WO2011110190A1

WO2011110190A1 - A cellobiose compound from herbal extracts having apoptotic activity

Info

Publication number: WO2011110190A1
Application number: PCT/EP2010/001431
Authority: WO
Inventors: Romina Znoj
Original assignee: Romina Znoj
Priority date: 2010-03-08
Filing date: 2010-03-08
Publication date: 2011-09-15

Abstract

The present invention refers to a compound isolated from a combination of herbal extracts of Capsicum chinense and Allium neapolitanum having apoptotic activity in cancer cells, which compound was identified as cellobiose. In particular, the present invention refers to a cellobiose compound for use in the prevention and treatment of cancer. The present invention further refers to a pharmaceutical composition comprising a cellobiose compound. The present invention also refers to a use of a cellobiose compound for inducing apoptosis in a cell or a tissue system in vitro.

Description

A CELLOBIOSE COMPOUND FROM HERBAL EXTRACTS HAVING APOPTOTIC ACTIVITY

The present invention refers to a compound isolated from a combination of herbal extracts of Capsicum chinense and Allium neapolitanum having apoptotic activity in cancer cells, which compound was identified as cellobiose. In particular, the present invention refers to cellobiose for use in the prevention and treatment of cancer.

Background of the invention

As evident from historical knowledge, archaeological discovery and epidemiological data cancer incidence in man is lower in certain geographical regions than in others, and this is attributed to the consumption of certain plants. Such plants and their extracts have been found effective in the prevention of malignant diseases when used as food or tea, or when active components out of them are applied as food supplements, e.g. in the form of tablets. Moreover, there are several examples where isolated active compounds originally discovered in plants turned out to be effective drugs in the treatment of a manifested cancer. A prominent example is paclitaxel, established for the treatment of e.g. breast cancer, which was first isolated from a yew tree (Taxus brevifolia).

EP 0 092 226 refers to the extract of plants of the Hypoxidaceae family, in particular of the genus Hypoxis and the genus Spiloxene, for the treatment of cancer. However, the compound eventually identified, to which the anti-cancer activity was ascribed, does not seem to be selective for cancer cells.

WO 2008/11 1918 (claiming priority of SI 22466) discloses a combination of extracts obtained from plants of the genus Capsicum and the genus Allium which triggers apoptosis in cells of various cancer cell lines derived from neuronal (SH-SY5Y), breast (MCF-7), cervical (HeLa), and liver (HepG2) carcinomas. It was suggested that the herbal extracts may be useful in the treatment of cancer. However, an active compound of these extracts has not been described. There exists a constant need to effectively treat various types of cancer, preferably by killing the cancer cells while leaving healthy cells widely unaffected. One of the mechanisms most frequently used to induce the dying of cancer cells is triggering apoptosis (programmed cell death) or making use of cytotoxic effects.

Thus, it was an object of the present invention to provide new active compounds which selectively induce apoptosis in cancer cells. In particular, it was an object of the present invention to identify such new active compounds in plants.

Summary of the invention

The object of the present invention is solved by a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for use as a medicament in a subject.

The object of the present invention is further solved by a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for the prevention or treatment or after-care of a disorder supposed to benefit from inducing apoptosis in a subject.

The object of the present invention is further solved by a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for the prevention or treatment or after-care of cancer in a subject.

In one embodiment, the cancer is manifested in the form of a solid tumor.

In one embodiment, the cancer is selected from the group consisting of cancer of the central nervous system, preferably a neuroblastoma; breast cancer, preferably a breast ductal carcinoma; cervical cancer, preferably a cervical adenocarcinoma; and liver cancer, preferably a hepatocellular carcinoma.

In one embodiment, the cellobiose compound is used or formulated in combination with one or more additional active compounds, preferably one or more anti-cancer drugs. The object of the present invention is further solved by a pharmaceutical composition comprising a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof, and further comprising a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition is formulated for oral, enteral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation.

In one embodiment, the pharmaceutical composition is in a unit dosage form selected from the group comprising pills, tablets, caplets, capsules (each including immediate release, timed release and sustained release formulations), powders, granules, sterile parenteral solutions or suspensions (including syrups and emulsions), metered aerosol or liquid sprays, drops, ampoules, autoinjector devices or suppositories. Further considered are liposome delivery systems or transdermal patch systems. Also considered is the delivery of a prodrug.

