WO2011139172A1 - Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same - Google Patents

Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same Download PDF

Info

Publication number
WO2011139172A1
WO2011139172A1 PCT/RS2010/000004 RS2010000004W WO2011139172A1 WO 2011139172 A1 WO2011139172 A1 WO 2011139172A1 RS 2010000004 W RS2010000004 W RS 2010000004W WO 2011139172 A1 WO2011139172 A1 WO 2011139172A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
childhood
radical
leukemia
compounds
Prior art date
Application number
PCT/RS2010/000004
Other languages
French (fr)
Inventor
Goran Milosavljevic
Nada Ivkovic
Vele Tesevic
Vlatka Vajs
Lvana Aljancic
Vojin Senk
Dragan Povrenovic
Djuro Kutlaca
Milica Pesic
Jasna Bankovic
Nikola Tanic
Sabera Ruzdijic
Milka Jadranin
Nina Todorovic
Slobodan Milosavljevic
Original Assignee
Goran Milosavljevic
Nada Ivkovic
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Goran Milosavljevic, Nada Ivkovic filed Critical Goran Milosavljevic
Priority to RS20120482A priority Critical patent/RS58784B1/en
Priority to PCT/RS2010/000004 priority patent/WO2011139172A1/en
Publication of WO2011139172A1 publication Critical patent/WO2011139172A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/013—Esters of alcohols having the esterified hydroxy group bound to a carbon atom of a ring other than a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • C07C69/78—Benzoic acid esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/32—All rings being cycloaliphatic the ring system containing at least eleven carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/40—Ortho- or ortho- and peri-condensed systems containing four condensed rings

Definitions

  • the present invention relates to novel nontoxic diterpene compounds and their derivatives for use as medicaments, especially for the treatment of cancer, as well as dietary and pharmaceutical composition containing such diterpenes and derivates and their uses.
  • Cancer is a group of diseases (acute lymphoblastic leukemia (adult), acute aymphoblastic leukemia (childhood), acute myeloid leukemia (adult), acute myeloid leukemia (childhood), adrenocortical carcinoma, adrenocortical carcinoma (childhood), AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (childhood, cerebellar), astrocytoma (childhood cerebral), basal cell carcinoma (nonmelanoma), bile duct cancer (extrahepatic), bladder cancer, bladder cancer (childhood), bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain stem glioma (childhood), brain tumor (adult), central nervous system embryonal tumors (childhood), cerebellar astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), ependymoblasto
  • Cancer is caused by both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). Cancer is treated with surgery, radiation, chemotherapy, hormone therapy, biological therapy, and targeted therapy.
  • Cancer is expected to overtake heart disease as the number one killer of people around the world by the year 2010. By 2030, the number of new cancer cases is expected to rise to 27 million, with 17 million cancer deaths.
  • anticancer drugs are in research process and some of them are used in clinical practices (mechlorethamine, cyclophosphamide, busulfan, nitrosoureas, procarbazine, melphalan, methotrexate, 6-mercatopurine, fludarabine, capecitabine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil, gemcitabine, dactinomycin, daunorubicin, doxorubicin, epirubicin, bleomycin, mitomycin C, etoposide, irinotecan, topotecan, vincristine, vinblastine, paclitaxel, docetaxel, hydroxyurea, cisplatin, carboplatin, asparaginase, mitoxantrone, amsacrine, imatinib, erlotinib, sorafenib, estrogens, tamoxifen, prednisone,
  • Another obstacle in the cancer treatment is the acquirement of resistance after first-line therapy, which prevents the second-line chemotherapy to extend the life of patients.
  • Some cancer types possess inherent resistance to the large scale of chemotherapeutics.
  • the present invention relates to compounds having anti-cancer activity.
  • the invention also relates to the use of combination of those compounds in non-toxic concentrations.
  • Taxol or Docetaxel have used agents from plant of the Euphorbiaceae family which are useful in the treatment and prophylaxis of prostate cancer in mammalian and in particular human subjects (United States Patent 7378445).
