EP1572191A1 - Pharmaceutical composition containing brevifoliol for use in chemotherapeutic treatment of human beings - Google Patents
Pharmaceutical composition containing brevifoliol for use in chemotherapeutic treatment of human beingsInfo
- Publication number
- EP1572191A1 EP1572191A1 EP02781652A EP02781652A EP1572191A1 EP 1572191 A1 EP1572191 A1 EP 1572191A1 EP 02781652 A EP02781652 A EP 02781652A EP 02781652 A EP02781652 A EP 02781652A EP 1572191 A1 EP1572191 A1 EP 1572191A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- brevifoliol
- pharmaceutically acceptable
- acceptable carrier
- formula
- chemotherapeutic treatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- VWGCDYGRSUJYGJ-GPOPEEISSA-N [(2s,4r,5r,5as,6s,8s,9ar,10as)-5,6-diacetyloxy-2,8-dihydroxy-10a-(2-hydroxypropan-2-yl)-3,5a-dimethyl-9-methylidene-2,4,5,6,7,8,9a,10-octahydro-1h-benzo[g]azulen-4-yl] benzoate Chemical compound O([C@@H]1C2=C(C)[C@@H](O)C[C@]2(C[C@@H]2C(=C)[C@@H](O)C[C@@H]([C@]2([C@H]1OC(C)=O)C)OC(=O)C)C(C)(C)O)C(=O)C1=CC=CC=C1 VWGCDYGRSUJYGJ-GPOPEEISSA-N 0.000 title claims abstract description 62
- JQIPMLOMQMJUPJ-UHFFFAOYSA-N Brevifoliol Natural products CC(=O)OC1C2(C)C(OC(=O)C)CC(O)C(=C)C2CC2C(C(C)(C)O)C(O)C(C)=C2C1OC(=O)C1=CC=CC=C1 JQIPMLOMQMJUPJ-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 241000282414 Homo sapiens Species 0.000 title claims abstract description 31
- 230000000973 chemotherapeutic effect Effects 0.000 title claims description 18
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 31
- 201000011510 cancer Diseases 0.000 claims abstract description 31
- 239000003937 drug carrier Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 19
- 210000000481 breast Anatomy 0.000 claims description 12
- 210000001072 colon Anatomy 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 10
- 230000002611 ovarian Effects 0.000 claims description 10
- 206010006187 Breast cancer Diseases 0.000 claims description 7
- 208000026310 Breast neoplasm Diseases 0.000 claims description 7
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 5
- 208000003445 Mouth Neoplasms Diseases 0.000 claims description 4
- 208000029742 colonic neoplasm Diseases 0.000 claims description 4
- 241001330449 Taxus wallichiana Species 0.000 abstract description 21
- 229940123237 Taxane Drugs 0.000 abstract description 12
- 239000002246 antineoplastic agent Substances 0.000 abstract description 11
- 238000000338 in vitro Methods 0.000 abstract description 7
- 241000282412 Homo Species 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 23
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 22
- 229930012538 Paclitaxel Natural products 0.000 description 21
- 229960001592 paclitaxel Drugs 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 238000012360 testing method Methods 0.000 description 11
- 241000202349 Taxus brevifolia Species 0.000 description 7
- 230000001093 anti-cancer Effects 0.000 description 7
- 238000002955 isolation Methods 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 239000000741 silica gel Substances 0.000 description 6
- 229910002027 silica gel Inorganic materials 0.000 description 6
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- YWLXLRUDGLRYDR-ZHPRIASZSA-N 5beta,20-epoxy-1,7beta,10beta,13alpha-tetrahydroxy-9-oxotax-11-ene-2alpha,4alpha-diyl 4-acetate 2-benzoate Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](O)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 YWLXLRUDGLRYDR-ZHPRIASZSA-N 0.000 description 4
- 231100000002 MTT assay Toxicity 0.000 description 4
- 238000000134 MTT assay Methods 0.000 description 4
- 241001116498 Taxus baccata Species 0.000 description 4
- 238000009643 clonogenic assay Methods 0.000 description 4
- 231100000096 clonogenic assay Toxicity 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 102000029749 Microtubule Human genes 0.000 description 3
- 108091022875 Microtubule Proteins 0.000 description 3
- 241001116500 Taxus Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
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- 241000894007 species Species 0.000 description 3
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229940044684 anti-microtubule agent Drugs 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 231100000050 cytotoxic potential Toxicity 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 208000014829 head and neck neoplasm Diseases 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 230000017095 negative regulation of cell growth Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 201000000274 Carcinosarcoma Diseases 0.000 description 1
