column chromatography on silica gel gave 3 main fractions with Rf 0.90; 0.53; 0.30. (Mobile phase on Merck silica gel foils with ethyl acetate / toluene / acetone 11: 9: 1). The non-polar compound turned out to be 10-deacetyl-14 beta-hydroxybaccatin-7,10,14-trilaurate, the polar one as 10-deacetyl-14 beta-hydroxybaccatin-III-7,10-dilaurate; after comparison of the spectra, the medium-polar compound could be determined as the 10-deacetyl-14 beta-hydroxy-10,14-dilaurate.


 Properties of 14-OH-10-DAB-trilaurate:
 



  Yield 0.1 g, colorless crystals from methanol / trace diisopropyl ether at -12 ° C; Mp 87-92 DEG C.



  UV (CH2Cl2): lambda max 234 nm (epsilon 18 100)



  Circular dichroism (CH2Cl2, RT): 229.4 (DELTA epsilon 19.2), 257.6 (0), 299.6 (-7.9)



  IR 3575w OH); 2043w-m, 2853s (methylene and methyl groups), 1733ss, broad [carbonyls]



  CI-MS (with NH3): m / z 1124.5 (60%, [M + NH3] <+>); 1107.5 (23%, [M <+>]; 1089.5 (M <+> - 18]); 924.5 (100%, [M + NH3] <+> - lauric acid]); 907.4 (66% [M <+>] - lauric acid).



   <1> 1/4 <-> 7/81 / 8p (CDCl3, 600 MHz): H-2: 5.75 (d J = 7.3); H-3: 3.95 (d J = 7.1); H-5: 4.90 (dd); H-6: 1.6 and 2.5 (each m); H-7: 5.56 (dd); H-10: 6.29 (s); H-13: 4.62 (broad s), 2.1 and 2.3 (each m); CH3 (16): 0.98 *; CH3 (17): 1.12 *; CH3 (18): 2.08; CH3 (19): 1.75 (s); CH2-20: 4.18 (AB system); CH3CO-4: 2.31 (s).



   <1> <3> D <-> 7/81 / 8p (CDCl3): C-4: 80.5; C-5: 83.9; C-7: 71.1; C-10: 75.2; C-11: 133.1; C-12. 141.1; C-13: 76.7; C-14: 75.2; ArylCO: 165.7; CH3CO-4: 170.8; CO-9: 201.8; LaurylCO-7: 172.8; LaurylCO-10: 171.5; LauroylCO-14: 173.9.


 Properties of 7,10-dilaurate:
 



  Yield 1.02 g; colorless, very viscous \ l that slowly solidifies to a solid mass.



  UV (CH2Cl2): lambda max 233 nm (epsilon 13 200)



  IR 3600w and 3186w, broad (OH); 3043m, 2922ss (methylene and methyl groups), 1733ss and 1710s [carbonyls]



   <1> 1/4 <-> 7/81 / 8p (CDCl3, 600 MHz): H-2: 5.81 (d J = 7.2); H-3: 3.95 (d J = 7.1); H-5: 4.96 (broadened d, J = 8.3); H-6: 1.63 and 2.41 (each m); H-7: 5.58 (dd); H-10: 6.32 (s); H-13: 4.79 (d with fine splitting, J = 6.3); H-14: 4.04 (d, J = 6.4); CH3 (16): 1.18 * (s); CH3 (17): 1.07 * (s); CH3 (18): 2.10 (d, J = 1.1); CH3 (19): 1.81 (s); CH2-20: 4.25 (AB system); CH3CO-4: 2.31 (s).



   <1> <3> D <-> 7/81 / 8p (CDCl3): C-1: 76.6; C-2. 74.0: C-3: 46.4; C-4: 80.7; C-5: 83.9; C-7: 71.2; C-10: 75.3; C-11: 132.7; C-12: 146.9; C-13: 75.9; C-14: 74.0; C-15: 42.8; CH3 (16): 21.9 *; CH3 (17): 26.1 *; CH3 (18): 14.9: CH3 (19): 10.8; CH2-20: 76.2; ArylCO: 166.2; CH3CO-4: 170.5; LaurylCO-7: 173.2; LaurylCO-10: 173.2.


 Properties of baccatin III 10,14 dilaurate,
 



  Yield 60 mg; colorless crystals of diisopropyl ether at -14 ° C; M.p. 93-103 DEG C.



  UV (CH2Cl2): lambda max 234 nm (epsilon 15 800).


 2.0 Importance of Appropriate Solubilization of the Esters
 



  The esters of selected baccatin compounds of the formulas (I) to (III) surprisingly have an excellent antitumor and antiviral or virucidal activity and are also suitable for the treatment of eczema, psoriasis and metabolic disorders, especially when they are in accordance with the invention , spontaneously dispersible concentrates have been incorporated which, diluted with distilled water, 5% glucose solution or Ringer's solution, result in thermodynamically stable \ l-in-water ultramicro emulsions.



  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 the ETH, Zurich, Institute of Polymers (Prof. Dr. Pier Luigi LUISI and Prof. Dr. Peter SCHURTENBERGER). See "Mode-selective dynamic light scattering; theory versus experimental realization ", Thomas Gisler, et al., Applied Optics / Vol. 34, No. 181 20 June 1995, pp. 3546-3553.



  The present invention accordingly also relates to spontaneously dispersible concentrates with antitumoral and / or antiviral or virucidal esters of selected baccatin compounds of the formulas (I) to (III). The esters according to the invention with baccatin compounds of the formulas (I) to (III) are almost water-insoluble and highly agglomerated compounds. However, in order that such compounds diffuse through the membrane barrier of the tumor or host cells and become effective inside the cell, they first have to be solubilized in a suitable manner in the aqueous medium. By forming thermodynamically stable \ l-in-water ultramicroemulsions with the help of specially selected cosurfactants 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 new esters.



  All experimental observations on ultramicroemulsions designed in this way can be interpreted uniformly by the assumption that the selected surfactants and cosurfactants, taken as a balanced system, form organized aggregates, so-called micelles, in the aqueous phase. These have a more or less spherical shape, with a hydrodynamic radius of 2.2 to 3.0 nm. The surfactants and hydrotropes (cosurfactants) leave between the outer, aqueous phase and the inner, oily phase of the microemulsion [containing the esters of the formulas (I) to (III), dissolved in the biosensitive solubilizer], an interface is formed, which means that these two phases are not mixed. In the oily inner phase, i.e. In the micellar core, the molecules of the esters true to the invention are in monomeric or in oligomeric agglomerated form.



  The micelles of the inner phase containing baccatin ester 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 into the interior of the tumor or host cell, or through the protein envelope of viruses. The diffusion through the outer membrane of such cells takes place exclusively due to thermal molecular movements.



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

   Your speed or the strength of the drug transport through the membrane of the tumor or host cell is determined:
 
   1. the difference in concentration in the two compartments
   2. of the particle radius of the diffusing drug molecule or drug system
   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 of 12.564 cm <2>. In contrast, 10 <1> <8> Micelles with a hydrodynamic radius of only 10 <-> <6> cm (10 nm), which together have the same volume of 4.189 cm <3> make up, already a total phase surface of 1256.4 m <2> on.


 Micelles: ratio of volume to total surface
 
 <tb> <TABLE> Columns = 4
 <tb> Head Col 1: number of micelles
 <tb> Head Col 2: More hydrodynamic
 Radius of the micelles
 <tb> Head Col 3: volume
 of the micelles
 <tb> Head Col 4:

   Total surface
 of the micelles
 <tb> <SEP> 1 <SEP> 1 cm <SEP> 4.189 cm <3> <SEP> 12.564 cm <2>
 <tb> <SEP> 10 <3> <SEP> 0.1 cm = 1 mm <CEL AL = L> 4.189 cm <3> <SEP> 125.64 cm <2>
 <tb> <SEP> 10 <6> <SEP> 0.01 cm <SEP> 4.189 cm <3> <SEP> 1256.4 cm <2>
 <tb> <SEP> 10 <9> <CEL AL = L> 0.001 cm <SEP> 4.189 cm <3> <SEP> 12 564 cm <2>
 <tb> <SEP> 10 <1> <2> <SEP> 0.001 cm = 1 mm = 1000 nm <SEP> 4.189 cm <3> <CEL AL = L> 125 640 cm <2>
 <tb> <SEP> 10 <1> <5> <SEP> 0.00001 cm = 100 nm <SEP> 4.189 cm <3> <SEP> 1 256 400 cm <2>
 <tb> <SEP> 10 <1> <8> <CEL AL = L> 10 <-> <6> cm = 10 nm <SEP> 4.189 cm <3> <SEP> 1256.4 m <2>
 <tb> <SEP> 10 <2> <1> <SEP> 10 <-> <6> mm = 1 nm <SEP> 4.189 cm <3> <CEL AL = L> 12 564 m <2>
 <tb> </TABLE>
EMI11.1
 
 



  Conclusion: Due to the 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 or "spreading") and thus the bioavailability and bioactivity of the active ingredients, which The core of the micelles is present in monomeric or oligomeric agglomerated form, also greatly improved. This can allow a significant reduction in the 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 exponentially 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.