The object of the present invention is further solved by a use of a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment or after-care of a disorder supposed to benefit from inducing apoptosis in a subject.

The object of the present invention is further solved by a use of a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment or after-care of cancer in a subject.

The object of the present invention is further solved by a prevention or treatment or after-care of a disorder supposed to benefit from inducing apoptosis in a subject using a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof.

The object of the present invention is further solved by a prevention or treatment or after-care of cancer in a subject using a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof.

The object of the present invention is further solved by a use of a cellobiose compound or an effective derivative or a salt thereof for inducing apoptosis in a cell or tissue system in vitro. In one embodiment, the cell is selected from the group consisting of cells of the SH-SY5Y, MCF-7, HeLa, and HepG2 cell line or, alternatively, the cell is a native cell or the tissue system is a native tissue system obtained from a subject.

In one embodiment, the subject is a vertebrate, preferably a mammal, most preferably a human.

The term "effective derivative" refers to a compound of a modified cellobiose compound or a compound structurally related to the cellobiose compound which is capable of eliciting a response, i.e. an apoptotic or cytotoxic response, in a subject or in a cell or a tissue system in vitro.

For use as a medicament, the cellobiose compound or the effective derivative thereof preferably is in the form of a non-toxic "pharmaceutically acceptable salt", e.g. a sodium salt.

The cellobiose compound or the effective derivative or the pharmaceutically acceptable salt thereof is to be administered in a "therapeutically effective amount", i.e. an amount of the active compound that elicits a response, i.e. an apoptotic or cytotoxic response, in a subject or in a cell or a tissue system in vitro.

A "native cell" or a "native tissue system" means a primary cell or primary tissue system, e.g. cells which have not been immortalized.

In summary, the present invention provides cellobiose isolated from herbal extracts of Capsicum chinense and Allium neapolitanum for use as a medicament, in particular for use in the treatment of cancer. Cellobiose turned out to be the active compound of the herbal extracts inducing apoptosis (or cytotoxicity at higher concentrations) in cancer cell lines: SH- SY5Y (neuroblastoma), MCF-7 (breast ductal cancer), HeLa (cervical adenocarcinoma), and HepG2 (hepatocellular carcinoma). Remarkably, cellobiose did not cause a high rate of apoptotic or cytotoxic response within 3 hours in immortalized non-cancerous cell lines: HCF 306-05f (human cardiac fibroblasts), NHDF (normal human dermal fibroblasts) and HaCaT (human keratinocytes). Moreover, it was concluded that cellobiose acts via an intrinsic apoptotic pathway. Detailed description of the invention

The invention is illustrated by the following non-limiting examples and the accompanying figures in which

Figure 1 shows a chromatogram resulting from HPLC analysis of herbal extracts from Capsicum chinense and Allium neapolitanum. Time was measured in minutes (x-axis). A fraction having apoptotic activity, i.e. containing the active ingredient, was eluted from the column after 10.0 minutes (indicated by an arrow).

Figure 2A and 2B show the results of cell viability tests carried out with samples of eluted HPLC fractions. No. 1 : control; no. 2: dead cells, treated with 1% KC1; no. 3 and upward: individual HPLC fractions. On the graphs the viability of HepG2 cells treated with samples of a total of 91 individual HPLC fractions is shown. As can be seen, the viability of HepG2 cells was significantly reduced after treatment with fraction no. 7, i.e. this fraction contained the active ingredient (indicated by an arrow). Viability of the cells was tested by MTS test.

Figure 3 shows the results of a mass spectrometry analysis of the HPLC fraction containing the active ingredient. The graph at the top shows several peaks representing different molecules. The active ingredient, i.e. cellobiose, is represented by the peak at 342.1 m/z corresponding to a molecular weight of 342 g/mol. On the graph at the bottom the peaks representing the molecules are recorded in a different spectrum. The peak at 383.1 shows two molecules of protonized cellobiose together with two ions of sodium.

Figure 4 shows a basic NMR analysis. The typical signal corresponding to the chemical structure of cellobiose appears between 3 and 5 ppm.