  • the Euphorbiaceae family of plants covers a wide variety of plants including weeds of Euphorbia species. There have been a variety of inconclusive reports on the potential effects of the sap of these plants on a variety of conditions as well as promoting tumorigenesis and causing skin and ocular irritation.
  • a recent report describes selective cytotoxicity of a number of tiglilane diterpene esters from the latex of Euphorbia poisonii , a highly toxic plant found in Northern Nigeria, which is used as a garden pesticide.
  • One of these compounds has a selective cytotoxicity for the human kidney carcinoma cell line A-498 more than 10,000 times greater than that of adriamycin (reference 28).
  • African Burkitt's lymphoma A plant, Euphorbia tirucalli, reduces Epstein-Barr virus-specific cellular immunity. Anticancer Research 000004
  • Antineoplastic agents are ususally highly toxic, and there is a strong need to find one with high antineoplastic activity and low toxicity.
  • a pharmaceutical composition of very low toxicity containing diterpene compounds with antineoplastic activities was found in the Euphorbiaceae family of plants, including weeds of Euphorbia species, and particularly in Euphorbia dendroides, and is preferred in the practice of the present invention.
  • Reference herein to Euphorbia dendroides includes various varieties, strains, lines, hybrids or derivatives of this plant as well as its botanical or horticultural relatives.
  • the present invention may be practiced using a whole Euphorbiaceae plant or parts thereof including sap or seeds or other reproductive material. Generally, for seeds or reproductive material to be used, a plant or plantlet is first required to be propagated.
  • Reference herein to an Euphorbiaceae plant, a Euphorbia species or Euphorbia dendroides further encompasses genetically modified plants.
  • Genetically modified plants include transgenic plants or plants in which a trait has been removed or where an endogenous gene sequence has been down-regulated, up-regulated, mutated or otherwise altered including the alteration or introduction of genetic material which exhibits a regulatory effect on a particular gene. Consequently, a plant which exhibits a character not naturally present in an Euphorbiaceae plant, a Euphorbia species or Euphorbia dendroides further encompasses genetically modified plants.
  • Genetically modified plants include transgenic plants or plants in which a trait has been removed or where an endogenous gene sequence has been down-regulated, up-regulated, mutated or otherwise altered including the alteration or introduction of genetic material which exhibits a regulatory effect on a particular gene. Consequently, a plant which exhibits a character not naturally present in an
  • Euphorbiaceae plant or a species of Euphorbia or in Euphorbia dendroides is nevertheless encompassed by the present invention and is included within the scope of the above- mentioned terms.
  • the present invention contemplates hybrid plant cells or plants comprising hybrid plant cells formed by the fusion of two or more plant cells from different strains, species or genera and optionally regenerating a plant therefrom. Such hybrid plant cells are proposed to generate novel secondary metabolites having useful therapeutic properties.
  • the diterpenes are generally in extracts of the Euphorbiaceae plants.
  • An extract may comprise, therefore, sap or liquid or semi-liquid material exuded from, or present in, leaves, stem, flowers, seeds and bark or between the bark and the stem. Most preferably, the extract is from sap.
  • the extract may comprise liquid or semi-liquid material located in fractions extracted from sap, leaves, stems, flowers, bark or other plant material of the Euphorbiaceae plant.
  • plant material may be subject to physical manipulation to disrupt plant fibres and extracellular matrix material and inter- and intra-tissue extracted into a solvent including an aqueous environment.
  • the fractions may include aqueous or alcohol extracts.
  • Other extraction media are also contemplated including fractions prepared by BPLC or other fractionation systems. All such sources of the d iterpenes are encompassed by the present invention including diterpenes obtained by synthetic routes.
  • the chemical agents of the present invention may be in purified or isolated form meaning that the preparation is substantially devoid of other compounds or contaminating agents other than diluent, solvent or carrier or isofbrms of the agents.
  • the term "chemical agent” includes preparations of two or more compounds either admixed together or co-purified from a particular source.