 3.0 Marigenol TM 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 (III), or combinations of such esters,
 and 0 to 10% by weight of individual or several active pharmaceutical ingredients, as described below,
 1 to 26% by weight of a pharmaceutically compatible solvent or solvent mixture serving as 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, a radical scavenger or a penetration enhancer and carriers and / or diluents.



  In the present case, pharmaceutical active ingredients are to be understood as all active ingredients that can normally be used in human medicine. These include, for example:


 Beta blocker
 
 



  Pindolol [1- (4-indolyloxy) -3-isopropylamino-2-propanol]
 Propanolol [1-isopropylamino-3- (1-naphthyloxy) -2-propanol]
 Oxprenofol [1- (o-allyloxyphenoxy) -3-isopropylamino-2-propanol]
 Metoprolol (di- (+ -) - 1- (isopropylamino) -3- [p- 2-methoxyethyl) -phenoxy-2-propanol] -L (+) tartrateÜ]
 Labetalol [5- [1-Hydroxy-2 (1-methyl-3-phenylpropylaminoethylUsalicylamide]


 Diuretic
 
 



  Acetazolamide [5-acetamido-1,3,4-thiadiazole-2-sulfonamide]
 Hydrochlorothiazide [6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide]
 Chlorotalidone [1-oxo-3- (3-sulfamyl-4-chlorophenyl) -3-hydroxy-isoindoline]
 Metolazone [7-chloro-1,2,3,4-tetrahydro-2-methyl-4-oxo-3-o-tolyl-6-quinazoline sulfonamide]


 Weak sedative
 
 



  Diazepam [7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one]
 Medazepan [7-chloro-2,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one]


 Strong sedative
 



  Sulpiride [N- (1-ethyl-2-pyrrolidinyl-methyl) -2-methoxy-5-sulfamoyl-benzamide]


 Muscle relaxant
 
 



  Baclofen [beta - (aminoaethyl) -p-chlorohydrocinnamic acid)


 Antibiotics
 
 



  Sulfamethoxazole [5-methyl-3-sulfanifamido-isoxazole]
 Trimethoprim [2,4-diamino-5- (3,4,5-trimethoxybenzyl) pyrimidine]
 Chloramphenicol [D (-) - threo-2-dichloroacetamido-1- (4-nitrophenyl) -1,3-propanediol]
 Cefaclor [3-chloro-7-D (2-phenylglycine amido) cephalosporanic acid monohydrate]
 Cefradin [7- D-2-amino-2- (1,4-cyclohexadien-1-yl) acetamido) -3-methyl-cephalosporanic acid]
 Bacampicillin [1-ethoxycarbonyloxy-ethyl-6- (D-alpha-aminophenyl-acetate-amido) -penicillinate]
 Minocycline [7-diethylamino-6-deoxy-6-desmethyltetracycline]
 Sulfadoxin [N min - (5,6-Dimethoxy-4-pyrimidinyl) sulfanilamide]
 Sulfamethoxazole [3-methyl-3-sulfanil-amido-isoxazole]
 Sulfisoxazole [3,4-dimethyl-5-sulfanil-amido-isoxazole]
 Sulfadimethoxin [2,4-dimethoxy-6-sulfanilamido-1,3, diazine]


 Dermatologica
 
 



  Chloroquinaldol [5,7-dichloro-8-hydroxyquininaldine]
 Crotamiton [N-crotonyl-N-ethyl-o-toluidine]
 Diamthazole [6 (-) 2-dimethylamino-ethoxy- (beta -diaethylamino -) - benzothiazole-dihydrochloride]
 Flumethasone pivalate [6 alpha, 9-difluoro-11 beta, 17.21-trihydroxy-16 alpha-methyl-pregna-1,4-diene-3.20, dione-21-pivalate]
 Tretinoin [vitamin A acid]


 Corticosteroids
 
 



  Cortisone [17 alpha-21, dihydroxy-pregn-4-en-3,11,20-triene-21-acetate]
 Prednisone [11 beta -17,21-trihydroxy-pregna-1,4-diene-3,20-dione]
 Dexamethasone [9-fluoro-11 beta, 17,21-dihydroxy-16 alpha-methyl-pregna-1,4-diene-3,20-dione]
 Deoxycortone acetate [21-hydroxy-pregn-4-en-3,20-dione-acetate]


 Endocrine regulation
 
 



  Melatonin [N-2- (5-methoxy-1H-indol-3yl) ethyl-acetamide]; N-acetyl-5-methoxytryptamine


 Coronary
 
 

 

  Pentaerythrityl tetranitrate (PETN)
 Nitroglycerin (glyceryl trinitrate)
 Pindolol [1- (4-indolyloxy-3-isopropylamino-2-propanol]


 Cytostatica
 
 



  Melphalan [p-di- (2-chloroethyl) amino-L-phenylalanine]
 Procarbazin [p- (N min -methyl-hydrazinomethyl) -N-isopropyl-benzamide]
 Dactinomycin [actinomycin D]
 Polyestradiol phosphate
 Cyclophosphamide [N, N-bis (beta-chloroethyl) amino-1-oxa-3-aza-2-phosphocyclohexane-2-oxide]


 Anti-inflammatory agents
 
 



  Mefenamic acid [3-xylyl-2-aminobenzoic acid]
 Dexamethasone [9-fluoro-11 beta, 17,21-trihydroxy-16 alpha-methyl-pregna-1,4-diene-3,20-dione]
 Hydrocortisone [17 alpha-hydrocorticosterone]


 Coronary dilators
 
 



  Nifedipine [1,4-dihydro-2,6-dimethyl-4- (o-nitrophenyl) pyridine-3,5-dicarboxylic acid dimethyl ester]
 Isosorbide dinitrate [1,4; 3,6-dianhydrosorbitol-2,5-dinitrate]
 Nitroglycerin (glyceryl trinitrate)
 Dipyramidol [2,6-bis- (diethanolamino) -4,8-dipiperidino (5,4-dipyrimidine)]


 Peripheral vasodilators
 
 



  Cinepazid [4 - (- 3,4,5-Trimethoxy-cinnamoyl) -1-piperazine-acetic acid pyrrolidide]
 Cyclandelate [3,3,6-trimethyl-cyclohexyl almondate]
 Cinnazarin [1-trans-cinnamyl-4-diphenylmethyl-piperazine]
 Pentoxyfylline [3,7-dimethyl-1- (5-oxo-hexyl) xanthine]


 Antirythmic
 
 



  Procainamide [4-aminobenzoic acid-beta-diethylaminoethylamide]
 Disopyramide [4-diisopropylamino-2-phenyl-2- (2-pyridyl) -butyramide]


 Anti-gout agent
 
 



  Allopurinol [1H-pyrazolo- (3,4-d) pyrimidin-4-ol]


 Antiepileptics
 
 



  Phenytoin DiphenylhydantoinÜ; (5,5-diphenyl-2,4-imidazolidinedione)
 Carbamazepine [5-carbamoyl-5H-dibenz (b, f) azepine]


 Antihistamines
 
 



  Chlorophenamine [3- (p-chlorophenyl) -3- (2-pyridyl) propyldimethylamine]
 Clemastine hydrogen fumarate; [1-methyl-2- 2- (alpha-methyl-p-chloro-diphenylmethoxy) ethyl-pyrrolidine]
 Mequitazine [10- (3-quinuclidinylmethyl) phenothiazine]
 Alimemazine [10- (3-dimethylamino-2-methylpropyl) phenothiazine]


