SI22466A - Combination containing plant extracts and its use for treatment of various forms of cancer - Google Patents

Abstract

A combination containing plant extracts is described, having the following composition: extract of plants of the genus Capsicum, extract of plants of the genus Allium, HEPES and elementary sulphur, preferentially under addition of an antibiotic, in various ratios with respect to the purpose of application, where the components are optionally suspended in a physiological solution of common salt. The above combination is applicable as the agent for triggering apoptosis of cancer cells, without producing, at the same time, neither a high occurrence of apoptosis in immortalized cells nor injuries of healthy cells.

Description

A combination containing herbal extracts and its use for the treatment of various cancers

Technical area

The invention is in the field of medicine and relates to a combination containing plant extracts useful for the treatment of various cancers. Capsicum and Allium plant extracts in different concentrations can treat many different cancers, e.g. neural, uterine, breast, liver, etc.

A technical problem

There is an ongoing need to treat various types of cancer by causing the cancer cells to die. One of the most commonly used methods that allow cancer cells to die is the induction of apoptosis in these cells. In the present invention, we have sought to discover novel products that can trigger apoptosis in cancer cell lines, while avoiding the high incidence of apoptosis in immortalized cells and also not damaging healthy cells.

The state of the art

Historical and archeological data, as well as epidemiological data on the incidence of cancer in certain geographical areas, show that various plant extracts have been used sufficiently successfully to prevent cancer.

Otherwise, there are few solutions listed in the professional literature.

One known solution similar to the present invention is described in EP 92226. Plants of other genera are used, and the compound for which protection in EP 92226 is claimed does not appear to be selective for cancers / non-cancers cells.

Description of the solution

Efforts have been made to identify novel substances that can trigger apoptosis in cancer cell lines (eg human cancer cell neural cell line: SHSY5Y, breast cancer cell line MCF-7, human uterine cancer cell line: HeLa, a human HepG2 cancer cell line) would not, at the same time, trigger the high incidence of apoptosis in immortalized cells (eg human keratinocyte cell line: HaCaT, murine endothelial fibroblast cell line MEF) and would not damage healthy cells (eg cell line NHDF human fibroblasts).

Surprisingly, we were able to come up with a product that met the requirements above.

The first object of the invention is a combination containing plant extracts, characterized in that it has the following composition:

Capsicum herb extract, Allium herb extract, and as HEPES sulfur-based and elemental sulfur-based supports, preferably with antibiotic supplementation, in different proportions according to the purpose of use, the constituents being optionally suspended in saline.

Penicillin and streptomycin can be added as antibiotics and are administered at a concentration of 0.5% by volume each.

An average expert in the field will easily determine the ingredient ratios based on his or her knowledge and experience.

As examples of Capsicum plants, for example, Capsicum chinense.

As examples of plants of the genus Allium, for example, Allium sativum, raised in Egypt, Allium neapolitanum.

Another object of the invention is a process for the preparation of the combination of the invention, characterized in that the Capsicum and Allium plants are sun-dried and completely dried, powdered, suspended in saline with HEPES and elemental sulfur, and preferably antibiotics, allow the solids to settle for a couple of days, while shaking several times, then filtering the whole and filtrate either stored at 4 to 10 ° C or dried.

Another object of the invention is the combination defined above for use as an agent for triggering cancer cell apoptosis. In particular, the cancer cells are the human cancer cell line SH-SY5Y, the human breast cancer cell line MCF-7, the human uterine cancer cell line HeLa, and the human liver cancer cell line HepG2.

This was found in in vitro experiments, which were otherwise based on standard procedures, except that there were no animal components in the culture medium.

The combination of the invention is all possible combinations that maintain or have similar specificity and efficacy as will be described below.

The invention also relates to the use of said combination for the preparation of a therapeutic agent for the treatment of cancer, e.g. neural cancer, breast and uterine cancer, liver cancer, and this agent also contains a suitable carrier, solvent or other additive together with sulfur-based support substances. An experienced person in the field of cancer therapy will thus be able to establish a therapeutically effective amount.

Therefore, one of the objects of the invention is the combination defined above for use as a means of triggering apoptosis of cancer cells.