  • the chemical agent may also be a chemical fraction, extract or other preparation including sap from the Euphorbiaceae plant.
  • the chemical agents or extracts or fractions of the present invention may also be referred to as "drugs" or "actives" or “active ingredients”.
  • agent is not to imply a synthetic compound and may include a fraction obtainable from the sap of the Euphorbiaceae plant.
  • the term “obtainable” also includes “obtained”.
  • a “chemical agent” includes a purified form of one or more compounds or a chemical fraction or extract such as from the sap of an Euphorbiaceae plant and in particular a species of Euphorbia, and most preferably from Euphorbia dendroides or botanical or horticultural relatives or variants thereof.
  • one aspect of the present invention contemplates a method for the treatment or prophylaxis of cancer or a related condition, said method comprising the administration to said subject of a symptom-ameliorating effective amount of a chemical agent obtainable from a plant of the Euphorbiaceae family or a derivative or chemical analog thereof which chemical agent is a diterpene selected from compounds extracted from an Euphorbiaceae plant of which chemical agent or derivative or chemical analog is represented by any one of the general formulae I, II and I I I,
  • Formula I Formula II Formula III wherein R represents hydrogen atom, hydroxy group, aliphatic alkoxy group with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, or a radical represented by general formula RCOO-, wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group.
  • Supstituents R 2 , R 3 , R 4 , R 5 , R 6 and R 7 may be same or different and represent hydrogen atoms, aliphatic groups with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, or radicals represented by general formula RCO ⁇ , wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group.
  • R 1 in the formulae I, II and III represents hydrogen atom, hydroxy group, aliphatic alkoxy group with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, containing preferably from 1 to 30 carbon atoms, or a radical represented by general formula RCOO-.
  • Supstituents R 2 , R 3 , R 4 , R 5 , R 6 and R 7 in the formulae I, II and ⁇ represent preferably linear or branched, saturated or unsaturated aliphatic groups containing preferably from 1 to 30 carbon atoms, or a radical represented by general formula RCO .
  • the aliphatic group may be substituted with a halogen atom, a hydroxyl group, a carbonyl group, a carboxyl group, an amino group and an amide group.
  • Examples of carboxylic acids from which radicals RCOO- or RCO- are derived include saturated aliphatic acids containing from 1 to 16 carbon atoms, such as acetic acid, propionoc acid, butiric acid, zso-butiric acid, 2,3-dimethyl butiric acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and unsaturated aliphatic acids containing from 3 to 16 carbon atoms, such as 2,4-decadienoic acid.
  • saturated aliphatic acids containing from 1 to 16 carbon atoms such as acetic acid, propionoc acid, butiric acid, zso-butiric acid, 2,3-dimethyl butiric acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and unsaturated aliphatic acids containing from 3 to 16 carbon atoms, such as 2,4-decadienoic acid.
  • Examples of carboxylic acids from which RCOO- or RCO- is derived include aromatic carboxylic acids, such as benzoic acid, phtalic acid, salicylic acid, and antranilic acid.
  • Examples of carboxilic acid from which RCOO- or RCO- is derived include furanocarboxylic acid, thiophenecarboxilic acid, pyridinecarboxilic acid, such as nicotinic acid and wo-nicotinic acid.
  • the aromatic and heteroaromatic radicals may be substituted with halogen atoms, hydroxyl radicals, carbonyl radicals, carboxyl radicals, amino radicals, and amide radicals.
  • R 3 and R 5 are acetyl radicals
  • R 2 and R 4 are iso- butanoyl radicals
  • R 6 is nicotinyl radical (compound 2)
  • a compound represented by the general formula ⁇ , wherein R 1 is acetoxy radical, R 2 , R 3 , R 5 and R 7 are acetyl radicals, R 4 is /so-butanoyl radical, R 6 is nicotinyl radical (compound 6), is prepared by extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, and then purifying the extracts.
  • a compound represented by the general formula 111, wherein R 1 is hydroxy radical, R 2 hydrogen atom, R 3 benzoyl radical and R 4 z ' so-butanoyl radical, (compound 7), is prepared extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, and then purifying the extracts according to known procedures.