 Anti-nausea and dizziness medication
 
 



  Domperidone [5-chloro-1- 1- (3- (2-oxo-1-benzimidazolinyl] propyl) -4-piperidylÜ2-benzimidazolinone]
 Betahistine [2- 2-methylaminoethylpyridine]
 Metoclopramide [4-amino-5-chloro-N- (2-diethylaminoethyl) -2-methoxy-benzamide]


 Antihypertensive agents
 
 



  Reserpine [3,4,5-trimethoxybenzoyl-methylreserpat]
 Rescinnamine [3,4,5-trimethoxy-methylreserpat]
 Methyldopa L-alpha-methyldopaÜ; [L-3- (3,4-dihydroxyphenyl) -2-methylalanine]
 Clomidine hydrochloride [2,6-dichloro-N-2-imidazoidinylidene-benzamine hydrochloride]


 Sympathomimetica
 
 



  Isoproterenol [N-isopropyl-nor-adrenaline]
 Etilefrin [DL-1-alpha-ethylaminomethylÜ-m-hydroxy-benzyl alcohol]


 Expectorants
 
 



  Carbocysteine [(S-carboxymethyl) cysteine]
 Bromhexine [N-Cyclohexyl-N-methyl- (2-amino-3,5-dibromo-benzyl) amine HCL]
 L-ethylcysteine
 L-methylcysteine


 Oral antidiabetics
 
 



  Glibenclamide [N-4-2- (5-chloro-2-methoxy-benzamido) -ethyl-phenylsulfonyl-N min -cyclohexyl-urea]
 Tolbutamide [N- (4-tolylsulfonyl) -N min -n-butylurea]


 Cardiovascular agents
 
 



  Ubidecarenone
 Adenosine [6-amino-9-beta-D-ribo-furanosyl-9H-purine]


 Immunosuppressant
 
 



  Cyclosporin A


 Antitumor agent
 
 



  Taxol (Paclitaxel)
 Baccatin III
 10-deacetylbaccatin III
 14-OH-10-deacetylbaccatin-III
 Cyclosporin D-oxide


 Antivirals (AIDS)
 
 



  4-isoleucine cyclosporine
 Cyclosporin G
 3 beta-stigmast-5-en-3-laurate (beta-sitosteryl laurate)
 3 beta-stigmast-5-en-3-palmitate (beta-sitosteryl palmitate)
 3 beta-stigmast-5-en-3-stearate (beta-sitosteryl stearate)
 3 beta-stigmast-5-en-3-arachidate (beta-sitosteryl arachidate)
 3 beta-stigmast-6-en-3-behenate (beta-sitosteryl behenate)
 3 beta -stigmast-5-en-3-10-undecenoate (beta-sitosteryl-10-undecenoate)
 3 beta-stigmast-5-en-3-oleate (beta-sitosteryl oleate)
 3 beta -stigmast-5-en-3-all trans-retinate (beta-sitosteryl-all-trans-retinate).
 (3 beta, 22E) -Ergosta-5,7,22-trien-3-yl-valeric acid ester [ergosteryl-n-valerate, provitamin D2-n-valerate]
 (3 beta, 22E) -Ergosta-5,7,22-trien-3-yl-10-undecenoic acid ester [Ergosteryl-10-undecenoate, provitamin D2-undecenoate]
 (3 beta, 22E) -Ergosta-5,7,22-trien-3-yl-lauric acid ester [ergosteryl laurate,

   Provitamin D2-laurat]
 (3 beta, 22E) -Ergosta-5,7,22-trien-3-yl-all-trans-retinoic acid ester [ergosteryl retinate, provitamin D2 retinate]
 9,10-secoergosta-5,7,10, (19), 22-tetra-en-3-yl-valeric acid ester [ergocalciferyl-n-valerate, vitamin D2-n-valerate]
 9,10-Secoergosta-5,7,10, (19), 22-tetra-en-3-yl-10-undecenoic acid ester [Ergocalciferyl-10-undecenoate, vitamin D2-undecenoate]
 9,10-secoergosta-5,7,10, (19), 22-tetra-en-3-yl lauric acid ester [ergocalciferyl laurate, vitamin D2 laurate]
 9,10-Secoergosta-5,7,10, (19), 22-tetra-en-3-yl-all trans-retinoic acid ester [ergocalciferyl retinate, vitamin D2 retinate]
 9,10-secocholesta-5,7,10, (19), - trien-3-yl-valeric acid ester [cholecalciferyl-n-valerate, vitamin D3-n-valerate]
 9,10-secocholesta-5,7,10, (19), - trien-3-yl-10-undecenoate [cholecalciferyl undecenoate, vitamin D3 undecenoate]
 9,10-secocholesta-5,7,10, (19),

  -trien-3-yl lauric acid ester [cholecalciferyl laurate, vitamin D3 laurate]
 9,10-Secocholesta-5,7,10, (19), - trien-3-yl-all trans-retinoic acid ester (cholecalciferyl retinate, vitamin D3 retinate]
 Morin tri undecanoate
 Morin Tri Laurat
 Quercetin penta undecenoate
 Quercetin penta laurate



  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 and 6 on the one hand and 10 and 15 on the other. HLB values provide information about the hydrophilic and lipophilic properties of an emulsifier. See "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 acid ester surfactants, such as: the tristyrylphenotpolyoxyethylene-18-phosphoric acid ester TEA salt (triethanolamine salt) with the formula:
EMI17.1
 
 



  (Soprophor ™ FL, Rhone-Poulence);



  Diphasol TM 3873 (CIBA-GEIGY), or the identical Sermul TM EA 188 (SERVO), a mixed emulsifier, each consisting of 50% of the two compounds with the formulas:
EMI18.1
 
 



  Diphasol ™ 3873 (CIBA-GEIGY), an alkylphenol polyglycol ether phosphate surfactant
 Surfactant 508 (CIBA-GEIGY)
EMI18.2
 
 



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



  Butyl mono-4-ethoxy-phosphoric acid ester (Zerostat TM AT, CIBA-GEIGY), or
EMI18.3
 
 



  (Zerostat TM AN, CIBA-GEIGY)
 and on the other hand betaine compounds, i.e. amphoteric, salt and water-free imidazole derivatives, such as:
EMI18.4
 
 



  where Me [ <+>] stand for hydrogen, alkali and alkaline earth atoms and Rx for C1-32-alkyl or C2-32-alkenyl groups.



  So-called "multi-functional glucose derivatives" are also used, such as e.g. Glucate TM SS (methyl glucose sesquistearate) and Glucamate TM SSE-20 (PEG-20 methyl glucose sesquistearate, in the CTFA Classification) from Amerchol, Edison, NJ, USA Good results can also be achieved in some cases with the use of not -ionogenic compounds from the "Tween ™" series (Atlas Chem. Ind., Inc .; or ICI Specialty Chemicals), so-called polyoxyethylene sorbitan esters or "polysorbates" 20 to 85 compounds in the CTFA Classification [Fluka 93773 to 931783].



  Pharmaceutically acceptable solvents which can be used as hydrotropes or as co-emulsifiers can be used, e.g .:
 Esters of an aliphatic alcohol (C3-18) with an aliphatic carboxylic acid (C10-22) such as isopropyl laurate, hexyl laurate, decyl laurate, isopropyl myristate, isopropyl palmitate and lauryl myristate; Hydrocarbons with a straight carbon chain (C12-32) 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 (C6-22), such as propylene glycol monolaurate and propylene glycol monomyristate.



  Esters of an aliphatic alcohol (C12-22) with lactic acid, e.g. Myristyl or preferably lauryl lactate; Mono-, di- or triesters of glycerol with an aliphatic carboxylic acid (C6-22), e.g. Glyceryl caprylate, or Miglyol TM 812 neutral oil (oleum neutral).



  Esters of a poly (2-7) ethylene glycol glycerol ether with at least one free hydroxyl group with an aliphatic carboxylic acid (C6-22), e.g. aliphatic alcohols (C12-22), including Dodecanol, tetradecanol, oley alcohol, 2-hexyl decanol and 2-octyl decanol.



  Esters with at least one free hydroxyl group, from poly (2-10) glycol with an aliphatic carboxylic acid (C6-22), monoethers from a polyethylene glycol with an aliphatic alcohol (C12-18), e.g. Polyoxyethylene (C10) octyl ether.