The echo mode is tailored to provide the optimum desired response (eg, therapeutic response). It is particularly preferred to prepare parenteral active ingredients in the dosage unit for ease of administration and uniformity of dosage. It is appropriate to use the dosage unit as a single dosage for the treatment of persons; Each unit should contain a predetermined amount of the active substance, calculated according to the desired therapeutic effect, together with the required pharmaceutical carrier. The characteristics of the dosage unit of the invention depend and are directly determined in the following steps:

a) the unique characteristics of the active ingredient and the specific therapeutic effects to be achieved; and

b) restrictions on the preparation of the active substance for the treatment of sensitive individuals (sensitive to the active substance or to the auxiliary ingredients or both).

The terms parenteral dosing and parenteral dosing refer to non-enteral or topical dosage routes that are usually administered by injection or infusion. They include intravenous, intramuscular, intraarterial, intradermal, intraperitoneal and subcutaneous initiation or infusion.

The combination of the invention may be formulated into a pharmaceutical form together with a suitable carrier or solvent. Examples of suitable water-soluble and non-water-soluble carriers that can be used to formulate the pharmaceutical form include water, polysorbate 80, vegetable oils (e.g. olive oil) and injectable esters (e.g. ethyl oleate). The ingredients listed may also contain additives such as preservatives, solubility enhancers, emulsions and sprays. The absence of micro-organisms is ensured by sterilization techniques (eg filtration without heat) and by the addition of antibacterial and fungal agents (eg penicillin, streptomycin, sorbic acid, etc.). It is advisable to add isotonic agents such as sodium chloride and the like to the combination of the invention. In the following, prolonged absorption of the pharmaceutical ingredient for injection can be ensured by the addition of absorption inhibitors, e.g. with the addition of gelatin.

When dosing the ingredients of the present invention as medicaments to animals or humans, they can be dosed alone or in pharmaceutical form in a ratio of 0.005: 0.995 (99.5% of active ingredient and 0.5% of pharmaceutically acceptable carrier).

Similar to WO 2005/050213, and regardless of the dosage method of the combination of the present invention, which can be used in a suitable water-soluble or / and pharmaceutical formulation, it is formulated into a pharmaceutical dosage form using standard methods known to those skilled in the art. .

The specific dosage values of the active ingredients of the present invention in the pharmaceutical form may vary depending on the preparation of the active ingredient, which is effective and which achieves the desired therapeutic effect for the individual patient, in terms of composition and non-deleterious dosage route. The dose selected will depend on a number of pharmacokinetic factors that include the activity of each component of the invention, such as dosage regimen, timing of dosing, bioavailability of the substances themselves, duration of treatment, presence of other drugs (concomitant therapy), ingredients and / or materials used in in combination with an individual ingredient, age, sex, weight, endurance and general health, history of the treated patient, and similar factors commonly known in medical practice.

The doctor or veterinarian may start dosing the ingredients of the present invention in pharmaceutical form and use the amount of the substance that is the lowest effective therapeutic dose. Such an effective dose will usually depend on the factors described above.

Therapeutic doses can be dosed with medical devices commonly known in medical practice. The therapeutic dose of the invention can best be dosed with well-known inserts and carriers, as stated in US Pat. No. 4,447,233, which describes a drug dispensing infusion pump that dispenses the drug in a precise time sequence. The present invention can be metered by the device described in US Pat. No. 4,447,224, showing an insert in the form of a device for different flow infusion intended for continuous dosing of the drug.

Certain embodiments of the combinations of the invention may be prepared in such a way as to ensure proper distribution in the organism. For example, take the blood barrier (BBB), which makes it impossible for many highly hydrophilic agents to pass through. In order to ensure the passage of the therapeutic agents of the present invention (if desired), they can be introduced into the liposome. For liposome manufacturing methods, please refer to the following patents: US Nos. 4,552,811; 5,374548 and 5,399,331. Liposomes can contain one or more active substances that are selectively transferred to specific cells or organs, thereby increasing the targeted rate of erosion (V. V. Ranade, 1989).