  • compound 1 after hydrolysis may react with acid anhydrides, i.e., (R"CO) 2 0 in the presence of anhidrous pyridine to give ester compounds of the present invention (Fig 2).
  • NCI-H460, DLD-1 and U-87 MG were from ATCC (American Type Culture Collection).
  • NCI-H460/R cell line with multidrug resistant phenotype was originally selected from NCI-H460 cells and cultured in a medium containing ⁇ doxorubicin (DOX) (reference 35).
  • DOX doxorubicin
  • K-562 chronic myelogenous leukemia
  • K-562 cells were maintained in RPMI 1640 with 10% FCS (Fetal Calf Serum). This cell line was sub cultured at 72 h intervals using 0.1 % trypsin/EDTA and seeded into a fresh medium at 20.000 cells/cm 2 .
  • Human normal lymphocytes were used in order to determine the level of toxicity on healthy cells.
  • Cisplatin (CPt) is used as positive control for toxicity.
  • Inhibition of growth relative to untreated control was determined by the SRB (Sulforhodamine B) assay and MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. These assays test the relative ability of a compound to inhibit cell growth, but not survival of the cells. However, inhibition of growth may reflect cell death (necrosis or apoptosis) and/or cytostasis.
  • SRB assay The cellular proteins were stained with sulforhodamine B (SRB) assay that was performed with some changes. Briefly, the cells in 96-well plates were fixed in 50 % trichloroacetic acid (50 ⁇ /well) for 1 h at 4° C, rinsed four times in tap water and stained with 0.4 % (w/v) sulforhodamine B in 1% acetic acid (50 ⁇ /well) for 30 min at room temperature. The cells were then rinsed three times in 1% acetic acid (250 ⁇ ) to remove the unbound stain. The protein-bound stain was extracted with 200 ⁇ 10 mM Tris base (pH 10.5) per well.
  • SRB sulforhodamine B
  • IC 30 and IC 50 values were defined as the concentration of the drug that inhibited cell growth by 30% and 50%, respectively.
  • the inhibitory concentrations for the compounds were calculated by linear regression analysis using Excel software.
  • An IC50 value above 50 ⁇ indicates that there was insufficient cytotoxicity of the compound to achieve a 50% inhibition of cell growth at 50 ⁇ .
  • Figure 1 Effect of 1, 2, 3, 4, 5, 6, 7 and CPt on viability of normal human lymphocytes measured by MTT.
  • the healthy lymphocytes were preserved after 72h treatment with 1, 2, 3, 4, 5, 6 and 7 (at tested concentrations the percent of viable lymphocytes was about 90-95%).
  • CPt induced significant decrease in lymphocyte counts (57% of lymphocytes survived at 5 ⁇ and only 35% at 50 ⁇ ). 04
  • 4 is active against non-small cell lung carcinoma, colorectal adenocarcinoma, glioblastoma and leukemia (Table 1). 4 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).
  • 7 is active against non-small cell lung carcinoma, colorectal adenocarcinoma and leukemia (Table 1). 7 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).
  • 5 is active against non-small cell lung carcinoma, colorectal adenocarcinoma, glioblastoma and leukemia (Table 1). 5 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).
  • 6 is active against non-small cell lung carcinoma and chronic myelogenous leukemia (Table 1). 6 shows great potential for treatment of leukemia, but not for solid tumors. This cytotoxic effect is followed by low toxicity of 6 observed on healthy lymphocytes ( Figure 1). IC50 for 6 was above 50 ⁇ for resistant non-small cell lung carcinoma cell line, and therefore resistance factor was not shown in Table 2.
  • DLD-1 colorectal adenocarcinoma
  • U-87 MG glioblastoma
  • K-562 chronic myelogenous leukemia
  • Raising the dose of paclitaxel, vinblastine, etoposide, epirubicin or doxorubicin, as shown in Table 2 by any amount increases their toxicity by the same amount, and therefore increases negative side effects to the human organism.