  Heterocyclic compounds, e.g. 1-methyl-2-pyrrolidone.



  CHAPS (FLUKA 26 680; Merck Index 11.2034): 3 - [(3-cholamido propyl) dimethylammoniol propane sulfonate.



  Biosurfactant esters of the general formula (VII)
 



  R <5> -COO-R <6> (VII)
 



  where R <5> a C2-31 alkyl, a C3-31 alkenyl or alkapolyen group and R <6> Citronellyl, farnesyl, geranyl, isophytyl or phytyl.



  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 methylene chloride.



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



  Composition examples of spontaneously dispersible concentrates according to the invention, which contain active ingredients of the formulas (I) to (III) and which, when diluted with water or 5% glucose solution or physiological saline (Ringer's solution), thermodynamically stable ultramicroemulsions with a hydrodynamic radius of 2.2 up to 3.0 nm.



  a) 0.1 to 10% by weight of one or more active compounds of the formulas (I) to (III),
 0 to 10% by weight of one or more active pharmaceutical ingredients,
 1 to 25% by weight isopropyl myristate, isopropyl palmitate or neutral oil, e.g. Miglyol TM 812 (Dynamite Nobel or Hüls),
 0 to 45% by weight of a phosphoric acid ester surfactant, e.g. Diphasol TM 3873 (CIBA-GEIGY), surfactant 508 (CIBA-GEIGY), Zerostat TM AN or AT (CIBA-GEIGY), Tinovetin TM JU (CIBA-GEIGY), Soprophor TM FL (Rhône-Poulenc),
 5 to 90% by weight Invadin JFC 800% (CIBA-GEIGY) and / or Polysorbate 20-85 (ICI Specialty Chemicals),
 0 to 10% by weight of a vitamin or provitamin,
 0 to 10% by weight of a penetration enhancer, a stabilizer and / or auxiliaries.



  b) 0.5 to 2% by weight of one or more active substances according to (I) to (II),
 0 to 2% by weight of CHAPS or DMSO,
 5 to 25% by weight of one or more biosensitive esters of the general formula (VII)
 



  R <5> -COO-R <6> (VII)
 



  where R <5> a C2-31 alkyl, a C3-31 alkenyl or an alkapolyen group and R <6> mean citronellyl, farnesyl, geranyl, isophytyl or phytyl,
 30 to 45% by weight Invadin ™ JFC 800% and / or Tween ™ -20
 30 to 45% by weight of Soprophor ™ FL or Diphasol ™ 3873.



  c) 1% by weight of an active ingredient according to formula (I), (II) or (III),
 7% by weight of ethyl alcohol (C2H5OH), or 2-pentanol, and / or DMSO,
 20% by weight of citronellyl 10 undecenoate (C11: 1 citronellyl ester) or citronellyl 10 laurate (C12: 0 citronellyl ester)
 30% by weight Invadin TM JFC 800% and / or Tween TM -20,
 42% by weight Soprophor ™ FL.



  N.B .: Invadin TM JFC 800% (CIBA-GEIGY) is an anhydrous tert. Octylphenyl polyoxyethylene ether surfactant with 9 to 10 oxyethylene groups.
 Soprophor TM FL (Rhône-Poulenc) is a tristyrylphenol polyoxyethylene 18 phosphoric acid ester, TEA salt surfactant,
 Tween TM -20 (ICI Specialty Chemicals) is a non-ionogenic polyoxyethylene sorbitan ester compound: "Polysorbate" -20.


 3.1 Example of the pharmaceutical production of a system preparation with concentrates according to the invention in the form of "multiple units".
 
 <tb> <TABLE> Columns = 2 a) Granulation
 <tb> <SEP> Metolose TM 90 SH-4000 (Shin-Etsu Chemical) <SEP> 90.0 g
 <tb> <SEP> Avicel TM PH-101 <CEL AL = L> 80.3 g
 <tb> <SEP> Marigenol ™ concentrate according to the invention <SEP> 139.4 g
 <tb> <SEP> Aerosil TM 200 <SEP> 80.3 g
 <tb> <CEL AL = L> SIGMA <SEP> 390.0 g
 <tb> </TABLE>



  Granulate / form 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.



  b) MSR and retard equipment in a rotary bed with Aqoat TM AS-HG (Shin-Etsu Chemical) and talc



  c) Composition of finished granules / or. Micropellets
 <tb> <TABLE> Columns = 2
 <tb> <SEP> core material <SEP> 44%
 <tb> <SEP> concentrate according to the invention <SEP> 25%
 <tb> <CEL AL = L> MSR coating <CEL AL = L> 31%
 <tb> <SEP> SIGMA <SEP> 100%
 <tb> </TABLE>



  N.B .: MSR = gastric juice resistance. The pellets / granules according to a) can also be filled directly into capsules, which are made from Aqoat TM (HPMC-AS-M or HPMC-AS-N), and sealed with acetone / ethanol 1: 1 without filming and thus the functions adequately control the MSR and the delayed release (retard).


 4.0 Biological tests
 



  The antitumor effect of spontaneously dispersible concentrates with active ingredients according to the manufacturing examples and the work-up examples for their formation a) to c) is confirmed on the basis of the following test results:


 4.1 In vitro tests with suitable tumor cell lines
 



  A biological assay system has been developed that works with microtiter plates and dilution series. There are 10 each <4> / ml tumor cells in culture medium RPMI 1640 inactivated with 10% fetal calf serum (GIBCO); they are sown so leaky that they can grow during the assay in so-called non-confluent monolayers. The sample is added after 6 to 24 hours, with 100 ml per row, which is mixed with 100 ml medium in the 1st hole. Half of this is removed and placed in the following hole, again mixed with 100 ml medium, etc. A geometric dilution series n1 / 4 is formed.



  The samples are incubated in the plaque assay for 3 to 5 days at 37 ° C. with 31/4% 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 greatest cytotoxic dilution is determined.

   The quantitative evaluation can also be carried out by means of scanning and absorption measurement on the spectrophotometer.


 4.2 Testing for cell toxicity
 
 <tb> <TABLE> Columns = 5 4.21 Cell toxicity of the Marigenol TM concentrates
 tested on Py6 cells (polyoma virus infected 3T3 mouse fibroblasts
 <tb> Head Col 1: Tested concentrate
 with 1% active substance
 <tb> Head Col 2: salary
 <tb> Head Col 3: microemulsion
 ME 1: 100
 Active substance
 A.S.
 <tb> Head Col 4: exposure 24 h,
 in dilution
 effective up to 1:
 <tb> Head Col 5: exposure 48 h,
 in dilution
 effective up to 1:
 <tb> Head Col 6:

   Exposure 72 h,
 in dilution
 effective up to 1:
 <tb> <SEP> Taxol-W.S. <SEP> ME 1: 100
 A.S. <SEP> 64,000
 6.4 million <SEP> 256,000
 25.6 million <SEP> 256,000
 26.6 million
 <tb> <SEP> Baccatin III-W.S. <SEP> ME 1: 100
 A.S. <SEP> 128,000
 12.8 million <SEP> 128,000
 12.8 million <SEP> 1 million
 100 million
 <tb> <SEP> Baccatin III 7.13-
 Diundecenoate <SEP> ME 1: 100
 A.S. <SEP> 128,000
 12.8 million <SEP> 256,000
 25.6 million <SEP> 1 million
 100 million
 <tb> <SEP> Baccatin III 7.13-
 Dilaurate <SEP> ME 1: 100
 A.S. <SEP> 128,000
 12.8 million <SEP> 128,000
 12.8 million <SEP> 256,000
 25.6 million
 Dilutions:
 First line calculated on microemulsion
 Second line calculated on active substance content
  
 <tb> </TABLE>
 <tb> <TABLE> Columns = 5 4.22 Cell toxicity of the Marigenolo concentrates (continued) tested on Py6 cells (polyoma virus infected 3T3 mouse fibroblasts)
 <tb> Head Col 1:

   Tested concentrate
 with 1% active substance
 salary
 <tb> Head Col 2: microemulsion
 ME 1: 100
 or 1: 1000
 Active substance
 A.S.
 <tb> Head Col 3: exposure 48 h,
 effective in dilution up to 1:
 <tb> Head Col 4: exposure
 72/96 h, in
 dilution
 effective up to 1:
 * = 96 h
 <tb> Head Col 5: W.S.