The combination according to the invention should preferably be inserted into the liposome. In the enhanced model, liposomes are targeted at a specific target. At best, the therapeutic agent introduced into the liposome is directly injected into the site where the therapeutic effect is desired (eg, in the part of the body where the tumor is located). The ingredient must be fluid to the point that it is easy to squeeze when injected into the body. It must be resistant to the conditions of production and storage and must be protected against microbial contamination (eg contamination with bacteria and fungi).

Effective dosages and dosage regimens (as described in WO 2005/050213) for the combinations of the invention depend on the degree of disease and health status of the treated patient. The dosage regimen can be determined by a person with experience in the field.

The therapeutically effective dose for tumor therapy can be measured by objective tumor responses to therapy. The tumor response may be complete or partial. CR-complete response is defined as clinical, radiological or other relevant evidence of absence of disease. PR-partial response occurs when the size of a thickened tumor is reduced by more than 50%. Average time for escalation is a measure that indicates the duration of tumor response.

Therapeutically effective dosage for tumor therapy can also be measured by its ability to stabilize the progression of the disease. The ability of an ingredient to stop the development of cancer can be evaluated in an animal model and thus predict the efficacy for treating a human tumor. A therapeutically effective amount of a therapeutic ingredient may reduce the size of the tumor or otherwise reduce the symptoms in a person or animal. By one of the routine procedures in the art, such an amount can be determined according to the size of the subject, the degree of expression of the symptoms of the subject, and the specificity of the ingredients or method of dosage selected.

In view of the above facts, patients treated with the components of the invention may be further treated by therapeutic agents and methods that enhance or enhance the therapeutic effect of the components of the invention described (eg, photodynamic therapy; Plaetzer et al., 2003). In order to increase the effectiveness of derivative therapy of the abovementioned plant extracts, patients receiving derivatives of the abovementioned plant extracts should consume vegetarian food throughout the treatment.

To demonstrate whether the combination of the invention can induce apoptosis in cancer cells, we selected the appropriate cell lines, observed cell morphology under a light microscope, performed a cell viability test, performed a caspase activity test, calculated the number of apoptotic cells with a FACS Calibur flow cytometer, and performed the test triggering PARP.

In order to identify the pathways (intrinsic or extrinsic) through which the combination of the invention triggers apoptosis, we further used broad-spectrum inhibitors of caspases and cathepsins (z-VAD-fmk and E-64 d) and a DNA damage inducer (mitomycin C).

The invention is explained by the following non-limiting examples.

Example 1

The preparation of a combination containing a plant extract of the invention

The plants of Capsicum chinense, Allium sativum and Allium neapolitanum are sun-dried. When completely dried, grind them to powder. The powder is suspended in saline together with sulfur-based support materials, namely HEPES and elemental sulfur with the addition of streptomycin (final suspension concentration of 0.5% vol) and penicillin (final suspension concentration 0.5% vol) . Allow the suspension to stand for 1 week and shake repeatedly. It is then filtered. There is obtained a combination of the invention which can be formulated into pharmaceutical preparations.

One specific example:

450 mg of the powder of each of the abovementioned plants in brine to a concentration of 10 mg / mL;

HEPES 1% by solution; elemental sulfur 0.01% by solution.

The solution can be stored as such at 4 to 10 ° C and is effective for 2 months or dried and processed immediately before use.

Example 2

The process of handling cells

The solution of Example 1 was incubated with the cells being examined (cell type is defined in the figures below) for one week at room temperature and filtered. The solution was then added to a culture medium prepared by standard procedures except that it did not contain any constituents of animal origin. Cells were grown according to standard procedures described in Ausubel et al. Current Protocols in Molecular Biology. Wiley Interscience 1-4 (1998); Bonifacino et al. Current Protocols in Whole Biology. Wiley Interscience 1-3 (2003). The solution is then refrigerated at 4-10 ° C and is effective under these conditions for two months.

Example 3

In vitro experiments

Various tests were performed with the solution obtained in Example 2, namely viability tests, caspase-3 activity assay, flow cytometry, PARP assay.

Viability and caspase-3 activity assays were performed according to standard procedures described in Ausubel et al. Current Protocols in Molecular Biology. Wiley Interscience 1-4 (1998); Bonifacino et al. Current Protocols in Whole Biology. Wiley Interscience 1-3 (2003); Lockshin & Zakers. When You Die Die II. A Comprehensive Evaluation of Apoptosis and Programmed Whole Death. Wiley

Interscience 2: 37-58 (2004); Holdenrieder & Stieber. Apoptotic markers in cancer. Ciin Biochem 37: 605-617 2004.