- 241000208328 Catharanthus Species 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 208000004356 Hysteria Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010029216 Nervousness Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 208000006268 Sarcoma 180 Diseases 0.000 description 1
- 229910020169 SiOa Inorganic materials 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 125000002619 bicyclic group Chemical group 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000002026 carminative effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002026 chloroform extract Substances 0.000 description 1
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- 208000012839 conversion disease Diseases 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 150000004141 diterpene derivatives Chemical class 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 description 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 1
- 239000003172 expectorant agent Substances 0.000 description 1
- 230000003419 expectorant effect Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000037841 lung tumor Diseases 0.000 description 1
- 208000026037 malignant tumor of neck Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 230000025090 microtubule depolymerization Effects 0.000 description 1
- 230000000986 microtubule polymerisation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
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- 239000003208 petroleum Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 208000030761 polycystic kidney disease Diseases 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
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- 210000001519 tissue Anatomy 0.000 description 1
Definitions
- the present invention relates to a method for the chemotherapeutic treatment of human beings using brevifoliol. More particularly, the present invention relates to the use of brevifoliol for the treatment human cancer lines. The present invention also relates to the bioactivity testing of taxanes from the leaves of Himalayan Yew tree Taxus wallichiana against human cancer cell lines grown in- vitro and subsequent identification of brevifoliol [1] as anticancer agent useful in the treatment of various types of cancer in humans. The present invention also relates to a pharmaceutical composition containing brevifoliol and a pharmaceutically acceptable carrier for the chemotherapeutic treatment of human beings.
- Taxus brevifolia Nutt. was described by Wani M.C. et al (in 1971, J.Am.Chem.Soc. 93, 2325- 2327).
- This compound named taxol (also known in the literature as paclitaxel) demonstrated moderate in vivo activity against the P-388, P-1534, and L-1210 murine leukaemia, the Walker 256 carcino-sarcoma, sarcoma 180, and Lewis lung tumor test systems. Taxol has a wide spectrum of anticancer activity. It has been approved by the Food and Drug Administration of United States in 1992 for the treatment of ovarian cancer and again in 1994 for the treatment of breast cancers.
- taxol has garnered support as an anticancer agent, culminating in recent FDA approval of its use against breast and ovarian cancers.
- Taxol shows promise against refractory breast and ovarian cancers, which are difficult to treat and which are responsible for the deaths of 60,000 women every year (Chemical &Engineering News 1991, (Sept.), 11-18).
- antimicrotubule agents which block microtubule production, taxol promotes tubulin polymerisation and stabilizes microtubules against depolymerization (Schiff P.B., et al. 1979, Nature 277, 665-667).
- Microtubules are important subcellular target for chemotherapeutic agents.
- Antimicrotubule agents including the Ninca (Catharanthus) alkaloids, are extremely potent, requiring only a few molecules to disrupt the microtubular structure of cancer cells. The discovery of a new compound targeting these structures is of particular importance.
- Himalayan yew has a remarkable history of medicinal uses.
- the leaves of the Himalayan yew are used for treatment of hysteria, epilepsy, nervousness and as a lithic in calculus complaints while its non-poisonous fleshy arils have carminative, expectorant and stomachic properties (The Wealth of India (1976), p.132-134).
- Himalayan yew (Taxus wallichiana) also contains the potent anticancer drug taxol and its important precursor 10-deacetyl baccatin III (DAB).
- the aim of present invention was to isolate different types of taxanes other than taxol, from the leaves commonly called 'needles' of Taxus wallichiana and evaluate their anticancer potential.
- a taxane brevifoliol [1] was identified which showed promising anticancer activity against in- vitro grown human cancer cell lines.
