DE10261825A1 - Use of hypericin and / or its derivatives for tumor prevention - Google Patents

Abstract

The present invention relates to the use of hypericin and / or its derivatives for the manufacture of a medicament for tumor prevention. The invention also relates to the use of hypericin and / or its derivatives for the production of a food or nutritional supplement and the food or nutritional supplement comprising hypericin in a nutritionally effective amount. Hypericin can preferably be used in the form of a hypericin-rich vegetable dry extract, preferably from St. John's wort.

Description

The present invention relates to the use of hypericin and / or its derivatives for the preparation a drug for tumor prevention. The invention also relates to the use of hypericin and / or its derivatives for the manufacture of a food or nutritional supplement and the nutritional or nutritional supplement comprising hypericin, in a nutritionally effective Quantity. Hypericin can preferably be in the form of a hypericin-rich vegetable Dry extract, preferably from St. John's wort, can be used.

Given the limited to date therapeutic options experts believe that chemoprevention cancer through specific food, dietary supplements and specific drug compositions have enormous potential to improve life.

It is known that, for example, polycyclic aromatic hydrocarbons (PAHs) are disease-triggering substances. PAHs can be ingested from the environment (exhaust gases) or when smoking, but polycyclic aromatic hydrocarbons are also formed when barbecuing. In particular, they are held responsible for the development of mutagenic changes and malignant tumors. The metabolism of these substances in the body has been the subject of numerous research work and is based on the example of one of the most important representatives, benzo [a] pyrene (B [a] P), in 1 shown. Out 1 it becomes clear that the human enzyme cytochrome P450 1A1 (CYP1A1) plays an important role in the development of the disease.

The first stage of bioactivation des B [a] P leads over the CYP1A1-catalyzed formation of the B [a] P-7,8 epoxide and its hydrolysis by epoxy hydrolase (EH) to B [a] P-7,8-dihydrodiol (7,8-diol-B [a] P). In one second step is the resulting 7,8-diol-B [a] P by CYP1A1 to the genotoxic (±) -B [a] P-r-7, t-8-dihydro-diol-t-9,10 epoxy (diol epoxide 2, DE2) metabolized. The enzyme cytochrome P450 1A1 catalyzes both epoxidation steps for the activation of benzopyrenes to the diolepoxides, their DNA adducts are regarded as the actual cause of the disease. Shimada et al. describe in drug metab. Disposables. 29 (2001) 1176-1182 that the human cytochrome P450 1A1 (CYP1A1) the most important in the chemical Carcinogenesis is the enzyme involved. PAKs such as Benzo [a] pyrene induces and activates these and many other environmental toxins and procarcinogens to the actual carcinogens (Guengerich, F. P., Cancer Res. 48 (1988) 2946-2954). CYP1A1 is essentially an extrahepatic enzyme, in addition to the lungs it is mainly used in entire gastrointestinal tract (esophagus, stomach, small and large intestine), expressed in the placenta, brain and lymphocytes.

Out 1 However, it is also clear that CYP1A1 also catalyzes the hydroxylation of B [a] P, which leads to the phenols, mainly to 3-OH-B [a] P, which are not carcinogenic products and characterize the detoxification pathway.

As an important chemopreventive The compound that prevents the formation of DNA adducts is quercetin, than natural occurring flavonoid component of many fruits and vegetables that e.g. is also contained in St. John's wort. Kang et al. in "Nutrition and Cancer "35 (2) (1999) 175-179. that quercetin the formation of benzo [a] pyrene-induced DNA adducts in human Hep G2 cells inhibited by influencing the CYP1A1 gene expression. The authors conclude that quercetin has a long-lasting preventive effect on the chemical Cancerogenesis can have from people who are rich in vegetables and fruits Ingest food.