Flow cytometry was performed according to standard procedures described in Rahman et al. Introduction to Flow Cytometry. Serotec 2000; Ormerod. Flow Cytometry: A Practical Approach. 3 (2000); Macey. Flow Cytometry: Clinical Applications. 1 (1994); Givan. Flow Cytometry: First Principles. 2 (2002); Haugland. Handbook of Fluorescent Probes and Research Products. 9 (2002); Carleton & Janet. Immunophenotyping. 1 (2000); Watson. Introduction to Flow Cytometry. 1 (2004); Shapiro. Practical Flow Cytometry. 4 (2003).

PARP testing was performed following standard procedures described in Ausubel et al. Current Protocols in Molecular Biology. Wiley Interscience 1-4 (1998); Bonifacino et al. Current Protocols in Whole Biology. Wiley Interscience 1-3 (2003).

The results are shown in Figures 1 to 18.

We repeated each experiment 2-5 times to obtain results that were the same with a relative error of ± 5-10%.

Concentrations of sulfur-based support substances, that is, HEPES and elemental sulfur, ranged from 2% to 0.5% by volume for HEPES in samples, and from 0.1% to 1% by volume for elemental sulfur and penicillin and streptomycin were added at a final suspension concentration of 0.5 vol. %.

Explanation of pictures:

Figure 1 shows that the combinations of the invention with different concentrations of plant extracts did not damage healthy fibroblasts from the NHDF cell line. Concentrations increased from sample 1 from 1 pg / mL to 10 mg / mL.

Number of cells per hole / unit: 5000; incubation time with the combination of the invention: 24 hours.

Figure 2 shows that immortalized cells in the HaCaT cell line were able to retain viability above 90% when individual sample combinations of the invention were added with different concentrations of plant extracts. Concentrations decreased from sample 1 onwards, namely Capsicum from 90% to 20% and Allium from 90% to 20%. Number of cells per hole: 5000; incubation time with the combination of the invention: 72 hours.

Figure 3 shows the caspase activity of immortalized cells from the HaCaT cell line. The first sample represents buffer, the second sample represents buffer with substrate, the third sample represents untreated cells (control), the fourth and fifth sample represent cells treated with the highest concentrations of plant extracts according to the results of the viability test (the highest concentrations at which the cells are in the viability test showed more than 90% viability; these concentrations were: Capsicum and Allium 1 mg / ml, 0.1 mg / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml; HEPES 10 pg / mL and 1 pg / ml; the individual concentrations of Capsicum, Allium and HEPES were combined with each other in Capsicum: Allium ratios of 1: 1, 1: 4, 4: 1; HEPES sessions ranged from 2% to 0.5% by volume, or only Capsicum or Allium or HEPES only). Later, when treating cancer cells with the above-mentioned combinations, we found that the viability of the cancer cells decreased more effectively when combined with treatment with only single Capsicum, Allium, and HEPES components). It is evident that the caspase activity was even lower than in untreated cells, indicating that no apoptosis process occurred.

Figure 4 shows the percentage of apoptotic cells in the HaCaT cell line as measured by a FACS Calibur flow cytometer and using the Cellquest program (Becton Dickinson, Mountain view, CA, USA). The first diagram shows the untreated cells and the second diagram the cells that were treated at the same concentrations as in the viability test and in the caspase activity assay. It can be seen that there were no significant changes in the percentage of apoptotic cells between untreated and treated cells.

Figure 5 shows that the viability of cancer cells from the SH-SY5Y cell line decreased linearly as derivative concentrations increased. Number of cells per hole: 10,000; incubation time with the combination of the invention: 24 hours.

Figure 6 shows the caspase activity of cancer cells from the SH-SY5Y cell line. The first sample represents buffer, the second sample represents buffer with substrate, the third sample represents untreated cells (control), the fourth, fifth and sixth sample represent cells treated with the highest concentrations of derivatives according to the results of the viability test (highest concentrations in the viability test showed a viability between 60 and 80%: Capsicum 10 pg / mL + Allium 10 pg / mL at a ratio of 1: 1 + HEPES 0.5 vol%). It is evident that caspase activity increases in proportion to higher concentrations of plant extracts.