- the main object of the invention is to provide a composition for the chemotherapeutic treatment of human beings.
- the present invention provides bioactivity testing of various taxanes isolated from leaves of Himalayan Yew tree Taxus wallichiana against human cancer cell lines grown in- vitro and subsequent identification of brevifoliol [1] as anticancer agent useful in the treatment of various types of cancer such as ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells where the IC90 values are comparatively similar or even less than that of standard drug 'taxol'.
- Brevifoliol is used as a cancer chemotherapeutic agent.
- the present invention provides a composition for chemotherapeutic treatment of human beings, comprising a pharmaceutically effective amount of brevifoliol of the formula 1 and a pharmaceutically acceptable carrier.
- the amount of brevifoliol is in the range of 0.004 to 20 ⁇ g/ml.
- the present invention also provides a method for the chemotherapeutic treatment of human beings comprising administering to a patient suffering from cancer, a therapeutically effective amount of brevifoliol of the formula 1
- Formula 1 along with a pharmaceutically acceptable carrier.
- the concentration of brevifoliol is in the range of 0.004 to 20 ⁇ g/ml.
- the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
- the chemotherapeutic treatment is limited to ovarian, colon, breast and oral cancers.
- the invention also provides a method for treatment of human cancer lines comprising administering to a patient a therapeutically effective amount of brevifoliol of the formula 1
- Formula 1 along with a pharmaceutically acceptable carrier.
- the concentration of brevifoliol is in the range of 0.004 to 20 ⁇ g/ml.
- the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
- the human cancer lines are selected from the group consisting of ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells.
- the present invention also provides for the use of brevifoliol of the formula 1
- Formula 1 along with a pharmaceutically acceptable carrier for the chemotherapeutic treatment of human beings.
- the concentration of brevifoliol is in the range of 0.004 to 20 ⁇ g/ml.
- the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
- the chemotherapeutic treatment is limited to ovarian, colon, breast and oral cancers.
- the invention also provides for the use of brevifoliol of formula 1
- Formula 1 along with a pharmaceutically acceptable carrier for chemotherapeutic treatment of human beings.
- the concentration of brevifoliol is in the range of 0.004 to 20 ⁇ g/ml.
- the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
- the human cancer lines are selected from the group consisting of ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells. Detailed description of the invention
- the IC 90 data obtained by the MTT assay may overestimate the cell killing activity of a compound.
- the clonogenic assay for tumor cells which determines the actual cell death was performed to determine the cytotoxic potential of test compounds.
- Persons skilled in the art of anti-cancer drug discovery can perform both these assays.
- brevifoliol was highly inhibitory to ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells where the IC90 values were comparatively similar or even less than that of standard drug 'taxol'.
- PA-1 ovarian
- Caco-2 colon
- MCF-7 breast
- KB-403 oral cancer cells where the IC90 values were comparatively similar or even less than that of standard drug 'taxol'.
- brevifoliol can be used as cancer chemotherapeutic agent.
- Brevifoliol was first isolated from the leaves (also referred as 'needles') of the plant Taxus brevifolia (Baka et al Phytochemistry 30, p.1613-1614; 1991). The process of its isolation involved extracting the fresh leaves of Taxus wallichiana with ethyl alcohol to get an extract. The crude extract after concentration was diluted with water and partitioned between hexane, chloroform and ethyl acetate sequentially. The chloroform extract upon concentration yielded a dark brown residue. The resultant residue was subjected to column chromatography over silica gel and eluted with chloroform and chloroform-methanol gradient. Six fractions were collected and brevifoliol was isolated from fraction five by rechromatography over silica gel and eluting with hexane-ethyl acetate gradient.
- Brevifoliol has been isolated from other species of Taxus including the Himalayan yew tree Taxus wallichiana that is available in India. Recently, the structure of brevifoliol has been revised and it was shown to belong to 11 (15-1) abeo taxoid bicyclic skeleton of formula [1].