  • substances 2, 4, 7, 5 and 3 have in vitro shown results under 50 ⁇ while, at the same time, it showed no toxic effects on healthy human cells.
  • the novel substances may be used as dietary supplements or pharmaceutical compositions.
  • the dietary composition may be in form of food such as dairy products (yoghurts), in form of fortified food such as cereal bars and bakery items such as cakes and cookies, in form of dietary supplements such as tablets, pills, granules, dragees, capsules, and effervescent formulations, in form of non-alcoholic drinks such as soft drinks, sport drinks, fruit juices, lemonades, near-water drinks, teas and milk based drinks, in form of liquid food such as soups and dairy products (muesli drinks), in form of alcoholic drinks such as brandy, wme or beer, or in any other beverage.
  • dairy products yoghurts
  • fortified food such as cereal bars and bakery items such as cakes and cookies
  • dietary supplements such as tablets, pills, granules, dragees, capsules, and effervescent formulations
  • non-alcoholic drinks such as soft drinks, sport drinks, fruit juices, lemonades,
  • the pharmaceutical compositions may be administered in various forms, such as tablets, powders, granules, capsules, injections, suppositories, ointments and cataplasms.
  • the pharmaceutical composition containing the active compounds of the present invention may be formulated by using conventional carriers and additives such as vehicles and resolvents, bases, diluents, fillers, adjuvants, such as solvent adjuvants, emulsifying agents, dispersers, disintegrants, solubilizers, viscosity-increasing agents, and lubricants, and additives such as antioxidans, preservatives, flavoring agents and sweetening agents.
  • Euphorbia dendroides can be planted industrially, it gives lots of juice from which the basic diterpenes may be extracted. It is a long living plant, and only the aerial parts of it are used, so it need not be destroyed when taking parts of it for the process needed.
  • the chemical processes needed to extract the diterpenes which are presented by this invention are standard, whereas the processes needed to transform them are tested during the preparation of this application, and are fully feasible in ammounts needed for mass production of the compounds.

Abstract

The present invention provides a generic formulation of a family of novel diterpene compounds with antineoplastic activities. Contrary to existing pharmaceuticals used for anticancer treatment, the compounds exhibit extremely low toxicity, and, at therapeutical doses, even stimulate the proliferation of human lymphocytes. Compounds from the family have been found to show particularly selective cytotoxicity against several different cancer cell lines, while having immunostimulating effects on human lymphocytes. The compounds are useful in effective treatment of cancers, particularly leukemia.

Description

DITERPENE COMPOUNDS WITH ANTINEOPLASTIC ACTIVITIES AND
PHARMACEUTICAL COMPOSITIONS COMPRISING SAME
BRIEF DESCRIPTION OF INVENTION
The present invention relates to novel nontoxic diterpene compounds and their derivatives for use as medicaments, especially for the treatment of cancer, as well as dietary and pharmaceutical composition containing such diterpenes and derivates and their uses.
BACKGROUND OF THE INVENTION
Compounds extracted from plants have been used for centuries for medicinal purposes, and their exploration continues in modern times. Identification and characterization of newly extracted compounds and their therapeutic properties is vital in the on-going search for therapeutic agents to improve detrimental health conditions, such as cancer.