Naturally occurring vegetable Phenols such as tannic acid, Quercetin, myricetin and anthraflavonic acid are described by Das, M. et al. in "Cancer Res. 47 (3) (1987), 767-73 "as potent inhibitors the PAK-DNA adduct formation in the epidermis and lungs of SENCAR mice. The authors conclude that these plant phenols might prove appropriate to modify the risk of tumor induction by PAHs.

DE 100 16 771 A1 describes quercetin and structurally similar compounds for chemoprevention of intestinal diseases, especially colon carcinomas. US 6,008,260 beard resveratrol (stilbene-3,4 ', 5-triol) as an agent for cancer prophylaxis.

Object of the present invention was to find highly effective connections for tumor prevention.

It has now surprisingly been found that hypericin and its derivatives are highly potent chemopreventive Compounds for the prophylaxis of any disease associated with mutagenic changes go hand in hand, e.g. neoplastic transformations, and in particular for cancer prophylaxis.

To date, hypericin has been used in the literature to treat viral infections ( EP 0 332 679 ), for the photodynamic diagnosis / therapy of malignant diseases (WO 00/53170, WO 01/89576) and in combination with hyperforin for the treatment of skin cancer (WO 00/30660 A2).

Hypericin belongs to the naphthodian thrones and has the following structural formula:

Hypericin is a component of plants belonging to the species Hypericum (St. John's wort) [cf. A. Nahrstedt et al., Pharmacopsychiat. 30 (1997) (supplement) 129-134]. The pure substance hypericin can be isolated from plants or chemically synthesized. A whole series of synthetic routes are described (cf. e.g. US 2,707,704 . EP 0 432 496 ). It has now been found that hypericin is a potent inhibitor of the epoxidation of the 7,8-diol-B [a] P to the diolepoxides, that is to say the terminal step leading to the ultimately genotoxic and carcinogenic products. In addition, hypericin has little or no influence on the hydroxylation of B [a] P, which leads to the phenols and is the detoxification pathway (cf. 1 ).

Hypericin is, for example, superior to resveratrol, which is described as a cancer prophylactic, and although it does not inhibit the hydroxylation of B [a] P, it only slightly inhibits the epoxidation of 7,8-diol-B [a] P to the genotoxic diols (cf. , 2 ). 3 shows that hypericin, on the other hand, inhibits epoxidation extremely strongly, without having any significant influence on the hydroxylation pathway, at least not at concentrations up to 5 μM, which are far above the IC ₅₀ value of 0.5 μM for the inhibition of epoxidation.

To the activity of CYP1A1 in hydroxylation to characterize B [a] P to 3-OH-B [a] P, the AHH test (aryl hydrocarbon hydroxylase assay) (Nebert, D.W. et al., J. Biol. Chem. 243 (1968) 6242-6249). The implementation of the Testing is described in Example 1.

The epoxidation activity of CYP1A1 To investigate, was its effect on epoxidation according to the invention of the 7,8-diol-B [a] P to the diol epoxides, i.e. the second terminal epoxidation step examined. Carrying out this Epoxidation tests are described in Example 2. Comparative studies were taken for the EROD test, a classic test using an (unphysiological) Model substrates, for the determination of CYP1A1 activity. Here will investigated the O-deethylation of 7-ethoxyresorufin. The implementation of the EROD testing is in example 3 described.

It has been shown that the use of the EROD test alone can lead to an incorrect assessment of the inhibitory potency of a substance. Resveratrol was identified on the basis of EROD studies as a strong selective inhibitor of human CYP1A1 (Chun et al., Biochem. Biophys. Res. Commun. 262 (1999), 20-24), while the epoxidation test used here for the first time shows that resveratrol only slightly inhibits the epoxidation of 7,8-diol-B [a] P (cf. 2 .).