- brevifoliol was also isolated from the leaves of the plant following a process which involved extracting the dried and crushed needles of Taxus wallichiana with methanol for 72 hours and the extract was concentrated in vacuo. The concentrate was diluted with water and extracted with hexane and chloroform respectively. Concentration of the chloroform phase under vacuum left a residue, which was separated by column chromatography over silica gel.
- Cytotoxicity testing in vitro was done by the method of Woerdenbag et al.,1993; J.NatProd. 56 (6): 849-856). 2x10 3 cells/well were incubated in the 5% CO 2 incubator for 24h to enable them to adhere properly to the 96 well polysterene microplate (Grenier, Germany). Test compounds dissolved in 100% DMSO (Merck,Germany) in at least five doses were added and left for 6h after which the compound plus media was replaced with fresh media and the cells were incubated for another 48h in the CO 2 incubator at 37°C. The concentration of DMSO used in our experiments never exceeded 1.25%, which was found to be non-toxic to cells.
- IC 90 is the concentration ⁇ g/mL required for 90% inhibition of cell growth as compared to that of untreated control.
- the results described in Table 2 indicate that brevifoliol is active against all cancer cell lines except liver cancer (WRL-68).
- Table 2 Cytotoxic properties (IC 90 in ⁇ g/mL) of various taxanes isolated from T.wallichiana against human cancer cells.
- the clonogenic assay for tumor cells which determines the actual cell death was performed to determine the cytotoxic potential of test compounds.
- the principle of clonogenic assay is to investigate the ability of an individual cell to form a colony on a soft agar plate containing various concentrations of test compounds.
- IC 90 concentration of test compound resulting in 90 % of the control (untreated) colonies was denoted as IC 90 and was used as a parameter for cytotoxicity.
- the assay was performed as described previously except that the test compounds were added into the top soft agar and the cells were plated out to form colonies.
- Anthracycline derivative doxorubiciti and microtubule depolymerization inhibitor paclitaxel both established anticancer agents were included as standard reference drugs.
- Table 3 Cytotoxic properties (IC 90 in ⁇ g/mL) of various taxanes isolated from T.wallichiana against human cancer cells.
- Brevifoliol was crystallized from petroleum ether-acetone mixture as needles. Brevifoliol, mp 200-201°C, ]D -25°C (C 1, MeOH) was characterized on the basis of its reported spectral data and by direct comparison with an authentic sample (Chattopadhyay, S.K. et al , Indian J. Chemistry 35B, 175-177 (1996)). The reaction mechanism is given below:
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to the bioactivity of taxanes isolated from the leaves of Himalayan Yew tree Taxus wallichiana against human cancer cell lines grown in-vitro and subsequent identification of brevifoliol (1) as anticancer agent useful in the treatment of various types of cancer in humans.
Description
PHARMACEUTICAL COMPOSITION CONTAINING BREVIFOLIOL FOR USE IN CHEMOTHERAPEUTIC TREATMENT OF HUMAN BEINGS
Field of the invention
The present invention relates to a method for the chemotherapeutic treatment of human beings using brevifoliol. More particularly, the present invention relates to the use of brevifoliol for the treatment human cancer lines. The present invention also relates to the bioactivity testing of taxanes from the leaves of Himalayan Yew tree Taxus wallichiana against human cancer cell lines grown in- vitro and subsequent identification of brevifoliol [1] as anticancer agent useful in the treatment of various types of cancer in humans. The present invention also relates to a pharmaceutical composition containing brevifoliol and a pharmaceutically acceptable carrier for the chemotherapeutic treatment of human beings.
(1) Background of the invention
In 1971, a novel compound isolated from the bark of the northwest Pacific yew tree, Taxus brevifolia Nutt. was described by Wani M.C. et al (in 1971, J.Am.Chem.Soc. 93, 2325- 2327). This compound, named taxol (also known in the literature as paclitaxel) demonstrated moderate in vivo activity against the P-388, P-1534, and L-1210 murine leukaemia, the Walker 256 carcino-sarcoma, sarcoma 180, and Lewis lung tumor test systems. Taxol has a wide spectrum of anticancer activity. It has been approved by the Food and Drug Administration of United States in 1992 for the treatment of ovarian cancer and again in 1994 for the treatment of breast cancers. It has also been found to be effective against leukemia, and cancer of the head, neck, endometrium and lungs. Recently, it has also been used to treat polycystic kidney disease which accounts for ten percent of the kidney transplant among the dialysis patients (Nature p.750, 1994).
