JP2002503699A - Use of squeezed juices, digestive juices or extracts of meat plants to inhibit protein kinases - Google Patents

Abstract

  (57) [Summary] The present invention relates to the use of squeezed juices, digestive juices or extracts of carnivorous plants for the manufacture of a protein kinase inhibiting agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the inhibition of protein kinases using squeezed juices, digestive juices or extracts of meat plants.

[0002] Recent research in the field of drug actives has focused on the field of naturally produced drugs. Not only is the surprisingly large number of actives found in plants and natural microorganisms, but naturally produced drugs are often compared to synthetically produced actives, which cause severe and undesirable side effects. Often has the surprising advantage of being well tolerated. Carnivorous plants are an interesting example of a natural active substance source. Thus, for example, squeezed juice of certain carnivorous plants is used to control malignant diseases such as cancer, Crohn's disease, ulcerative colitis, herpes and AIDS. This is, for example, EP-A-2
49465, EP-A-219295 and DE-A-2920631.

[0003] At the same time, there is a wide range of diseases for which conventional dosing with synthetic active substances shows virtually no success. Gastric ulcers are an example of such a disease, for which expensive chemotherapeutic treatments are currently being given. Currently the most commonly used treatments are
For now, it is an improved triplet therapy. These treatments are described, for example, in the technical magazine "
Muench, med. WSCHR, 140 (1998), No. 7, pp. 90-93 and Deutsches AErztem.
agazin No. 51/52, December 1997, der Kassenarzt ", pp. 31-34. But these treatments, like similar chemotherapy in cancer,
The disadvantage is that it has serious side effects.

[0004] It is therefore an object of the present invention to provide an active substance or a combination of active substances which can suppress diseases which respond to the inhibition of protein kinase with as few side effects as possible.

[0005] This object is achieved by using pressed juices, digestive juices or extracts of carnivorous plants for the inhibition of protein kinases. Hereinafter, these are collectively referred to as liquid components.

[0006] Protein kinases play a central role in cell growth, division and differentiation.
Growth signals that bind in the form of growth factors to specific recognition structures (receptors) outside the cell membrane are transmitted to the cell nucleus via various kinase cascades, where they result in the modification of transcription signals. As a result, certain genes are activated or transcription of other genes is stopped.

[0007] Surprisingly, squeezed juices, extracts or digestive juices of carnivorous plants allow the inhibition of protein kinases, while at the same time controlling a variety of diseases that have hitherto been very difficult to treat Was found here.

[0008] Specifically, it has been found that the liquid fraction of carnivorous plants exhibits effects similar to conventional antibiotics, but with significantly less side effects. The carnivorous plant fluid has an effect on Gram-positive bacteria, Gram-negative bacteria, fungi and other microorganisms.

Specifically, the liquid fraction used according to the present invention comprises plasmodium, chlamydia, trypansomes, photosynthetic bacteria (phototrophic bacteria), staphylococci, streptococci, pneumococci, Klebsiella, Suitable for controlling Candida, mold, Aspergillus and bacterium pyocyaneus. Perhaps the microorganisms, bacteria and fungi mentioned above are inactivated by inhibition of protein kinases. Specifically, squeezed juices, extracts or digestive juices of carnivorous plants are suitable for controlling Helicobacter pylori, one of the most common causes of gastric ulcer.

[0010] A series of experiments further confirmed that the following microbial, fungal and bacterial control could be achieved using the flesh of carnivorous plants: Pseudomonas aeruginosa (
Pseudomonas aeruginosa, P. mirabilis (Proteus mirabi)
lis, mirabilis variant, Staphylococcus aureus
aureus, Staphylococcus aureus, Streptococcus pneumoniae
pneumoniae, Streptococcus pneumoniae, Escherichia coli, Staphylococcus epidermis, Group B streptococci (haemolytic), Streptococcus pyogenes, Streptococcus pyogenes (A)
), Candida albicans, Candida albicans, Aspergillus fumigatus, Pneumococcus mucosus, Bact. Pyocyaus
neus), Bacterium Proteus vulgaris,
Klebsiella oraeanae, Candida mycod., Candida tropicaus and Mucor racemosus.

[0011] The acquisition of pressed juice, digestive juice or extract of meat plants and preferred examples of presentation are described below.

The meat plant may be any meat plant. Preferably, Helianfora (Heli
amphora, genus Sarracenia, genus Darlingtonia, cephalotus (Cephalotus), Nepenthes (Ne)
phentes). Preference is also given to carnivorous plants of the family Drosera, especially Dionaea muscipula, or shreds.

[0013] Squeezed juices, extracts or digestive juices of carnivorous plants can be used.