   At first-
 concentration in
 Concentrate in mmol final concentration
 in the M.E. in pmol
 <tb> <SEP> Taxol-W.S. <SEP> ME 1: 100
 A.S. <SEP> 256,000
 25.6 million <SEP> 256,000
 25.6 million <SEP> 0.0117
 45.7
 <tb> <SEP> Baccatin III-W.S. <SEP> ME 1: 100
 A.S. <SEP> 80,000
 8 million <SEP> 160,000
 16 million <SEP> 0.0171
 106.9
 <tb> <SEP> 10-Dab Baccatin III
 W.S. <SEP> ME 1: 1000
 A.S. <SEP> 640,000
 64 million <SEP> 1 280 000 *
 128 million <SEP> 0.0190
 14.8
 <tb> <SEP> 14-OH-10-Dab-
 Baccatin III-W: S. <SEP> ME 1: 1000
 A.S. <SEP> 640,000
 64 million <SEP> 1 280 000 *
 128 million <SEP> 0.0184
 14.4
 <tb> <SEP> Baccatin III
 Monolaurate <SEP> ME 1: 1000
 A.S. <SEP> 160,000
 16 million <SEP> 640 000 *
 640 million <SEP> 0.0130
 20.3
 <tb> <SEP> Baccatin III
 Monopalmitate <SEP> ME 1: 1000
 A.S. <SEP> 320,000
 32 million <SEP> 640 000 *
 64 million <SEP> 0.0124
 19.4
 <tb> <SEP> Baccatin III 7.13-
 Dilaurate <SEP> ME 1:

  100
 A.S. <SEP> 40,000
 4 million <SEP> 80,000
 8 million <SEP> 0.0105
 131.2
 <tb> </TABLE>
 <tb> <TABLE> Columns = 5
 <tb> Head Col 1: Tested concentrate
 with 1% active substance
 salary
 <tb> Head Col 2: microemulsion
 ME 1: 1000
 Active substance
 A.S.
 <tb> Head Col 3: exposure 48 h,
 in dilution
 effective up to 1:
 <tb> Head Col 4: exposure 96 h,
 in dilution
 effective up to 1:
 <tb> Head Col 5: W.S.

   At first-
 concentration in
 Concentrate in mmol final concentration
 in the M.E. in pmol
 <tb> <SEP> 10-deacetyl baccatin
 III-7,10-dilaurate <SEP> ME 1: 1000
 A.S: <SEP> 32,000
 32 million <SEP> 32,000
 32 million <SEP> 0.0130
 40.6
 <tb> <SEP> 14-OH-10-deacetyl
 Baccatin III 7, 10, 14
 Trilaurate <SEP> ME 1: 1000
 A.S. <SEP> 16,000
 16 million <SEP> 32,000
 32 million <SEP> 0.0092
 28.8
 Dilutions:
 First line calculated on microemulsion
 The second line on the active substance content calculates the concentration information for the concentrate:
 in mmol (initial concentration) or in pMol (highest cell-toxic dilution)
  
 <tb> </TABLE>
 



  N.B .:
 Composition of the 1% concentrates:
 1% by weight of active substance
 12% by weight of C11: 1-citronellyl ester
 87% by weight Invadin JFC 800% / Soprophor FL 1: 1


 5.0 Analytical evidence
 


 5.1 Identification of baccatin III double ester active substance capillary zone electrophoresis using a P / ACE 2100 device from Beckman Instruments or a BioFocus integrator from Bio-Rad Laboratories.
 
 <tb> <TABLE> Columns = 2 conditions:
 <tb> <SEP> buffer pH = 7.0 <SEP> 50 mM Na-phosphate 100 mM boric acid 50 mm SDS filtered 0.2 μm
 <tb> <SEP> 5 ml of methanol are added to 20 ml of buffer
 <tb> <SEP> capillary: HP bubble cell 15 µm DIAMETER 37 cm
 <tb> <SEP> injection: 20 psi * sec. Run 15 kV, measurement at 192 nm
 <tb> <SEP> pressure injection 10 sec.
 <tb> <CEL CB = 2 AL = L> detection limit 0.5 ppm, very high resolution
 <tb> </TABLE>



  The peak appears for the concentrate after 22 minutes (surfactant effect!), For the pure taxol substance or the ester after 44 minutes.



  As an alternative, a so-called "fused silica capillary": FS 75 μm, 50 cm can also be used.
 Run 15 kV - 70 mu A, measurement at 195/220 nm
 The clear peak appears after 57 minutes!


 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 approx. 45-60 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 trains around. A small amount of canthaxanthin, which has a clear fluorescence, can be added to the concentrate as a marking substance. Phenosafranin, Tinopal TM GS or Uvitex TM CF or EBF (CIBA-GEIGY) are also suitable. The analytical proof that these vacuoles contain the baccatin III esters as active substance is also carried out by means of micellar capillary zone electrophoresis "MECC" (Beckman Instruments, P / ACE System 2100 or the BioFocus integrator from Bio-Rad).



  See also: Koji Otsuka et al .: Separation of lipophile compounds by micellar electrokinetic chromatography with organic modifiers, Electrophoresis, 1994, 15, 1280-83 (VCH Verlagsgesellschaft mbH, Weinheim).



  Additional control by means of measurement with UV-Maldi-Ms spectroscopy, using 2,6-dihydroxyacetophenone as a matrix.


 6.0 Comparative tests with human tumor cell lines
 
 <tb> <TABLE> Columns = 7 vitality test / 1% marigenol concentrate leukemia K 562 / controls 50 037 cpm
 1 x 10 <4> cells per hole
 Proliferation test (tritium: 1 mu Ci / well H <+>)
 <tb> Head Col 1:

   preparation
 <tb> Head Col 2:10 <-> <3>
 <tb> Head Col 3:
 <tb> Head Col 4:10 <-> <4>
 <tb> Head Col 6 AL = L: 10 <-5>
 <tb> Head Col 5: with
 <tb> Head Col 6: cpm
 <tb> Head Col 7:%
 <tb> Head Col 8: cpm
 <tb> Head Col 9:%
 <tb> Head Col 10: cpm
 <tb> Head Col 11:%
 <tb> <SEP> Taxol-W.S. <SEP> 679 <SEP> 1.3 <SEP> 830 <SEP> 1.6 <SEP> 3553 <CEL AL = L> 7.1
 <tb> <CEL AL = L> Baccatin III-W.S. <SEP> 680 <SEP> 1.3 <SEP> 757 <SEP> 1.5 <SEP> 3188 <SEP> 6.3
 <tb> <CEL AL = L> Baccatin III diundecenoate <SEP> 466 <SEP> 0.9 <SEP> 665 <SEP> 1.3 <SEP> 4770 <SEP> 9.5
 <tb> <CEL AL = L> Baccatin III dilaurate <SEP> 585 <SEP> 1.1 <SEP> 651 <SEP> 1.3 <SEP> 3060 <SEP> 6.1
 <tb> </TABLE>
 <tb> <TABLE> Columns = 7 vitality test / 1% marigenol concentrates Leukemia Daudi line / controls 23 090 cpm
 <tb> Head Col 1:

   preparation
 <tb> Head Col 2:10 <-> <3>
 <tb> Head Col 4 AL = L: 10 <-> <4>
 <tb> Head Col 6 AL = L: 10 <-> <5>
 <tb> Head Col 3: with
 <tb> Head Col 4: cpm
 <tb> Head Col 5:%
 <tb> Head Col 6: cpm
 <tb> Head Col 7:%
 <tb> Head Col 8: cpm
 <tb> Head Col 9:%
 <tb> <SEP> Taxol-W.S. <SEP> 552 <SEP> 2.3 <SEP> 672 <SEP> 2.9 <SEP> 16 090 <CEL AL = L> 69.6
 <tb> <CEL AL = L> Baccatin III-W.S. <SEP> 708 <SEP> 3.0 <SEP> 795 <SEP> 3.4 <SEP> 14 190 <SEP> 61.4
 <tb> <CEL AL = L> Baccatin III diundecenoate <SEP> 293 <SEP> 1.2 <SEP> 653 <SEP> 2.8 <SEP> 9636 <SEP> 41.7
 <tb> <CEL AL = L> Baccatin III dilaurate <SEP> 346 <SEP> 1.4 <SEP> 455 <SEP> 1.9 <SEP> 8851 <SEP> 38.3
 <tb> </TABLE>



  Work carried out by Dottoressa Anna Rita GUARINI, Università degli Studi di Torino, Clinica medica, August 21-24, 1995.