In more than twenty years since the initial report of its isolation, structure elucidation, and bioactivity, taxol has garnered support as an anticancer agent, culminating in recent FDA approval of its use against breast and ovarian cancers. There are two main reasons for the attention directed toward this drug. First, it shows promise against refractory breast and
ovarian cancers, which are difficult to treat and which are responsible for the deaths of 60,000 women every year (Chemical &Engineering News 1991, (Sept.), 11-18). Second, it exhibits a mode of action, which is unique among cancer chemotherapeutic agents. Unlike known antimicrotubule agents, which block microtubule production, taxol promotes tubulin polymerisation and stabilizes microtubules against depolymerization (Schiff P.B., et al. 1979, Nature 277, 665-667). Microtubules are important subcellular target for chemotherapeutic agents. Antimicrotubule agents, including the Ninca (Catharanthus) alkaloids, are extremely potent, requiring only a few molecules to disrupt the microtubular structure of cancer cells. The discovery of a new compound targeting these structures is of particular importance.
Despite its promise, there is a problem with taxol. This highly fiinctionalised diterpene is isolated from inner bark of relatively rare and slow growing Pacific yew tree Taxus brevifolia, and few related species in extremely small quantities (<0.02% dry wt) (Chemical &Engineering News 1991, (Sept.), 11-18). Himalayan yew, Taxus wallichiana Zucc. is a tree or a large shrub distributed in the north temperate zone of the Indian subcontinent (The Wealth of India (1976), A dictionary of Indian raw materials and industrial products, Vol. X, CSIR publication, New Delhi, P.132-134.). In contrast to the European yew (T. baccata Linn.), the Himalayan yew has a remarkable history of medicinal uses. The leaves of the Himalayan yew are used for treatment of hysteria, epilepsy, nervousness and as a lithic in calculus complaints while its non-poisonous fleshy arils have carminative, expectorant and stomachic properties (The Wealth of India (1976), p.132-134). In addition to the above medicinal properties, Himalayan yew (Taxus wallichiana) also contains the potent anticancer drug taxol and its important precursor 10-deacetyl baccatin III (DAB).
The currently practiced procedures for isolating taxol from bark have the disadvantages of being fatal to the source, being very difficult to carry out, and producing low yields. For example, (Nidensek et al 1990; Journal of Natural Products 53, 1609-1610) a 0.01% yield from a large-scale isolation starting with 806 lbs. or more of Taxus brevifolia bark has been reported. Similar procedures have been reported which comparably produce low yields, ranging from as low as 0.004%, up to about (but not above) 0.017%. A yield of 0.01% translates into 1 g being isolated from 10 kg of the bark, or 1 kg of taxol from 10,000 kg (approx.22,000 lbs) of the bark. A mature tree is said to yield 20-25 lbs. of bark, and this means that nearly 800-1000 trees are needed to produce a kilogram of taxol. Reported yields of taxol from various species of yew tree range from 50 mg/kg to 165 mg/kg (i.e., 0.005- 0.017%). At present, bark of Taxus brevifolia is still being used as the major source of taxol. Because of (a) the low (0.01% or less) yields of taxol from the bark, (b) the relative
unavailability of any other useful analogues, and (c) the need to cut the slow-growing trees to harvest the bark, it was decided that the bark was not an attractive source for taxol. Therefore, besides isolation from the bark, there are currently three avenues that are being pursued for the future production of taxol: (1) isolation from renewable plant parts, e.g., the ornamental yew clippings and needles; (2) semi- synthesis of taxol; (3) production of taxol by tissue culture procedures and (4) to find new taxanes from needles which are a renewable source of supply with anticancer properties.