Since digestive juice can be collected by tapping, the use of digestive juice has the advantage that it is not necessary to destroy the plant to obtain it. In this case, the yield can be considerably increased by constant feeding of the plant. Usually, carnivorous plants are fed a piece of meat of the same tissue over an extended period of time. This also has the consequence of stabilizing the composition of the digestive juice, that is to say that in the same type of plant the individual components of the digestive juice are present in similar proportions. Without feeding, this is not the case, and since the active substance is present at different concentrations in each plant, its therapeutic effect can only be achieved by mixing the digestive juices of several plants. Does not lead to Stimulation of secretion can be performed to increase the amount of digestive secretions provided by the plant. Spraying uric acid on plants is particularly suitable for this, and it has been found that uric acid residues have no detrimental effect. Other agents suitable for stimulating secretion are bactopeptone, califora (ka
lifora), glutamic acid, yeast extract, peptone, taurine, proline and asparagine. Digestion fluid is conveniently collected by aspirating with a pipette or by making a small stab wound inside the fold and then collecting the secretion with a micropipette.

However, squeezed juice can also be used according to the invention. The juice is advantageously produced using fresh plants, i.e. fresh plants before the onset of the enzymatically induced wilting process. Usually, harvested plants are first washed to remove extraneous components. The washed plants are then shredded, ground or crushed and squeezed wet. It is advantageous to treat them with pressurized steam for a few seconds before pressing in order to inactivate the enzymes, achieve disintegration by partial destruction of the cell wall and to facilitate the subsequent clarification procedure by albumin precipitation. is there. Similarly, ultra-rapid freezing can be performed.

For the pressing, a continuous operation type fly press, a hydraulic filter press, and a continuous screw press (screw press) are generally used. The parameters to be set for these tasks are well known to the expert. The squeezed juice can then be processed by conventional methods such as filtration, centrifugation, separation and the like. If necessary, it is then subjected to ultra-high temperature sterilization before filling in sterile bottles. It is then preferably cooled as quickly as possible to a temperature below 70 ° C.

[0017] Extracts obtained from carnivorous plants can also be used. In that case, the following extractants and methods can be used.

Extractants: cold water, hot water, dilute acetic acid, sweet wines, ethanol-water mixtures, ethanol and fatty oils or neutral oils and supercritical gases such as supercritical CO ₂ .

Extraction method: Static immersion, stirring immersion, turbulent extraction, digestion, percolation, repercolation, evacolation, diacolation, combination of immersion and percolation , Ultrasonic extraction, countercurrent extraction, and extraction with separators, centrifuges and decanters.

The equipment to be used for this and the commonly used process parameters are well known to the expert.

The product obtained by tapping, squeezing and extracting digestive juices can be further processed in the usual way. Specifically, these liquid components can be freeze-dried (particularly, freeze-dried under reduced pressure). For this purpose, ordinary additives such as mannitol and glycerol can be used during freeze-drying under reduced pressure. The equipment and process parameters required for this are well known to the expert. In addition, various additives that have a favorable effect on the therapeutic effect upon administration, such as formic acid, benzoic acid, citric acid, malic acid, cyanidin-3-glucoside, sodium, magnesium, potassium, calcium, chlorine, naphthoquinone, L- Arginine, L-asparagine, L-serine, L-glutamic acid, L-alanine, L-threonine, L-lysine, glycine, L-tyrosine, L-phenylalanine, L-valine, L-leucine and L-isoleucine, etc. It can be added to these liquid components.

The carnivorous plant fluid can be administered in various ways.

The liquid can be used directly, orally or topically. Alternatively, they may be diluted in advance with a harmless solvent. Examples of suitable solvents are water, sweet wines, ethanol-water mixtures and salt solutions.

Usually they have a dry residue of 0.1 to 10% by weight, preferably 0.5 to 5% by weight,
It is particularly preferably diluted to 0.8 to 2.5% by weight.

Further, the liquid component of the meat plant can be orally administered in the form of tablets or capsules. The equipment and formulations required for this are well known to the expert. In addition, topical application in the liquid form itself or in the form of an ointment is also possible. The equipment, materials and recipes required for the manufacture of ointments are well known to the expert.

It is also possible to produce parenteral preparations from carnivorous plant fluids. They can be administered intramuscularly, subcutaneously or intravenously. The equipment, additives, and recipes required for the manufacture of such parenteral formulations are well-known to the expert.

The following experiments illustrate the invention. 1. Protein Kinase Inhibition Samples: Carnivora® squeeze diluent and Carnivora® squeeze neat from Carnivora Research.

Until the start of the experiment, both samples were kept unopened in the refrigerator. On the day of the first experiment, both samples were pre-diluted 1:10 and 1: 100 with water and DMSO, respectively, to obtain 1:10 and 1: 100 initial solutions of both samples in 10% DMSO. A further 1:10 dilution was performed each time upon addition to the test system. Both samples were tested at 1: 100 and 1: 1000 dilutions. DMSO in this assay
The concentration was 1%. The pre-diluted initial solution was stored at 4 ° C. and used for all measurements over 10 days.