 7.0 General compatibility of the Marigenol TM preparations
 
 <tb> <TABLE> Columns = 7 Effect on blood count Toxicity of Marigenol TM concentrates in the Balb / c-mouse% of the blood cells
 <tb> Head Col 1: preparation
 <tb> Head Col 2: L
 <tb> Head Col 3: M
 <tb> Head Col 4: N
 <tb> Head Col 5: E
 <tb> Head Col 6:

   B
 <tb> <SEP> G17 <SEP> 10 <-> <7>
 10th <-> <5>
 10th <-> <3> <SEP> 70 +/- 6
 77 +/- 6
 69 +/- 10 <SEP> 11 +/- 3
 6 +/- 3
 7 +/- 5 <SEP> 13 +/- 4
 11 +/- 4
 22 +/- 8 <SEP> 6 +/- 4
 5 +/- 4
 2 +/- 2 <SEP> 0
 1 +/- 1
 0
 <tb> <SEP> G41 <SEP> 10 <-> <7>
 10th <-> <5>
 10th <-> <3> <SEP> 77 +/- 6
 78 +/- 4
 80 +/- 6 <SEP> 6 +/- 3
 10 +/- 2
 8 +/- 2 <SEP> 13 +/- 5
 10 +/- 4
 10 +/- 6 <SEP> 3 +/- 3
 1 +/- 1
 12 +/- 1 <SEP> 0
 1 +/- 1
 0
 <tb> <SEP> G44 <SEP> 10 <-> <7>
 10th <-> <5>
 10th <-> <3> <SEP> 74 +/- 17
 74 +/- 6
 76 +/- 5 <SEP> 10 +/- 1
 9 +/- 4
 6 +/- 4 <SEP> 20 +/- 9
 14 +/- 7
 16 +/- 8 <SEP> 1 +/- 1
 4 +/- 3
 2 +/- 1 <SEP> 0 <SEP> 0
 0
 <tb> <SEP> G55 <SEP> 10 <-> <7>
 10th <-> <5>
 10th <-> <3> <SEP> 78 +/- 4
 69 +/- 10
 77 +/- 5 <SEP> 10 +/- 4
 11 +/- 3
 6 +/- 4 <SEP> 10 +/- 4
 18 +/- 4
 14 +/- 2 <SEP> 2 +/- 1
 1 +/- 1
 2 +/- 1 <SEP> 0
 0
 1 +/- 1
 <tb> <SEP> controls (physical buffer)

   <SEP> 76 +/- 5 <SEP> 8 +/- 2 <SEP> 15 +/- 4 <SEP> 1 +/- 1 <CEL AL = L> 0
 <tb> </TABLE>
 



  G 17 2% concentrate with C5: 0-iso-cholesteryl ester (cholesteryl-iso-valerate)
 G 41 2% concentrate with C11: 1-ergosteryl ester (ergosteryl-10-undecenoate)
 G 44 2% concentrate with C18: 2-cholecalciferyl ester (C18.2-D3; vitamin D3 linolate)
 G 55 2% concentrate with C4: 1 cholecalciferyl ester (C4: 1-D3; vitamin D3 crotonate)
 Dilutions:
 10th <-> <7> = 0.1 ppm concentrate; 0.002 ppm active substance
 10th <-> <5> = 10 ppm concentrate; 0.200 ppm active substance
 10th <-> <3> = 1000 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. Guido FORNI, Dott <a> Stefania VAI, Università di Torino, Dipartimento di Scienze Cliniche e Biologiche, Ospedale San Luigi Gonzaga, I-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 each of 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: 13 groups of 5 per animal



  Result: There are no significant differences to 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.


 7.1 Examination of the blood picture
 
 <tb> <TABLE> Columns = 5 Effect of Marigenol TM concentrates on normal human lymphomonocytes
 Cells: normal human phagocytic white blood cells (macrophages);
 2 x 10 <5> cells per well stimulation of controls: 1% PHA (phytohemagglutinin)
 <tb> Head Col 1: preparation
 <tb> Head Col 2: dilution
 <tb> Head Col 3: Test 1
 <tb> Head Col 4: Test 2
 <tb> Head Col 5: Test 3
 <tb> Head Col 6: controls PHA 1%
 <tb> Head Col 7:

   (stimulated)
 <tb> Head Col 8: cpm
 2682
 80 981
 <tb> Head Col 9: cpm
 3386
 61 966
 <tb> Head Col 10: cpm
 3386
 61 966
 <tb> <SEP> concentrates as aqueous
 Microemulsions <SEP> 10 <-> <1>
 10th <-> <2>
 10th <-> <3>
 10th <-> <4>
 10th <-> <5>
 10th <-> <6> <SEP> 1076
 1396
 1431
 1386
 1656
 2536 <SEP> 3826
 3726
 2141
 3221
 2888
 2105 <SEP> 3936
 3845
 2939
 2752
 3093
 3046
 Test 1: 2% concentrate containing C12: 0-cholesteryl ester
 Test 2: 2% concentrate containing C16: 0-ergosteryl ester
 Test 3: 2% concentrate containing C5: 0-cholecalciferyl ester (vitamin D3-iso-valerate)
  
 <tb> </TABLE>



  Tests performed by Dottoressa Anna GUARINI, Università di Torino, Clinica medica, I-10 26 TORINO, March 21-24, 1994.


 7.2 Comparative tests with different processing
 
 <tb> <TABLE> Columns = 2
 <tb> <SEP> A <SEP> Taxol-W.S., Formulated as a spontaneously dispersible Marigenol ™ concentrate, which forms an aqueous ultramicroemulsion
 <tb> <SEP> B <SEP> Taxol-W.S., Formulated as a conventional concentrate that only forms an aqueous macroemulsion
 <tb> <SEP> C <SEP> Baccatin-III-W.S., Formulated as a spontaneously dispersible Marigenol ™ concentrate, which forms an aqueous ultramicroemulsion
 <tb> <SEP> D <SEP> Baccatin-III-W.S., Formulated as a conventional concentrate that only forms an aqueous macroemulsion
 <tb> </TABLE>
 <tb> <TABLE> Columns = 4
 <tb> Head Col 1: Tested concentrate
 with 1% active substance
 <tb> Head Col 2:

   Mikoro macro
 Emulsion 1: 100
 Active substance A.S.
 <tb> Head Col 3: exposure 24 h,
 in dilution
 effective up to 1
 <tb> Head Col 4: exposure 48 h,
 effective in dilution up to 1
 <tb> <SEP> A: Taxol-W.S. <SEP> EM 1: 100
 A.S. <SEP> 128,000
 12.8 million <SEP> 512,000
 51.2 million
 <tb> <SEP> B: Taxol-W.S. <SEP> EM 1: 100
 A.S: <SEP> <4000
  <400,000 <SEP> <4000
  <400,000
 <tb> <SEP> C: Baccatin III-W.S. <SEP> EM 1: 100
 A.S. <SEP> 128,000
 12.8 million <SEP> 256,000
 25.6 million
 <tb> <SEP> D: Baccatin III-W.S. <SEP> EM 1: 100
 A.S. <SEP> <4000
  <400,000 <SEP> <4000
  <400,000
 <tb> </TABLE>



  It should be noted that the formation of the concentrate according to the invention is far superior in effect to the conventional formulation. In the short exposure time of 24 h, their bioreactivity is 30 times and at 48 h exposure more than 125 times what has been usual and achievable.



  Marigenol ™ concentrates formulated with C11: 1 citric acid ester as co-emulsifier and with a 1: 1 surfactant mixture Invadin JFC 800% / Soprophor FL.

 

  Conventionally formulated concentrates with isopropyl myristate as co-emulsifier and with Cremophor TM EL (BASF), but without ethyl alcohol.



  In both cases, the same proportionate ratio W.S .: coemulsifier: surfactants assumed.