IC _{5 0} values were determined for hypericin and comparatively for quercetin and α-naphthoflavone. By definition, the IC ₅₀ value is the inhibitor concentration which leads to 50% inhibition of the reaction. The IC ₅₀ value of hypericin was determined to be 0.5 μM, while that of quercetin, also a plant phenol, which has a preventive effect on chemical cancerogenesis, is 1.5 μM. Even the IC ₅₀ value of α-naphthoflavone (α-NF, Alpha-NF), a well-known selective CYP1A1 inhibitor, is 0.8 μM. The inventors determined the latter value for the first time for the inhibition of the CYP1A1-dependent epoxidation of 7,8-diol-B [a] P. In 4 the reciprocal IC ₅₀ values for hypericin, quercetin and α-naphthoflavone are shown for comparison of the relative inhibitory potential of these substances.

Enzyme kinetic studies (inhibitor kinetics) have shown that hypericin is a potent non-competitive inhibitor of CYP1A1 epoxidation activity, with an inhibition constant (K _i value) of approximately 0.6 μM. In contrast, quercetin is a mixed-type inhibitor (competitive plus non-competitive inhibition) with a K _i = 2.4 μM.

It was thus found that hypericin and / or its derivatives are beneficial for the prevention of PAH-induced tumors can be used, in particular for prevention of lung carcinoma, breast carcinoma, portiocarcinoma, colon carcinoma, skin carcinoma and brain cancer. The substances are also for prevention other diseases induced by PAK can be used, e.g. to Prevention of neoplastic transformations.

Based on the above Results are also the use of hypericin and / or its Derivatives to inhibit the epoxidation activity of the enzyme CYP1A1 present invention. According to the invention, hypericin derivatives in particular come Pseudohypericin, protohypericin, pseudoprotohypericin, cyclopseudohypericin and emodinanthron in question. Hypericin and its are preferred Derivatives in the form of their salts with organic or inorganic acids, e.g. used as acetates or phosphates.

To produce a medicament, these compounds or mixtures of these compounds are formulated in a manner known per se with suitable auxiliaries and / or carriers and / or diluents to give solid dosage forms, to injection or infusion solutions or to topical dosage forms. The oral one Administration of the active compounds according to the invention is preferred. Parenteral administration is of course also possible. The preparation of the pharmaceutical formulations is not a problem for the person skilled in the art. A method for the pharmaceutical preparation of infusion or injection solutions of hypericin or hypericin derivatives is, for example, in DE 39 12 433 A1 described.

It is also possible according to the invention to use a plant extract rich in hypericin, in particular from St. John's wort, particularly preferably from Hypericum perforatum, instead of the pure hypericin / hypericin derivative. The production of such an extract is, for example, in DE 39 35 772 A1 described. Commercially available St. John's wort dry extracts, for example Esbericum ^® capsules (Schaper and Brümmer, Salzgitter Germany), are also hypericin-rich. For the purposes of the invention, hypericin-rich means that at least 0.3 to 0.35% by weight of hypericin is contained in the dry extract. In St. John's wort, hypericin and pseudohypericin occur side by side, so that hypericin-rich St. John's wort extracts always contain the pseudohypericin in addition to hypericin, which also works in the sense of the invention.

As is known, commercially available St. John's wort extracts, including also the hypericin-rich, for the treatment of mood disorders and nervous Unrest is used, and by large parts, especially the older population are used, according to the invention, at the same time it becomes a tumor preventative Effect achieved.

Object of the present invention is also a method of tumor prevention in which the above described pharmaceutical preparations, human or animal body applied in an amount that is sufficient and effective, the epoxidation activity to inhibit the enzyme CYP1A1. It has been shown that plasma concentrations of approx. 13 μg Hypericin / L, which is more common Administration of 900 mg St. John's wort dry extract / day on average in stationary Condition can be achieved (Johne et al., In Clin. Pharmacol. Ther. (1999) 338-345) more than 90% inhibition of CYP1A1 in the in vitro experiment to lead. Based on this, the dose of hypericin or hypericin derivative should usually be approx. 10 to 200 μg / day amount, preferably from 70 to 200 μg / day.