Figure 5 shows an NMR analysis with specific filters. Here, the NMR analysis is recorded with more specific and accurate filters, which enables to see and confirm the chemical structure of cellobiose shown in Figure 7. Figure 6 shows NMR mathematical correlations. Here, the typical correlations between the individual atoms of cellobiose can be seen. This is the final and most accurate step enabling to set the perfect and complete chemical structure shown in Figure 7.

Figure 7 shows the chemical structure of the active ingredient, i.e. cellobiose.

Figure 8A and 8B show the apoptotic morphology of cells of the cell lines HepG2 (Fig.

8A, top), HeLa (Fig. 8A, bottom), HaCaT (Fig. 8B, top), and NHDF (Fig. 8B, bottom) after incubation with the HPLC fraction containing cellobiose (0.1 mg/mL) for three hours (pictures at the right hand side; left: control). A typical apoptotic morphology with the shrinkage of cells and some already detached cells is indicated by arrows. In contrast to the crude plant extract, the effect of the HPLC fraction containing the active ingredient occurred after only 3 hours of treatment (results with the crude extract not shown). Note that there were only few cells with apoptotic morphology in HaCaT and even less in NHDF cells. However, if we added to the HPLC fraction containing cellobiose some additional molecules (e.g. sulphur, glucose), the apoptotic effect was delayed for up to 24 hours (results not shown).

Figure 9 shows that the HPLC fraction containing cellobiose induced different levels of viability in cells of the cell lines HCF 306-05f (top left), MCF-7 (top right), HeLa (mid left), NHDF (mid right), HepG2 (bottom left), and HaCaT (bottom right) after incubation for three hours. After evaluation of cell viability by means of cytotoxic features under the light microscope, cell viability was determined by MTS test. The concentration of individual fraction samples containing cellobiose ranged from 10 mg/mL to ^g/mL. No. 1 : control (untreated cells); no. 2-6 or 2-5: decreasing concentrations (10 mg/mL to 1 μg/mL). Quantity of cells: 10.000 cells/well. The results obtained with the HPLC fraction were similar to those obtained with the crude plant extract, but the onset was faster and was shown after 3 hours in all cell lines (results with the crude extract not shown). However, the effect on all types of cells was the same after 24 hours of incubation with complete destruction of the cells, independent from concentration.

10 shows the caspase activity in cells of the cell lines HepG2 (top left), HeLa (top right) and HaCaT (bottom left) after incubation with the HPLC fraction containing cellobiose (0,1 mg/mL) for three hours. In HeLa and HepG2 cells an activation of caspase-3 was observed. No. 1 : buffer control; no. 2: buffer and substrate control; no. 3: cell control (viable cells, untreated); no. 4: caspase activity. Caspase activity is indicated as RFU arbitrary units. Quantity of cells: 10,000 cells/well.

Figure 1 1 shows that the caspase activity induced by cellobiose was blocked by z-VAD- fmk. For the experiments, cells of the cell lines HepG2 [top left; no. 1 : control; no. 2: z-VAD-fmk (10 μΜ); no. 3: dead cells; no. 4: HPLC fraction (0.1 mg/L); no. 5: HPLC fraction (0.01 mg/mL); no. 6: HPLC fraction (0.1 mg/mL + z-VAD-fmk (10 μΜ); no. 7: HPLC fraction 7 (0.01 mg/mL) z-VAD-fmk (10 μΜ)], HeLa [top right; no. 1 : control; no. 2: z-VAD-fmk (10 μΜ); no. 3: dead cells; no. 4: HPLC fraction (0.1 mg/L); no. 5: HPLC fraction (0.1 mg/mL + z-VAD-fmk (10 μΜ)] and HaCaT [bottom left; no. 1 : control; no. 2: z-VAD-fmk (10 μΜ); no. 3: dead cells; no. 4: HPLC fraction (0.1 mg/L); no. 5: HPLC fraction (0.1 mg/mL + z-VAD-fmk (10 μΜ)]) were used. As can be seen, caspase-3 activity was completely (100%) blocked after incubation of the cells with the HPLC fraction containing cellobiose (0.1 mg/mL) for three hours in the presence of caspase-inhibitor z-VAD-fmk. Cell viability was measured by MTS test.