  The analytical review of the different formulations at the Institute for Polymers at E.T.H. in Zurich gave the following measurement data:
 <tb> <TABLE> Columns = 2 micellar size
 <tb> Head Col 1: preparation
 <tb> Head Col 2: micelle size in nm
 <tb> <SEP> A: Taxol-W.S. <SEP> 2.2-2.3 without filter
 <tb> <SEP> B: Taxol-W.S. <CEL AL = L> 5-6 60-100 strong scatter wide distribution 10% filter
 <tb> <SEP> C: Baccatin III-W.S. <SEP> 2.2-3.0 without filter, but 75 DEG angle
 <tb> <SEP> D: Baccatin III-W.S. <SEP> 4-12 strong scatter, wide distribution 10% filter
 <tb> <CEL AL = L> ester compounds:
 <tb> <SEP> Q: Quercetin penta undecenoate <SEP> 2-3
 <tb> <SEP> QE: beta-oestradiol di-oleate <CEL AL = L> 2-3
 <tb> <SEP> Q: Apigenin trilaurate <SEP> 2-3
 <tb> <SEP> L: Genistein trilaurate <SEP> 2-3
 <tb> </TABLE>



  The aqueous emulsion was measured 1: 100 (CMC limit for the homogeneous distribution of the surfactants) of the 1% concentrates by means of dynamic light scattering (DLS) in 3 angular positions (60 DEG, 90 DEG and 120 DEG), with 10 individual measurements each, on a specially equipped "Fiberoptic Spectrometer" of the Institute for Polymers, ETH, Zurich. For a description of the methodology and the device cf. "Mode-selective dynamic light scattering: theory versus experimental realization". Thomas Gisler et al., Applied Optics / Vol. 34, No. 18120, June 1995.


 8.0 Testing for protection and antiviral activity against sensitive MT4 cells infected with the AIDS virus HIV ("Human Immunodeficiency Virus")
 



  Tests were carried out at the Institute for Infectious Diseases at the University of Turin (Director: Prof. Dr. Paolo GIOANNINI), by Dott. Alberto BIGLINO, Head of the Infectious Diseases Department at Ospedale Generale di Asti, U.S.L. 19, with the collaboration of Dott <a>. Brunella FORNO and Dott <a>. Annamaria POLLONO. June / July 1994 and March / April 1995.


 8.1 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", were grown from a 24-hour culture at a concentration of 2 x 10 <5> / ml suspended, 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 CCID50 / ml) or sham-infected with pure medium, incubated for 90 min at 37 ° C. and then mixed with 1 ml of pure medium reset the initial cell concentration and bring the HIV concentration to 100 CCID50 / ml. [CCID50 / ml = 50% cell culture infective dose].



  100 μl volumes of the MARIGENOL TM concentrates used in the test, diluted to aqueous ultramicroemulsions 10 <-> <3> and then additionally diluted by 10 <-> <3> to 10 <-> <5> with medium RPMI 1640, were introduced three times in flat-bottomed microtiter plates with 96 holes. 100 μl of the HIV-infected or the sham-infected MT4 culture were introduced into each hole in order to set up 4 test series:
 - simple culture (controls; test for viability of the MT-4 cells: 10 <5> cells / ml or 2 x 10 <4> / hole)
 - Culture + HIV (virus CPE control; concentration 50 CCID50 / ml or 10 CCID50 / well)
 - Culture + active substance in microemulsion
 - Culture + HIV + active substance microemulsion in dilutions 10 <-> <3> to 10 <-> <5> (= 1000 ppm, 100 ppm and 10 ppm, or 10 ppm, 1 ppm and 0.1 ppm W.S. content)



  Incubate the cultures at 37 ° C in a 5% CO2 + 95% humid atmosphere. 5 days after the infection, 100 .mu.l of the supernatant are removed in each well and the viability of the cells is checked using a methyl tetrazole salt (MTT, Sigma, 25 .mu./hole in 5 mg / ml solution) in a 2-hour incubation test, the this is followed by a solubilization with 100 μl DMSO / hole and the photometric reading for optical density at 550 nm. Residual values of cell 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 host times has so far been achieved with a 1% concentrate of beta-sitosteryl palmitate at a concentration of 10 <-> <4> (= 100 ppm concentrate in aqueous ultramicroemulsion) achieved; The results with 1% concentrates containing beta-sitosteryl caproylate, beta-sitosteryl laurate, beta-sitosteryl arachidate or beta-sitosteryl behenate are also very noteworthy.


 7.2 Effect on the ability to be infected with HIV (virulence, direct antiviral or virucidal effect against the acquired immune deficiency, the human "acquired immune deficiency syndrome: AIDS").
 



  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 CCID50 / ml and incubated for 3 hours at +40 ° C with a Marigenol TM concentrate, as microemulsions in complete medium to the concentrations 10 <-> <2> to 10 <-> <5> diluted, 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 CCID50 method (CCID50 = cell culture infective dose 50%). Two serial dilutions of each of the 6 virus suspensions were made briefly, of which 200 μl each were incubated with MT-4 cell pellets for 90 min, as indicated above (see 7.1). 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 ml 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 infects 4 out of 8 samples in a row (50%).

   The contents of a hole are considered infected if the O.D. 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-10-undecenoate and beta-sitosteryl-palmitate, a very clear inhibition of replication and an 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.
 <tb> <TABLE> Columns = 2 Evaluation of the test results:
 <tb> Head Col 1: test substrate
 <tb> Head Col 2:

   Residual values in the HIV titer (CCID50)
 (Average of 3 cultures)
 <tb> <SEP> HIV strain 21/4 ("Clinical isolate") + medium <SEP> 300
 <tb> <SEP> HIV strain 4/5 ("Clinical isolate") + medium <SEP> 200
 <tb> <SEP> HIV (2 strains) + carrier concentrate <SEP> 200 (mean titer from 2 strains)
 <tb> <SEP> HIV (2 strains) + ergosteryl 10 undecenoate <SEP> 25 (mean titer from 2 strains)
 <tb> <SEP> HIV (2 strains) + beta-sitosteryl palmitate 1000 ppm <SEP> 2 (mean titer from 2 strains)
 <tb> <SEP> HIV (2 strains) + beta-sitosteryl palmitate 100 ppm <SEP> 50 (mean titer from 2 strains)
 <tb> <SEP> HIV (2 strains) + beta = sitosteryl palmitate 50 ppm <SEP> 76 (mean titer from 2 strains)
 <tb> <SEP> HIV (2 strains) + beta-sitosteryl palmitate 10 ppm <SEP> 120 (mean titer from 2 strains)
 <tb> </TABLE>



  Residual values of HIV-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 / 603.50); J.Virol.Methods, 1987; 16, 171-85



  A. Bergamini, C.F. Perna et al., "A tetrazolium-based, colorimetric assay for quantification of HIV-induced catopathogenicicity in monocyte-macrophages, exposed to macrophage colony-stimulating factor". J.Virol.Methods, 1992, 40 (3), 275-86.



  Paul Wallerstein: The AIDS Dilemma; Research in a dead end. Rombach Aktuell, 1988, Freiburg i.Br.



  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. Darnell, H. Lodish, D. Baltimore; Scientific American Books, Chapter 5: Viruses, Structure and Functions, pp. 177-188. New York, 1990 (W.H. Freeman & Co.)


 8.0 Other viruses
 


 8.1 Human 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 for 4 hours at +4 ° C. with different concentrations of the test substance C16: 0-beta sitosteryl ester, as a 1% concentrate and then diluted to an aqueous microemulsion 10 <-> <3>, 10 <-> <4> and 10 <-> <5>, incubated and then inoculated as pretreated virus suspensions on cultures of human, embryonic lung fibroblasts. Approach as 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 h.



  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 after 6 hours in the infected cells) 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-time 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:
 
 <tb> <TABLE> Columns = 4 Virucidal effects of the beta sitosteryl palmitate (formulated as Marigenol TM concentrate)
 <tb> Head Col 1: controls: number of
 Antigens (anti P-72)
 <tb> Head Col 2: concentration
 Microemulsion
 1: 1000
 <tb> Head Col 3: concentration
 Microemulsion
 1:10 000
 <tb> Head Col 4: Microemulsion concentration 1: 100,000
 <tb> <SEP> 115,000 <SEP> 0 <SEP> 2 <SEP> 82,000
 <tb> </TABLE>



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



  Tests carried out by Dottoressa Rossana CAVALLO, Istituto di Microbiologia, Università di Torino, July-November 1995.