The aim of present invention was to isolate different types of taxanes other than taxol, from the leaves commonly called 'needles' of Taxus wallichiana and evaluate their anticancer potential. In the course of these investigations a taxane brevifoliol [1] was identified which showed promising anticancer activity against in- vitro grown human cancer cell lines. Objects of the invention
The main object of the invention is to provide a composition for the chemotherapeutic treatment of human beings.
It is another object of the invention to provide a method for the chemotherapeutic treatment of human beings using brevifoliol, a taxane isolated from various Taxus species.
It is another object of the invention to provide a method for the treatment of human cancer lines using brevifoliol, a taxane isolated from various Taxus species. Summary of the invention
The present invention provides bioactivity testing of various taxanes isolated from leaves of Himalayan Yew tree Taxus wallichiana against human cancer cell lines grown in- vitro and subsequent identification of brevifoliol [1] as anticancer agent useful in the treatment of various types of cancer such as ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells where the IC90 values are comparatively similar or even less than that of standard drug 'taxol'. Brevifoliol is used as a cancer chemotherapeutic agent.
Accordingly, the present invention provides a composition for chemotherapeutic treatment of human beings, comprising a pharmaceutically effective amount of brevifoliol of the formula 1 and a pharmaceutically acceptable carrier.
In one embodiment of the invention, the amount of brevifoliol is in the range of 0.004 to 20 μg/ml.
The present invention also provides a method for the chemotherapeutic treatment of human beings comprising administering to a patient suffering from cancer, a therapeutically effective amount of brevifoliol of the formula 1
Formula 1 along with a pharmaceutically acceptable carrier.
In one embodiment of the invention, the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
In yet another embodiment of the invention, the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
In yet another embodiment of the invention, the chemotherapeutic treatment is limited to ovarian, colon, breast and oral cancers.
The invention also provides a method for treatment of human cancer lines comprising administering to a patient a therapeutically effective amount of brevifoliol of the formula 1
Formula 1 along with a pharmaceutically acceptable carrier.
In one embodiment of the invention, the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
In yet another embodiment of the invention, the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
In yet another embodiment of the invention, the human cancer lines are selected from the group consisting of ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells.
The present invention also provides for the use of brevifoliol of the formula 1
Formula 1 along with a pharmaceutically acceptable carrier for the chemotherapeutic treatment of human beings.
In one embodiment of the invention, the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
In yet another embodiment of the invention, the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
In yet another embodiment of the invention, the chemotherapeutic treatment is limited to ovarian, colon, breast and oral cancers.
The invention also provides for the use of brevifoliol of formula 1
Formula 1 along with a pharmaceutically acceptable carrier for chemotherapeutic treatment of human beings.
In one embodiment of the invention, the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
In yet another embodiment of the invention, the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
In yet another embodiment of the invention, the human cancer lines are selected from the group consisting of ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells. Detailed description of the invention
As a part of the studies on the isolation of anticancer compounds a number of taxanes were isolated from the needles of the plant Taxus wallichiana and tested for their anti-cancer properties against six human cancer cell lines in- vitro. The bioactivity testing was done at two stages. In the initial step, MTT assay was performed from which the inhibitory concentration (IC90) the concentration (μg/mL) of the taxanes required for 90% inhibition of cell growth was deduced. Further, the compounds, which showed cell growth inhibitory activity was evaluated in the clonogenic assay. The MTT assay cannot discriminate between growth inhibition and cell death; it is an overall measurement of cell proliferation (Woerdenbag et al, 1993; J.Nat.Prod. 56 (6): 849-856). Therefore the IC90 data obtained by the MTT assay may overestimate the cell killing activity of a compound. Hence, the clonogenic assay for tumor cells which determines the actual cell death was performed to determine the cytotoxic potential of test compounds. Persons skilled in the art of anti-cancer drug discovery can perform both these assays.
The data obtained in these bioassays against human cancer cells indicated that brevifoliol was highly inhibitory to ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells where the IC90 values were comparatively similar or even less than that of standard drug 'taxol'. Thus brevifoliol can be used as cancer chemotherapeutic agent.