Protein kinases used: Cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 2 (CD
K2) Platelet-derived growth factor β receptor tyrosine kinase (PDGF-Rβ-TK
Epidermal growth factor receptor tyrosine kinase (EGF-R-TK) ERBB2 receptor tyrosine kinase ERBB2-TK Fibroblast growth factor receptor 1 tyrosine kinase (FGF-R1-TK) Insulin-like growth factor receptor type 1 Tyrosine kinase (IGF-1-RT)
K) Insulin receptor tyrosine kinase Ins-R-TK protein kinase C subtype γ (PKC-γ) protein kinase C subtype ε (PKC-ε) Janus kinase 2 (JAK2)

These protein kinases were cloned from appropriate cellular RNA using RT-PCR. Next, the isolated DNA fragment was subcloned into a baculovirus transfer vector to prepare a recombinant baculovirus. The proteins were expressed in Sf9 insect cells using these recombinant baculoviruses. The kinases were purified from insect cell lysates using glutathione agarose gel.

All kinase assays were performed in 96-well microtiter plates at 100 μl
l. The reaction was performed with the amount of the reaction solution. When measuring PKC activity, the test mixture is 50 mM
HEPES-NaOH (pH 7.5), 1 mM EDTA, 1.25 mM E
GTA, 5 mM MgCl ₂ , 1.32 mM CaCl ₂ , 5 μg / ml phosphatidylserine, 1 μg / ml 1,2-diolein, 1 mM DTT, 0.1 μm
[ ³³ P] -ATP, 50 ng of recombinant kinase, and 0.5 μg of histone H1 as substrate. For all other kinases, the test mixture was 50 mM
HEPES-NaOH (pH 7.5), 3 mM MgCl ₂ , 3 mM MnCl ₂ , 3 μM sodium orthovanadate, 1 mM DTT, 0.1 μM ATP, 100 ng of recombinant kinase and appropriate substrate.

The reaction mixture was incubated at 30 ° C. for 80 minutes. The reaction was stopped by the addition of 100 μl H ₃ PO ₄ (2%). Then the plates were washed with H ₂ O 3 times.
Washed twice. The amount of [ ³³ P] -phosphate incorporated into the relevant substrate was determined by a scintillation counter for a microtiter plate. The results are summarized in the following table.

[0033]

[Table 1]

[0034] These results clearly show that neat and diluted juice of Carnivora both cause inhibition of protein kinase.

II. Inhibition of Helicobacter pylori A study on eradication of Helicobacter pylori was performed in a rat duodenal ulcer model. In this ulcer model, rats were used which colonized Helicobacter pylori predominantly. After oral administration of the ulcer-inducing agent cysteamine hydrochloride, duodenal ulcers and gastric ulcers could be detected 100% both visually and microscopically 48 hours after administration.

[0036] Carnivora® squeezed juice (2% dry residue) was added to 0.06, 0.6
And orally at three doses of 2.83 ml / kg body weight (BW) three times daily for 20 days after ulcer induction. Reference group (tetracycline (6 mg / kg-B
W), metronidazole (15 mg / kg-BW) and basic bismuth nitrate (
30 mg / kg-BW)) and a control group (isotonic saline solution, 0.9% NaCl)
The same dosing regimen was used.

At 2, 5, 8, 14, and 21 days after ulcer induction, each group was examined for ulcer healing by gross evaluation and measurements of Helicobacter pylori were also performed. The following parameters were targeted: average rating score (mAS) based on the severity of duodenal and gastric ulcers, percentage of ulcers (% U), ulcer index (UI) (mAS x%
U), ulcer area, weight and number of deaths.

This model showed the expected effect of the combined use of three reference substances. The ulcer index decreased, the lesion area of duodenal or gastric ulcer decreased, and the incidence of Helicobacter pylori decreased.

The three doses of Carnivora juice tested showed very good dose-dependent effects. The ulcer index decreased, the lesion area of duodenal or gastric ulcer was reduced, or the ulcer was completely removed. The incidence of Helicobacter pylori could also be reduced, and eradication of this organism was achieved at the highest dose. Further efficacy of the test substance was manifested by a reduction in spontaneous death occurring during the first 3 days after administration of the ulcer-inducing agent and by a greater weight gain than the control group over the 0-21 day test period.

The effect of Carnivora juice shown here confirms its excellent effect on this model.

[0041]

[Table 2]

[0042]

[Table 3]

[0043]

[Table 4]

[0044]

[Table 5]

[0045]

[Table 6]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW

Claims (6)

[Claims] 1. Use of a pressed juice, digestive juice or extract of a meat plant for producing a protein kinase inhibitory drug. 2. The use according to claim 1, wherein the carnivorous plant belongs to the genus Helianfora, the genus Sarracenia, the genus Darlingtonia, the genus Cephalotas or the genus Nepenthes. 3. The use according to claim 1, wherein the carnivorous plant belongs to the family Araceae. 4. Use according to at least one of the preceding claims, wherein the extract is an aqueous extract. 5. Use according to at least one of the preceding claims for inhibiting photosynthetic bacteria, Plasmodium, Chlamydia, Trypanosomes, Staphylococci, Streptococci, Candida, Mold and Aspergillus. 6. A method of treating mammals comprising administering a squeezed juice, digestive juice or extract of a carnivorous plant to inhibit protein kinase.