Object of the present invention continue to use hypericin and / or its derivatives for the production of a degradation of toxic and procarcinogenic substances promoting Food or nutritional supplement as well as the food or nutritional supplement, the hypericin and / or its derivatives in a nutritionally effective amount. It deals is a food or nutritional supplement that inhibits the epoxidation activity of the enzyme CYP1A1 inhibits. The hypericin can preferably also be in the form of a hypericin-rich herbal dry extract, preferably from St. John's wort, in foodstuffs according to the invention or nutritional supplements be included.

The food or nutritional supplement according to the invention For example, a food powder made from milk powder, carbohydrates, Vitamins, minerals and lipids that are at least 0.3% by weight Contains Hyperin / Hypericin derivative. It is self-evident possible, also any other food powder with hypericin / hypericin derivatives to enrich.

The invention is described in more detail below. without restricting it to the stated embodiments.

Show it:

1 : Activation of Benz [a] pyrene by CYP1A1 to Its Ultimately Carcinogenic Form;

2 : Inhibition of human CYP1A1 activities by resveratrol;

3 : Inhibition of human CYP1A1 activities by hypericin;
The EROD activity cannot be determined on the strong intrinsic fluorescence of hypericin.

4 : Inhibitory potential of hypericin, quercetin and α-NF;
Effect on the 7,8-diol-B [a] P epoxidation activity of human CYP1A1.
The reciprocal IC ₅₀ values were shown for all three inhibitors.

embodiments materials

Quercetin, B [a] P, 7-ethoxyresorufin, Resorufin, alpha naphthoflavone, Dilaurylphosphatidylcholine (DLPC) were obtained from SIGMA (Deisenhofen, BR Germany) bought. 3-OH-B [a] P, 7,8-dihydrodiol-B [a] P, the Tetrols r-7, t-8, t-9, c-10-tetrahydroxy-7,8,9,10-tetrahydro-B [a] P (RTTC), r-7, t-8, t-9, t-10-tetrahydroxy-7,8,9,10-tetrahydro-B [a] P (RTTT), r-7, t-8, c-9, t-10-tetrahydroxy-7,8,9,10-tetrahydro-B [a] P (RTCT), r-7, t-8, c-9, c-10-tetrahydroxy-7,8,9,10-tetrahydro-B [a] P (RTCC) at NCI Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MI, USA).

Cloning, heterologous expression and purification of the recombinant CYP1A1

Human CYP1A1 and P450 reductase were heterologously expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system and then isolated and purified. The cDNAs were cloned into baculovirus transfer vectors pBlueBac4.5 (Invitrogen, Netherlands) as described by Schwarz et al. in Phar macogenetics 10 (2000) 519-530. An N-terminal 72 bp SphI-SexAI fragment was then replaced by the same with the following modifications: the coding sequence upstream of the stop codon was expanded by a coding sequence for a thrombin cleavage site and 6xHis residues. The entire modified CYP1A1 sequence was then cloned into the vector pAcMP3 (Pharmingen, USA) (under the control of the 'late basic protein promoter' P _cor ). The pAcMP3 plasmid modified in this way was amplified in E. coli and used for the preparation of recombinant baculoviruses (according to the protocol of the manufacturer Pharmingen). The expression took place in 400 ml Sf9 spinner cultures over approx. 55 h, after which the cells were harvested, lysed and the CYP1A1 protein isolated and purified as a 6xHis fusion protein as described by Modi et al. in Biochemistry 35 (1996) 4540-4550, but with the modification that a mixture of emulsions 913 (2%) and cholate (0.2%) was used for the solubilization of the membranes. CYP1A1 was electrophoretically homogeneous with a specific P450 content of 11 nmol / mg protein. The expression of the reductase was carried out analogously, its purification was carried out according to the method described by Tamura et al. in Arch. Biochem. Biophys 293 (1992) 219-223.