Figure 12 shows the results of a flow cytometry analysis. The figure shows the number of apoptotic HepG2 (top) and HeLa (bottom) cells after incubation with the HPLC fraction containing cellobiose. The maximum response was obtained with a concentration of the HPLC fraction of 0.1 mg/mL. As can be taken from the figure, 27.4% of apoptotic HepG2 (top right; top left: control, 6.9%) and 18.0% of apoptotic HeLa (bottom right; bottom left: control, 4.7%) cells where obtained, i.e. enough to confirm the onset of apoptosis.

Figure 13 shows mitochondrial disruption induced by the HPLC fraction containing cellobiose at a concentration of 0.1 mg/mL. As can be taken from the figure, the percentages of disrupted mitochondria were 35.8% with HepG2 (top right; top left: control, 10.1%) and 21.3 % with HeLa (bottom right; bottom left: control, 6.4%) cells, i.e. enough to confirm the onset of apoptosis.

Figure 14 shows the results of a Western blot analysis. As can be seen, incubation of HepG2 (top) and HeLa (bottom) cells with the HPLC fraction containing cellobiose (0.1 mg/mL) resulted in expression of proapoptotic Bcl-2 proteins and tBid. The expression of proapoptotic protein Bax and tBid is shown. Actin served as a loading control.

Figure 15 shows the proposed scheme of mode of action of the plant extracts and cellobiose, respectively. It is concluded from the experimental results that apoptosis is triggered via an intrinsic apoptotic pathway. The plant extract and cellobiose, respectively, causes cell stress which leads to an imbalance between pro- and anti- apoptotic Bcl-2 proteins in favour of pro-apoptotic Bcl-2 proteins (e.g. Bax). These later on causes disruption of mitochondria, what leads to caspase-9 activation and in the end to caspase-3 -crucial executor of apoptosis. Meanwhile, caspase-8 is also activated, which cleaves Bid into tBid and therefore amplifies the expression of proapoptotic Bcl-2 proteins. From the experiments it is deducted that the lysosomes also get disrupted at the same time as the disruption of mitochondria occur.

EXAMPLE 1 : Preparation of herbal extracts

A herbal extract from Capsicum chinense and Allium neapolitanum was prepared as described in SI 22466 and WO 2008/1 1 1918.

Plants of Capsicum chinense and Allium neapolitanum were dried in the sun. After they were completely dry, they were separately ground into powder. The powder (450 mg) was suspended in a saline (up to a concentration of 10 mg/mL) together with supportive substances on the basis of sulphur, namely HEPES and elementary sulphur, under the addition of streptomycin and penicillin. The concentrations of supportive substances in the samples ranged from 2% to 0.5 % by vol for HEPES and from 0.1% to 1% by vol for elementary sulphur, and, additionally, penicillin and streptomycin were added up to a final concentration in the suspension of 0.5% by vol each. The suspension was left to stand for one week, was occasionally shaken and finally filtered.

EXAMPLE 2: Cell culture and cell-related analysis

The cells were grown according to standard methods. MTS tests, viability tests with the pancaspase inhibitor z-VAD-fmk and Western blot analysis for tBid and the pro-apoptotic Bcl-2 protein Bax were carried out according to standard methods as outlined in Ausubel et al., Current Protocols in Molecular Biology. Wiley Interscience 1-4 (1998); Bonifacino et al., Current Protocols in Cell Biology. Wiley Interscience 1 -3 (2003); Lockshin & Zakeri, When Cells Die II. A Comprehensive Evaluation of Apoptosis and Programmed Cell Death. Wiley Interscience 2:37-58 (2004); Holdenrieder & Stieber, Apoptotic Markers in Cancer. Clin Biochem 37:605-617 (2004).

Flow cytometry was carried out according to standard methods disclosed 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).

EXAMPLE 3 : Identification of the active compound

HPLC and NMR analysis were performed according to the standard procedures used in the field of chemistry for identification of molecules (Hornak, J.P. The Basics of NMR, (2008); http://www.pharm.uky.edu/ASRG/HPLC/HPLCMYTRY.html).

The herbal extracts were subjected to HPLC. The fractions obtained were tested with the cultivated cell. In order to identify the fraction(s) containing the active ingredient(s), an MTS test was used. The fraction having activity was analyzed using mass spectrometry and, if not sufficiently pure, was further analyzed with a more accurate HPLC procedure. Again, the cells were treated with the HPLC fractions and the active fraction was identified with an MTS test. The active fractions were analyzed by mass spectrometry again to determine the molecular weight of the compounds contained therein. The last step was NMR by which the structure of the molecule was determined and, together with mass spectrometry, the active molecule was identified.