Figure 7 shows the percentage of apoptotic cells in the SH-SY5Y cell line measured with a FACS Calibur flow cytometer and using the Cellquest program (Becton Dickinson, Mountain view, CA, USA). The first diagram shows the untreated cells and the second diagram the cells that were treated at the same concentrations as in the viability test and in the caspase activity assay. Many tumor cells are apoptotic, as shown by determining the percentage of apoptotic cells.

Figure 8 shows the results of testing for PARP activation. It is evident that PARP triggered, as evidenced by the protein determination techniques in all three samples of treated SH-SY5Y cells with the concentrations of plant extracts mentioned in Figures 5 and 6.

Figure 9 shows the viability of cancer cells from the MCF-7 cell line, which decreased as the concentrations (see text in Figure 3) of the plant extracts increased. Given that these cells do not contain caspase 3, we did not perform a test to determine caspase activity. Number of cells per hole: 10,000; incubation time with the combination of the invention: 24 hours.

Figure 10 shows the percentage of apoptotic cells in the MCF-7 cell line alternated with a FACS Calibur flow cytometer and using the Cellquest program (Becton Dickinson, Mountain view, CA, USA). The first diagram shows the untreated cells and the second diagram the cells treated at the same concentrations as in the viability test (Figure 9) (Capsicum 1 pg / mL + Allium 1 pg / mL at 1: 1+ HEPES ratio

0,5 vol. %). The tumor cells were mostly apoptotic and necrotic, as shown by determining the percentage of apoptotic and necrotic cells.

Figure 11 shows the viability percentage of MCF-7 cells treated with the combination of the invention, as indicated in Figure 9, and at the same time with the broad-spectrum inhibition of caspases (z-VAD-fmk), cathepsins (E - 64 d) and mitomycin C (sample 1: Capsicum 1 pg / mL + Allium 1 pg / mL at 4: 1 + HEPES 0.8 vol% ratio; sample 2: Capsicum 1 pg / mL + Allium 1 pg / mL at 4: 1 + HEPES 0.8 vol%, z-VAD-fmk at a concentration of 10 pM; sample 3: Capsicum 1 pg / mL + Allium 1 pg / mL at a 4: 1 ratio + HEPES 0.8 vol%, E - 64 dv at a concentration of 15 pM; sample 4: Capsicum 1 pg / mL + Allium 1 pg / mL at a ratio of 4: 1 + HEPES 0.8 vol, mitomycin C at a concentration of 2 pg / mL). The results indicate that the combination of the invention acts in the apoptosis pathway where caspases are involved, since the viability after treatment with the inhibitors mentioned above is greater. In the treatment of cells with the combination of the invention and mitomycin C, the viability was lower than in the treatment of the cells with the combination of the invention only, indicating that the combination of the invention does not work in the same way as mitomycin C (mitomycin C induces DNA damage and thus induces apoptosis).

Figure 12 shows that the number of apoptotic cancer cells in the HeLa cell line was less than 80% in only two samples. Concentrations that caused a lower viability of cancer cells than 80% (second sample Capsicum 0.1 mg / mL + Allium 0.1 mg / mL in a 1: 1 ratio + HEPES 0.8 vol.% And fourth Capsicum sample 1 pg / mL + Allium 1 pg / mL at 4: 1 ratio + HEPES 1 vol%; the first sample is untreated cells as a control and the third sample is Capsicum 40 pg / mL + Allium 1 pg / mL at 9: 1 ratio + HEPES 0 , 8 vol.%) Were used below for further experiments. Number of cells per hole: 10,000; incubation time with the combination of the invention: 48 hours.

Figure 13 shows the caspase activity of cancer cells from the HeLa cell line. The first sample represents buffer, the second sample represents buffer with substrate, the third sample represents untreated cells (control), the fourth and fifth sample represent cells treated with the second and third highest concentrations of derivatives according to the results of the viability test (second and third highest concentrations, in which cells in the viability test showed a viability of between 50 and 80%: Capsicum 0.1 mg / mL + Allium 0.1 mg / mL in a 1: 1 ratio + HEPES 0.8 vol% and Capsicum 1 pg / mL + Allium 1 pg / mL in 4: 1 + HEPES ratio (1% vol.). It is clear that caspase activity is present in these cells, which are treated with the concentrations of plant extracts mentioned above, which further indicates the presence of apoptosis.