Brevifoliol was first isolated from the leaves (also referred as 'needles') of the plant Taxus brevifolia (Baka et al Phytochemistry 30, p.1613-1614; 1991). The process of its isolation involved extracting the fresh leaves of Taxus wallichiana with ethyl alcohol to get an extract. The crude extract after concentration was diluted with water and partitioned between hexane, chloroform and ethyl acetate sequentially. The chloroform extract upon concentration yielded a dark brown residue. The resultant residue was subjected to column chromatography over silica gel and eluted with chloroform and chloroform-methanol gradient. Six fractions were collected and brevifoliol was isolated from fraction five by rechromatography over silica gel and eluting with hexane-ethyl acetate gradient.
Brevifoliol has been isolated from other species of Taxus including the Himalayan yew tree Taxus wallichiana that is available in India. Recently, the structure of brevifoliol has been revised and it was shown to belong to 11 (15-1) abeo taxoid bicyclic skeleton of formula [1]. For this invention, brevifoliol was also isolated from the leaves of the plant following a
process which involved extracting the dried and crushed needles of Taxus wallichiana with methanol for 72 hours and the extract was concentrated in vacuo. The concentrate was diluted with water and extracted with hexane and chloroform respectively. Concentration of the chloroform phase under vacuum left a residue, which was separated by column chromatography over silica gel. Fraction eluted with chloroform-methanol (98:5) contained brevifoliol, which was further purified by rechromatography over silica gel and eluted with chloroform-methanol (99:2). Fractions containing brevifoliol were combined and concentrated and recrystallized from pet-ether and ethyl acetate mixture to get brevifoliol as needles (Chattopadhyay et al (1996) Indian J. Chemistry 35B, 175-177).
The following examples further illustrate the invention and should not be construed as limiting the scope of the invention. EXAMPLES Example-1: In-vitro anticancer MTT assay
The following six human cancer cell lines were procured from the Cell Repository of the National Center for Cell Sciences (NCCS) at Pune. Their corresponding ATCC No. and the organ from which they were isolated are also mentioned in Table 1 below: Table 1: Description of human cancer cell lines and their ATCC Nos.
Cytotoxicity testing in vitro was done by the method of Woerdenbag et al.,1993; J.NatProd. 56 (6): 849-856). 2x103 cells/well were incubated in the 5% CO2 incubator for 24h to enable them to adhere properly to the 96 well polysterene microplate (Grenier, Germany). Test compounds dissolved in 100% DMSO (Merck,Germany) in at least five doses were added and left for 6h after which the compound plus media was replaced with fresh media and the cells were incubated for another 48h in the CO2 incubator at 37°C. The concentration of DMSO used in our experiments never exceeded 1.25%, which was found to be non-toxic to cells. Then, 10 μl MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma M 2128] was added, and plates were incubated at 37°C for 4 h. 100 μl
dimethyl sulfoxide (DMSO, Merck, Germany) were added to all wells and mixed thoroughly to dissolve the dark blue crystals. After a few minutes at room temperature to ensure that all crystals were dissolved, the plates were read on a SpectraMax 190 Microplate Elisa reader (Molecular Devices Inc., USA), at 570 nm. Plates were normally read within 1 h of adding the DMSO. The experiment was done in triplicate and the inhibitory concentration (IC) values were calculated as follows: % inhibition =[l-OD (570 nm) of sample well / OD (570 nm) of control well] x 100. IC90 is the concentration μg/mL required for 90% inhibition of cell growth as compared to that of untreated control. The results described in Table 2 indicate that brevifoliol is active against all cancer cell lines except liver cancer (WRL-68). Table 2: Cytotoxic properties (IC 90 in μg/mL) of various taxanes isolated from T.wallichiana against human cancer cells.
Example-2: Soft Agar Assay For Colony Formation
The clonogenic assay for tumor cells which determines the actual cell death was performed to determine the cytotoxic potential of test compounds. The principle of clonogenic assay is to investigate the ability of an individual cell to form a colony on a soft agar plate containing various concentrations of test compounds.
Cells not able to form colonies are considered clonogenically dead (Beekman et al 1997; J.NatProd. 60(4): 325-330). The concentration of test compound resulting in 90 % of the control (untreated) colonies was denoted as IC90 and was used as a parameter for cytotoxicity. The assay was performed as described previously except that the test compounds were added into the top soft agar and the cells were plated out to form colonies.