The results are shown in Figs. 1 to 15. Each experiment was repeated 2 to 5 times and the obtained results were the same with a relative error of ± 5-15%. In conclusion, it has been shown that cellobiose has a selective apoptose-inducing effect on cells of the cancer cell lines SH-SY5Y, HeLa, MCF-7, and HepG2, but does not have a significant effect on immortalized cells of the non-cancerous cell lines NHDF, HCF 306-05f and HaCaT.

According to the fact that similar molecules identified in the past (Arcamone, F. Doxorubicin Disaccharide Analogue: Apoptosis-Related Improvement of Efficacy In Vivo. Journal of the National Cancer Institute 89 (16): 1217 (1997); Fuster, M.M. et al. A Disaccharide Precursor of Sialyl Lewis X Inhibits Metastatic Potential of Tumor Cells. Cancer Research 63: 2775- 2781 (2003)) showed very promising results and are used in cancer therapy as common and established treatment, or even as "gold standard", it is reasonable to believe that cellobiose may have similar or even better effects. It is clearly known from the literature cited above and other sources that disaccharides are used for molecular modelling of old chemotherapeutic medicines to improve their efficacy and lower sides effects. Also in the field of anaesthesia sugars are new very potent, effective medicines with fever or none side effects (for example Bridion^® with the generic name sugammadex; Naguib, M. Sugammadex. Another Milestone in Clinical Neuromuscular Pharmacology. Anaesthesia & Analgesia, Vol. 104 (3): 575-581 (2007)). On this basis, it can also be predicted to have class effect of all disaccharides as this was also shown to be true in the past (for example saltans in the field of cardiology for hypertension treatment, diabetic nephropathy and congestive heart failure; http://en.wikipedia.org/wiki/Angiotensin_II_receptor_antagonist).

EXAMPLE 4: Determination of the signalling pathway induced by cellobiose

In order to determine the pathway (intrinsic or extrinsic) used by cellobiose in triggering apoptosis, various dyes and the pancaspase inhibitor z-VAD-fmk were used in order to show the integrity of certain organelles involved in the specific cellular signalling pathway (MitoTracker Red CMXRos). Cells with damaged organelles were counted with the FACS Calibur flow cytometer Becton Dickinson. It was shown that cellobiose triggers the same intrinsic apoptotic pathway as the plant extracts described in SI 22466 and WO 2008/11 1918.

Claims

1. A cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for use as a medicament.

2. A cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for the prevention or treatment or after-care of a disorder supposed to benefit from inducing apoptosis.

3. A cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof for the prevention or treatment or after-care of cancer.

4. The cellobiose compound or effective derivative or pharmaceutically acceptable salt thereof according to claim 3, wherein the cancer is manifested in the form of a solid tumor.

5. The cellobiose compound or effective derivative or pharmaceutically acceptable salt thereof according to claim 3 or 4, wherein the cancer is selected from the group consisting of cancer of the central nervous system, preferably a neuroblastoma; breast cancer, preferably a breast ductal carcinoma; cervical cancer, preferably a cervical adenocarcinoma; and liver cancer, preferably a hepatocellular carcinoma.

6. The cellobiose compound or effective derivative or pharmaceutically acceptable salt thereof according to any of the preceding claims, which is used or formulated in combination with one or more additional active compounds, preferably one or more anti-cancer drugs.

7. A pharmaceutical composition comprising a cellobiose compound or an effective derivative or a pharmaceutically acceptable salt thereof, and further comprising a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, which is formulated for oral, enteral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation.

9. Use of a cellobiose compound or an effective derivative or a salt thereof for inducing apoptosis in a cell or a tissue system, preferably a cancer cell or a cancer tissue system, in vitro.

10. The use according to claim 9, wherein the cell is selected from the group consisting of cells of the SH-SY5Y, MCF-7, HeLa, or HepG2 cell lines.

11. The use according to claim 9, wherein the cell is a native cell or the tissue system is a native tissue system obtained from a subject, preferably a mammal, most preferably a human.