example 1

Implementation of the Epoxidationstests

This test (epoxidation of 7,8-diol-B [a] P) serves to demonstrate the terminal epoxidation during activation of B [a] P to the B [a] P-diolepoxides 1 and 2 (DE1 and DE2), whereby DE2 is the ultimate carcinogenic metabolite that leads to cancerogenesis through DNA adduct formation. The 7,8-diol-B [a] P epoxidation was detected by HPLC separation and spectrophotometric detection of the products in accordance with published methodology (Yang et al., In Cancer Res. 35 (1975) 3642-3650; Schwarz et al. , in Carcinogenesis 22 (2001) 453-459), except for the use of a reconstituted enzyme system consisting of the purified enzymes (CYP1A1 and P450 reductase) and lipid (DLPC). The reconstituted system, consisting of 200 pmol purified CYP1A1 (28 nmol / ml), 900 pmol purified reductase (56 nmol / ml) and 0.5 mg DLPC (5 mg / ml), was dissolved in 200 μl buffer (50 mM Tris / HCl, 100 mM NaCl, pH 7.5) incubated on ice for 10 min. For the test, 10 μl aliquots of this enzyme-lipid mix (corresponding to 10 pmol CYP1A1) per reaction with the substrate 7,8-diol-B [a] P (final concentration: 2 μM) and the corresponding inhibitor concentration in 200 μl buffer incubated, then diluted to 990 μl and preincubated again at 37 ° C. for 1 min. The reaction was started by adding 10 μl of NADPH (16.6 mg / 200 μl) and carried out in a water bath at 37 ° C. for 15 min. The reaction was then stopped by adding 100 μl of 1M K-phosphate buffer (pH 3.5) and the suspension was left for hydrolysis at 4 ° C. for 1 h. After addition of 10 μg of 1,1'-bi-2-naphthol (10 μg / 10 μl of methanol) as an internal HPLC standard, the products and substrate were extracted twice using ethyl acetate. After evaporation of the solvent, the product and substrate were taken up in 100 μl of methanol, homogenized in an ultrasonic bath and injected into the HPLC system. For the IC ₅₀ and kinetic studies, stock solutions with a suitable inhibitor concentration in DMSO were prepared to ensure that the DMSO concentration was <0.5%. The controls (without inhibitor, 100% activity) were carried out with the appropriate amount of DMSO.

Separation and identification of the Products and their analysis were carried out using reversed-phase HPLC according to the method described by Schwarz et al., in Carcinogenesis 22 (2001) 453-459 and Shou et al. in Mol. Carcinogenesis 10 (1994) 159-168) Methodology performed. Although both diol epoxides DE1 and DE2) were analyzed as products, relate to all those listed and discussed in the present work activities and education rates, including of kinetic analysis, on DE2, as it is the ultimate carcinogenic Product represents and is formed at a significantly higher rate than DE1 becomes.

Example 2

Implementation of the AHH tests

This test (aryl hydrocarbon hydroxylation, AHH) is an assay to quantify the CYP1A1-catalyzed hydroxylation of B [a] P to 3-OH-B [a] P. The reaction characterizes the detoxification route (cf. 1 ). The B [a] P hydroxylation was quantified by fluorescence detection of the products according to the classic AHH test (Nebert and Gelboin in J. Biol. Chem. 243 (1968) 6242-6249). The reconstituted CYP1A1 system (enzyme-lipid mix) was prepared as described for the epoxidation test (example 1). However, 20 μl aliquots (corresponding to 20 pmol CYP1A1) were used per assay and dissolved in 250 μl final volume together with the substrate B [a] P (final concentration: 5 μM) and the corresponding inhibitor concentration. Preincubation and reaction were carried out as described in Example 1. The reaction was quenched with 1 ml acetone (cold) and the products and substrate extracted with 3.25 ml n-hexane. 1 ml aliquots of the organic phase were mixed with 3 ml of 1N NaOH and the 3-OH-B [a] P formed was detected fluorometrically (excitation: 396 nm, emission: 519 nm, spectrofluorimeter RF5000-PC, Shimadzu, Japan). The quantification was carried out using a calibration curve, which was recorded under the same conditions with authentic 3-OH-B [a] P.