 8.2 Herpes virus (herpes simplex, HSV)
 



  The antiviral / virucidal effect of an aqueous microemulsion of suitable Marigenol ™ concentrates is determined using a confluent monolayer culture of Vero cells (i.e. "African green monkey kidney cells"). The host cells are pretreated for 3 hours at 4 ° C. with the microemulsion or with the culture medium alone and then with a constant dose of 10 <4> Herpes simplex viruses, so-called "pIaque forming units" = PFU infected. 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% 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 "plaques" of lysed cells which still form 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% C20: 0 beta sitosteryl ester
 1% quercetin penta-10 undecenoate
 1% C16: 0 beta sitosteryl ester
 1% C12: 0-ergosteryl ester


 Evaluation:
 
 <tb> <TABLE> Columns = 5
 <tb> Head Col 1: dilution
 <tb> Head Col 2: C20: 0- beta -Sitoester:
 (Arachidate)
 <tb> Head Col 3: Quercetin-Penta-
 Undecenoat
 <tb> Head Col 4: C16: 0- beta -Sitoester:
 (Palmitate)
 <tb> Head Col 5: C-12: 0 Ergoester (Laurat)
 <tb> <SEP> 1: 1000 <SEP> cell lysis <SEP> cell lysis <SEP> cell lysis <CEL AL = L> cell lysis
 <tb> <SEP> 1:10 000 <SEP> 76% <SEP> 48% <SEP> 24% <SEP> 62%
 <tb> <SEP> 1: 100,000 <SEP> 5% <CEL AL = L> 0% <SEP> 0% <SEP> 0%
 <tb> </TABLE>



  The results show a clear direct effect on the Vero host cells infected with HS virus. At a concentration of 10 <-> <4> - beta-sitosteryl arachidate, the number of plaques appears to be reduced by 76% in comparison with the controls. The HSV titer is significantly reduced.



  At this concentration of the tested substances, there is no lysis of eternalized lymphocytes of the MT-4 type, nor of the Vero cells themselves. (See above under 8.0)



  Tests carried out by Dottoressa Rossana CAVALLO, Istituto di Microbiologia, Università di Torino, July 1995.


 8.3 Herpes virus (herpes simplex, HSV)
 



  Repeat the test with fluorescent labeling as in the CMV test. See 8.1 above
 <tb> <TABLE> Columns = 4 Virucidal effects of the beta sitosteryl arachidate (formulated as Marigenol TM concentrate)
 <tb> Head Col 1: controls: number
 the infected cells)
 <tb> Head Col 2: concentration
 Microemulsion
 1: 1000
 <tb> Head Col 3: concentration
 Microemulsion
 1:10 000
 <tb> Head Col 4: Microemulsion concentration 1: 100,000
 <tb> <SEP> 38 <SEP> 0 <SEP> 0 <SEP> 20
 <tb> </TABLE>



  Tests carried out by Dottoressa Rossana CAVALLO, Istituto di Microbiologia, Università di Torino, November-December 1995.


    

Claims (9)

    1. An ester of baccatin-III, of 10-deacetylbaccatin-III or of 14-OH-10-deacetylbaccatin-III according to one of the general formulas (I), (II) or (III) EMI39.1          (I) = baccatin III; R <1> = R <3> = H, R <2> = COCH3, R <4> = H  (II) = 10-deacetylbaccatin-III; R <1> = R <2> = R <3> = R <4> = H  (III) = 14-hydroxy-10-deacetylbaccatin-III; R <1> = R <2> = R <3> = H, R <4> = OH      wherein in formulas (I) and (II) mono- or diesters at C (7) and / or C (13) with C6-32-alkylcarbonyl, C6-32-alkenylcarbonyl, C6-32-alkapolyencarbonyl or retinoyl as ester components and in the formula (III) esters at C (7), C (13) and / or C (14) with these ester components are claimed. 2nd  A process for the preparation of an ester according to claim 1, characterized in that a compound of the formulas (I) to (III) according to the fat-tail principle with a long-chain, activated, aliphatic fatty acid in abs. Pyridine and 1,2-dichloroethane, and an addition of 4-dimethylaminopyridine and AgCN esterified under a protective gas and the product is then filtered, washed and purified by chromatography over two stages. 3rd  Spontaneously dispersible concentrate, which dilutes with water, with 5% glucose solution or with Ringer's solution, results in thermodynamically stable ultramicroemulsions that have micelles with a hydrodynamic radius of 2.2 to 3.0 nm, determined by the fact that the following components are combined to form a pharmaceutically usable system:  0.1 to 10% by weight of an ester of baccatin III, 10-deacetylbaccatin-III or 14-OH-10-deactylbaccatin-III according to one of the formulas (I) to (III) according to claim 1, or a combination such esters,  0 to 10% by weight of one or more active pharmaceutical ingredients,  1 to 40% by weight of a hydrotrope, or  Co-emulsifier-serving, pharmaceutically acceptable solvent or solvent mixture,  0.1 to 90% by weight of a pharmaceutically acceptable surfactant or surfactant mixture,  0 to 10% by weight of a vitamin or provitamin,  0 to 10 wt .-% of a stabilizer, a radical scavenger or a penetration enhancer and carriers and / or diluents. 4. Spontaneously dispersible concentrate according to claim 3, containing a compound according to one of the formulas (I) to (III) according to claim 1, as an agent for the preparation of pharmaceutical preparations with improved bioavailability for the treatment of tumors, eczema and psoriasis. 5.  Spontaneously dispersible concentrate according to claim 3, containing a compound according to one of the formulas (I) to (III) according to claim 1, as an agent for the preparation of pharmaceutical preparations with antiviral and / or virucidal activity and for increased absorption of exogenous activators. 6.  Spontaneously dispersible concentrate according to claim 3, characterized in its features that the following components are combined:  0.5 to 5% by weight of one or more esters of baccantine-111 according to one of the formulas (I) to (III) according to claim 1,  5 to 30% by weight of isopropyl myristate, isopropyl palmitate, 3 - [(3-cholamidopropyl) dimethylammonio] propane sulfonate or neutral oil (neutral oleum), or one or more biosensitive esters of the general formula (VII):      R <5> -COO-R <6> (VII)      where R 5 is a C2-31 alkyl, a C3-31 alkenyl or a C3-31 alkapolyen group and R 6 is citronellyl, farnesyl, geranyl, isophytyl or phytyl,  0 to 45% by weight of a pharmaceutically acceptable phosphoric ester surfactant,  5 to 90 wt .-% of anhydrous tert.  Octylphenyl polyoxyethylene ether surfactant with 9 to 10 oxyethylene groups and / or of polyoxyethylene sorbitan monolaurate. 7. Spontaneously dispersible concentrate according to claim 3, characterized in that the following components are combined:  0.5 to 5% by weight of one or more esters of baccatin III according to formulas (I) to (III) according to claim 1,  0 to 5% by weight of 3 - [(3-cholamidopropyl) dimethylammonio] propane sulfonate surfactant,  5 to 30% by weight of one or more biosensitive esters of the general formula (VII):      R <5> -COO-R <6> (VII)      where R 5 is a C2-31 alkyl, a C3-31 alkenyl or a C3-31 alkapolyen group and R 6 is citronellyl, farnesyl, geranyl, isophytyl or phytyl,  35 to 45 wt .-% of anhydrous tert.  Octylphenyl polyoxyethylene ether surfactant with 9 to 10 oxyethylene groups,  35 to 45% by weight of an alkylphenol polyglycol ether phosphate surfactant or the tristyrylphenol polyoxyethylene-18-phosphoric acid ester triethanol amine salt surfactant. 8. Therapeutic system preparation which contains 1 to 95 wt .-% of the spontaneously dispersible concentrate according to one of claims 6 or 7 and up to 10 wt .-% of a pharmaceutically acceptable additive, solvent or stabilizer or a mixture thereof and which in unit dosage form is present as micropellets, granules, dragées, suppositories, ampoules or as capsules. 9. Therapeutic system preparation according to claim 8, which contains 44 parts of core material for the granules or  Pellet formation, 25 parts of a concentrate according to the invention and 31 parts of enteric-coated and retarding coating with hydroxypropyl methyl cellulose acetate succinate.