Cancer is a group of diseases (acute lymphoblastic leukemia (adult), acute aymphoblastic leukemia (childhood), acute myeloid leukemia (adult), acute myeloid leukemia (childhood), adrenocortical carcinoma, adrenocortical carcinoma (childhood), AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (childhood, cerebellar), astrocytoma (childhood cerebral), basal cell carcinoma (nonmelanoma), bile duct cancer (extrahepatic), bladder cancer, bladder cancer (childhood), bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain stem glioma (childhood), brain tumor (adult), central nervous system embryonal tumors (childhood), cerebellar astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), ependymoblastoma (childhood), ependymoma (childhood), medulloblastoma (childhood), medulloepithelioma (childhood), pineal parenchymal tumors of intermediate differentiation (childhood), supratentorial primitive neuroectodermal tumors and pineoblastoma (childhood), visual pathway and hypothalamic glioma (childhood), brain and spinal cord tumors (childhood), breast cancer, breast cancer and pregnancy, breast cancer (childhood), breast cancer (male), bronchial tumors (childhood), Burkitt lymphoma, carcinoid tumor (childhood), carcinoid tumor (gastrointestinal), carcinoma of head and neck, central nervous system embryonal tumors (childhood), central nervous system lymphoma (primary), cerebellar astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), cervical cancer, cervical cancer (childhood), childhood cancers, chordoma (childhood), chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T- cell lymphoma, embryonal tumors (central nervous system, childhood), endometrial cancer, ependymoblastoma (childhood), ependymoma (childhood), esophageal cancer, esophageal cancer (childhood), Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastric (stomach) cancer (childhood), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gastrointestinal stromal cell tumor (childhood), germ cell tumor (extracranial, childhood), germ cell tumor (extragonadal), germ cell tumor (ovarian), gestational trophoblastic tumor, glioma (adult), glioma (childhood) brain stem glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer (adult, primary), hepatocellular (liver) cancer (childhood, primary), Hodgkin lymphoma (adult), hodgkin lymphoma (childhood), hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposi sarcoma, kidney (renal cell) cancer, kidney cancer, laryngeal cancer, laryngeal cancer (childhood), leukemia (acute lymphoblastic, adult), leukemia (acute lymphoblastic, childhood), leukemia (acute myeloid, adult), leukemia (acute myeloid, childhood), leukemia (chronic lymphocytic), leukemia (chronic myelogenous), hairy cell, lip and oral cavity cancer, non-small cell lung cancer, small cell lung cancer, non- Hodgkin adult lymphoma, non-Hodgkin childhood lymphoma, macroglobulinemia (Waldenstrom), melanoma, Merkel cell carcinoma, mesothelioma (adult malignant), mesothelioma (childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, chronic myeloid leukemia (adult acute), myeloid leukemia (childhood acute), myeloma (multiple), myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer (childhood), neuroblastoma, oral cancer (childhood), oral cavity cancer, lip and oropharyngeal cancer, ovarian cancer (childhood), ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (childhood), papillomatosis (childhood), parathyroid Cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary tumor, pleuropulmonary blastoma, prostate Cancer, rectal cancer, renal pelvis and ureter transitional cell cancer, respiratory tract carcinoma involving the NUT gene on chromosome 15, rhabdomyosarcoma (childhood), salivary gland cancer, salivary gland cancer (childhood), soft tissue sarcoma (adult), soft tissue sarcoma (childhood), uterine sarcoma, small intestine cancer, squamous neck cancer with occult primary (metastatic), supratentorial primitive neuroectodermal tumors (childhood), testicular cancer, throat cancer, thymoma and thymic carcinoma, thymoma and thymic carcinoma (childhood), thyroid cancer, thyroid cancer (childhood), trophoblastic tumor (gestational, unknown primary site), carcinoma of adult (unknown primary site), cancer of childhood, unusual cancers of childhood, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, vaginal cancer (childhood), vulvar cancer, Wilms tumor, women's cancers) characterized by uncontrolled growth and spread of abnormal cells. The loss of differentiation, increased rate of growth, invasion of surrounding tissue and metastasis can result in death. Cancer is caused by both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). Cancer is treated with surgery, radiation, chemotherapy, hormone therapy, biological therapy, and targeted therapy.
Cancer is expected to overtake heart disease as the number one killer of people around the world by the year 2010. By 2030, the number of new cancer cases is expected to rise to 27 million, with 17 million cancer deaths.