Anthracycline derivative doxorubiciti and microtubule depolymerization inhibitor paclitaxel (Sigma Chem. Co., St. Loius, USA) both established anticancer agents were included as standard reference drugs.
Table 3: Cytotoxic properties (IC 90 in μg/mL) of various taxanes isolated from T.wallichiana against human cancer cells.
Example 3: Chemical Process for the Isolation of Brevifoliol
Dried and crushed needles of Taxus wallichiana (1 kg) were extracted with methanol (3x3 L) for 72 hours and concentrated in vacuo. The concentrate was diluted with water and extracted with hexane (1L) and chloroform (1L) respectively. Concentration of the chloroform phase under vacuum left a residue (12g), which was separated by column chromatography over silica gel. Fraction eluted with chloroform-methanol (95:5) contained brevifoliol, which was further purified by re-chromatography over silica gel and eluted with chloroform-methanol (98:2). Fractions containing brevifoliol were concentrated under vacuum and brevifoliol was obtained as amorphous solids, (50 mg).
Brevifoliol was crystallized from petroleum ether-acetone mixture as needles. Brevifoliol, mp 200-201°C, ]D -25°C (C 1, MeOH) was characterized on the basis of its reported spectral data and by direct comparison with an authentic sample (Chattopadhyay, S.K. et al , Indian J. Chemistry 35B, 175-177 (1996)).
The reaction mechanism is given below:
MeOH
Crushed needles ► MeOH ext.
Hexane/H20
H20 (discarded)
CHCI3
CHCI3 H20 (Discarded)
SiOa/ CHCI3-MeOH (95:5)
Brevifoliol rich fraction
Rechromatography SKV CHCI3-MeOH (98:2) Brevifoliol
Claims
1. A pharmaceutical composition for the chemotherapeutic treatment of human beings comprising a therapeutically effective amount of brevifoliol of formula 1
Formula 1 along with a pharmaceutically acceptable carrier.
2. A composition as claimed in claim 1 wherein the concentration of brevifoUol is in the range of 0.004 to 20 μg/ml.
3. A method for the chemotherapeutic treatment of human beings comprising administering to a cancer patient, a therapeutically effective amount of brevifoliol of formula 1
Formula 1 along with a pharmaceutically acceptable carrier.
4. A method as claimed in claim 3 wherein the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
5. A method as claimed in claim 3 wherein the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
6. A method as claimed in claim 3 wherein the chemotherapeutic treatment is limited to ovarian, colon, breast and oral cancers.
7. A method as for treatment of human cancer lines comprising administering to a patient a therapeutically effective amount of brevifoliol of the formula 1
along with a pharmaceutically acceptable carrier.
8. A method as claimed in claim 7 wherein the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
9. A method as claimed in claim 7 wherein the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
10. A method as claimed in claim 7 wherein the human cancer lines are selected from the group consisting of ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells.
11. Use of brevifoliol of the formula 1
Formula 1 along with a pharmaceutically acceptable carrier for the chemotherapeutic treatment of human beings.
12. Use as claimed in claim 11 wherein the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
13. Use as claimed in claim 11 wherein the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
14. Use as claimed in claim 11 wherein the chemotherapeutic treatment is limited to ovarian, colon, breast and oral cancers.
15. Use of brevifoliol of formula 1
Formula 1 along with a pharmaceutically acceptable carrier for chemotherapeutic treatment of human cancer lines selected from the group consisting of ovarian (PA-1), colon (Caco-2), breast (MCF-7) and oral (KB-403) cancer cells.
16. Use as claimed in claim 15 wherein the concentration of brevifoliol is in the range of 0.004 to 20 μg/ml.
17. Use as claimed in claim 15 wherein the pharmaceutically acceptable carrier does not interfere with the activity of brevifoliol.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IB2002/005399 WO2004054569A1 (en) | 2002-12-16 | 2002-12-16 | Pharmaceutical composition containing brevifoliol for use in chemotherapeutic treatment of human beings |
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