Figure 14 shows the percentage of apoptotic cells in the HeLa cell line as measured by a FACS Calibur flow cytometer and using the Cellquest program (Becton Dickinson, Mountain view, CA, USA). The first diagram shows the untreated cells and the second diagram the cells that were treated at the same concentrations as in the viability test and in the caspase activity assay. Capsicum sample used 0.1 mg / mL + Allium 0.1 mg / mL in a 1: 1 ratio + HEPES 0.8 vol. %. The tumor cells were mostly apoptotic, as shown by determining the percentage of apoptotic cells.

Figure 15 shows the viability percentage of HeLa cells treated with the combination of the invention and simultaneously with a broad spectrum inhibitor of caspases (z-VAD-fmk), cathepsins (E - 64 d) and mitomycin C (sample composition as in Example 11 except that Capsicum is 1 pg / mL + Allium 1 pg / mL at a ratio of 4: 1 + HEPES (1 vol%). The results indicate that the combination of the invention triggers apoptosis via a caspase enzyme cascade, since the viability after treatment with the above inhibitors is greater. In the treatment of cells with the combination of the invention and mitomycin C, the viability was lower than in the treatment of the cells with the combination of the invention only, indicating that the combination of the invention does not work in the same way as mitomycin C (mitomycin C induces DNA damage and thus induces apoptosis).

Figure 16 shows that immortalized cells in the MEF cell line (murine endothelial frbroblasts) averaged a viability of more than 30% when individual sample combinations of the invention with different concentrations were added to them. Concentrations decreased from sample 1 onwards. Number of cells per well: 5000, incubation time with the combination of the invention: 24 hours.

Figure 17 shows that healthy cells in the MEF wt cell line (wild-type mouse endothelial fibroblasts) averaged a viability above 60% when added to individual sample combinations of the invention at different concentrations. This indicates the selective action of the combinations (see also Figure 16). Concentrations decreased from sample 1 onwards. Number of cells per well: 5000, incubation time with the combination of the invention: 24 hours.

Figure 18 shows that the viability of cancer cells in the HepG2 (human liver cancer cell) cell line decreased as the concentrations of the combinations of the invention increased. The first sample represents the lowest concentration and the last one the higher concentration of the combinations according to the invention, as already mentioned in the above examples. Number of cells per well: 10,000, incubation time with the combination of the invention: 24 hours.

The above explanation shows that the combinations of the invention act selectively on cancer cells from SH-SY5Y, HeLa, MCF-7, and HepG2 cell lines in which they induce apoptosis but have no major effect on immortalized cells from the HaCaT cell line and have a very high little or no effect on healthy cells from the NHDF cell line.

It is understood that the disposal of the present invention is the exclusive right of the holder and can only be used with the agreement of the holder.

Claims (6)

1. A combination containing plant extracts, characterized in that it has the following composition: Capsicum plant extract, Allium plant extract, HEPES and elemental sulfur, preferably with the addition of antibiotics, in different proportions according to the purpose of use, the ingredients being optionally suspended in brine. Process for the preparation of a combination according to claim 1, characterized in that the Capsicum and Allium plants are sun-dried and ground to powder, suspended in saline with HEPES and elemental sulfur and preferably antibiotics, allow the solids to settle for a couple of days, while shaking them repeatedly, then filter and collect the filtrate, either stored at 4 to 10 ° C or dried. The combination of claim 1 for use as a cancer apoptosis triggering agent. Combination according to Claims 1 and 3, characterized in that the cancer cells are the human cancer cell line SH-SY5Y, the human breast cancer cell line MCF-7, the human uterine cancer cell line HeLa and the cell line the human cancer cell line HepG2. 5. A pharmaceutical formulation comprising the combination as claimed in claim 1, with the proviso that the patient is vegetarian. Formulation according to claim 5, characterized in that it is incorporated into liposomes.