Although many anticancer drugs are in research process and some of them are used in clinical practices (mechlorethamine, cyclophosphamide, busulfan, nitrosoureas, procarbazine, melphalan, methotrexate, 6-mercatopurine, fludarabine, capecitabine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil, gemcitabine, dactinomycin, daunorubicin, doxorubicin, epirubicin, bleomycin, mitomycin C, etoposide, irinotecan, topotecan, vincristine, vinblastine, paclitaxel, docetaxel, hydroxyurea, cisplatin, carboplatin, asparaginase, mitoxantrone, amsacrine, imatinib, erlotinib, sorafenib, estrogens, tamoxifen, prednisone, flutamide, leuprolide, goserelin), there is no cure for cancer yet. Moreover, the cure rates of drugs for the treatment of cancers are unacceptably low, and the side effects of these drugs are severe. Therapeutic regimes with two cytotoxic drugs give better result compared to the use of only one drug, but the application of combination chemotherapy is usually followed by increase in toxicity. The toxic effects of the drugs on the cancer tissues only marginally exceed their toxic effects on normal, healthy cells of the body that should be protected from the effects of the drugs. Chemotherapy research in cancer treatment has been largely devoted to the search for drugs providing toxin specificity to destroy neoplastic tissue in the body without exceeding toxic exposure levels injurious to healthy tissues. For most cancer types, success has been quite limited.
Another obstacle in the cancer treatment is the acquirement of resistance after first-line therapy, which prevents the second-line chemotherapy to extend the life of patients. Some cancer types possess inherent resistance to the large scale of chemotherapeutics.
The discovery of new anti-cancer drugs that have more than one of the following characteristics is an imperative in our modern times: (1) ability to inhibit the growth of cancer cells, (2) ability to prevent cancer cell invasion to other tissues, (3) acceptable levels of toxicity to healthy cells, (4) effectiveness against cancer cells that are resistant to other drugs, and (5) a mechanism that differs from the action of conventional drugs, so the chance of the cancer cells to develop cross-resistance is reduced when the new drug is applied in combination with an existing drug.
The present invention relates to compounds having anti-cancer activity. The invention also relates to the use of combination of those compounds in non-toxic concentrations.
Up to now, not many diterpenes acting as anticancer agent were known. One of them was extracted from Taxus brevifolia, named Taxol or Docetaxel (references 2-23).Others have used agents from plant of the Euphorbiaceae family which are useful in the treatment and prophylaxis of prostate cancer in mammalian and in particular human subjects (United States Patent 7378445).
The Euphorbiaceae family of plants covers a wide variety of plants including weeds of Euphorbia species. There have been a variety of inconclusive reports on the potential effects of the sap of these plants on a variety of conditions as well as promoting tumorigenesis and causing skin and ocular irritation. A recent report describes selective cytotoxicity of a number of tiglilane diterpene esters from the latex of Euphorbia poisonii , a highly toxic plant found in Northern Nigeria, which is used as a garden pesticide. One of these compounds has a selective cytotoxicity for the human kidney carcinoma cell line A-498 more than 10,000 times greater than that of adriamycin (reference 28). Euphorbia hirta plants and extracts thereof 010 000004
5
have been considered for a variety of purposes, including tumor therapy (EP 0 330 094), AIDS-related complex and AIDS (HU-208790) and increasing immunity and as an anti- fungoid agent for treatment of open wounds (DE-4102054).
Thus, while there are isolated reports of anti-cancer activity of various Euphorbia preparations (reference 28), not only are the compounds present in at least one Euphorbia species reported to be carcinogenic (references 30-32), but at least one species has a skin-irritant and tumor- promoting effect (reference 33) and another species reduces EBV-specific cellular immunity in Burkitt's lymphoma (reference 34).
On the other hand, till now, several types of diterpenes were isolated from Euphorbia dendroides plant, having the effect of aiding antineoplastic activities of certain cytostatic drugs (references 24-27). So for, no substances isolated from Euphorbia dendroides were known to work as an anticancer agent.
REFERENCES:
1. Mathers, Colin D, Cynthia Boschi-Pintb, Alan D Lopez and Christopher JL Murray (2001). Cancer incidence, mortality and survival by site for 14 regions of the world. Global Programme on Evidence for Health Policy Discussion Paper No. 13 (World Health Organization)
2. Lyseng- Williamson KA, Fenton C. Docetaxel: a review of its use in metastatic breast cancer. Drugs 2005;65(17):2513-31.