Example 3

Implementation of the EROD tests

This test (7-ethoxyresorufin-O-deethylation, EROD) is the well-known standard assay for the characterization of activity from CYP1A1, which helps to convert the substrate to resorufin fluorescence spectroscopy (Burke and Mayer in Chem. Biol. Int. 45 (1983) 243-258). The reconstituted CYP1A1 system was described as in Example 1 prepared. 10 ul aligote (corresponding to 10 pmol CYP1A1) were mixed with the substrate (final concentration: 0.5 μM) and the corresponding inhibitor concentration in 200 μl, to 790 μl diluted and 1 min at 37 ° C preincubated before the reaction was started by adding 10 μl of NADPH. To 15 min at 37 ° C the reaction was stopped by adding 1.5 ml acetone (cold). The suspension was then centrifuged at 3000 rpm for 5 min and the product resorufin formed by measuring its fluorescence detected (excitation: 530 nm, emission: 585 nm). The quantification was carried out using a calibration curve under the same conditions was created with authentic resorufin.

Example 4

Determination of the IC ₅₀ values, determination of the inhibition constants for hypericin and quercetin

For the determination of the IC ₅₀ values, the activities (for example the rate of DE2 formation) were measured as a function of the inhibitor concentration, as described in Examples 1-3. The activities measured were based on the control activity (100%) in the absence of the inhibitor. The IC ₅₀ value was then determined by linear regression of the percentage activities vs. the logarithmically transformed inhibitor concentrations. The inhibition constants for hypericin and quercetin were determined by analyzing the inhibitor kinetics, ie by measuring the enzyme kinetics for different inhibitor concentrations. For hypericin, the analyzes were carried out with a substrate concentration of 2 μM for four inhibitor concentrations (0, 1, 2 and 5 μM). For quercetin, the measurements were carried out at the same substrate concentration for the following inhibitor concentrations: 0, 1, 5 and 10 μM. After that, the conversion rates were first analyzed by Lineweaver-Burk plots and the type of inhibition (competitive, non-competitive, uncompetitive or mixed type) before the kinetic constants, including the inhibition constants K _i , by non-linear regression using SIGMA-PLOT-2001 and ENZYME KINETICS module 1.1 (software from SPSS Science Software, Erkrath, BR Germany).

Claims (10)

Use of hypericin and / or its derivatives for the manufacture of a drug for tumor prevention. Use according to claim 1, characterized in that that a hypericin-rich herbal dry extract is used is preferably a St. John's wort extract. Use of hypericin and / or its derivatives for the manufacture of a medicament for inhibiting the epoxidation activity of the enzyme CYP1A1 (Cytochrome P450 1A1). Use according to claim 3, characterized in that that a hypericin-rich herbal dry extract is used is preferably a St. John's wort extract. Procedure for tumor prevention by administration of a preventive pharmaceutical composition in an amount sufficient is the epoxidation activity to inhibit the enzyme CYP1A1, the composition of which is effective Amount of hypericin and / or its derivatives includes. A method according to claim 5, characterized in that the composition to be applied is hypericin-rich vegetable dry extract, preferably a St. John's wort extract. Use of hypericin and / or its derivatives for the manufacture of a food or nutritional supplement. Use according to claim 7, characterized in that that a hypericin-rich herbal dry extract is used is preferably a St. John's wort extract. Food or food supplements comprising a nutritionally effective Amount of hypericin and / or its derivatives. Food or nutritional supplements according to claim 9, comprising a hypericin-rich vegetable dry extract, preferably a St. John's wort extract.