3. Clarke SJ, Rivory LP. Clinical pharmacokinetics of docetaxel. Clin Pharmacokinet 1999;36(2):99-114
4. Anonymous. Oncology Tools: Approved Claims for microtubule inhibitors. US Food and Drug Administration. (17 Sep 2006). Last modified 22 Jun 1998.
5. Anonymous. Taxotere.com for Healthcare Professionals: About. Sanofi-aventis U.S.
LLC. (17 Sep 2006). Last modified Jul 2005.
6. Anonymous. Taxotere Docetaxel concentrate for infusion. Medsafe. (25 Sep 2006).
Last modified 6 Feb 2006.
7. Snyder JP, Nettles JH, Cornett B, Downing KH, Nogales E. The binding conformation of Taxol in b-tubulin: A model based on electron crystallographic density. PNAS. 2001;98(9):5312-16. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. London: Churchill Livingstone; 2003. p. 100-1
Anonymous. Drugdex Evaluations: Docetaxel. Thomson MICROMEDEX. (26 Sep 2006). Last modified 2006.
Anonymous. Taxotere.com for Healthcare Professionals: Efficacy and Safety. Sanofi- aventis U.S. LLC. (24 Sep 2006). Last modified Jul 2005.
Anonymous. New Zealand Pharmaceutical Schedule. Wellington: PHARMAC; 2006. p. 133.
Urien S, Barre J, Morin C, Paccaly A, Montay G, Tillement JP. Docetaxel serum protein binding with high affinity to alpha 1-acid glycoprotein. Invest New Drugs. 1996;14(2):147-51.
Baker SD, Zhao M, Lee CKK, Verweij J, Zabelina Y, Brahmer JR, et al. Comparative pharmacokinetics of weekly and every-three-weeks docetaxel. Clin Cancer Res. 2004;10(6):1976-83.
Anonymous. Taxotere.com for Healthcare Professionals: Pharmacokinetics. Sanofi- aventis U.S. LLC. (23 Sep 2006). Last modified Jul 2005.
Guitton J, Cohen S, Tranchand B, Vignal B, Droz JP, Guillaumont M, et al. Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(17):2419-26.
Yvon AC, Wadsworth P, Jordan MA. Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells. The American Society for Cell Biology. 1999;10:947-959.
Anonymous. Docetaxel: Clinical Pharmacology. RxList. (24 Sep 2006). Last modified 29 Jun 2006.
Eisenhauer EA, Vermorken JB. The taxoids: Comparative clinical pharmacology and therapeutic potential. Drugs. 1998;55(l):5-30.
Tannock IF, de Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med. 2004;351(15):1502-12.
Goyle S, Maraveyas A. Chemotherapy for colorectal cancer. Dig Surg. 2005;22(6):401-14.
Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. London: Churchill Livingstone; 2003. p. 694-8. Pierre Potier, chemist, 1998 CNRS Gold Medalist
Vogel CL, Bellet RE, inventors; Aventis Pharma S.A., assignee. Use of docetaxel for treating cancers. United States patent US20016333348. 2001 Dec 25.
Corea, G.; Di Pietro, A.; Dumontet, C; Fattorusso, E.; Lanzotti, V Jatrophane diterpenes from Euphorbia spp. as modulators of multidrug resistance in cancer therapy. Phytochemistry Reviews (2009), 8(2), 431-447.
Barile, Elisa; Corea, Gabriella; Lanzotti, Virginia Diterpenes from Euphorbia as potential leads for drug design. Natural Product Communications (2008), 3(6), 1003- 1020.
Corea, Gabriella; Fattorusso, Ernesto; Lanzotti, Virginia; Taglialatela-Scafati, Orazio; Appendino, Giovanni; Ballero, Mauro; Simon, Pierre-Noel; Dumontet, Charles; Di Pietro, Attilio. Modified jatrophane diterpenes as modulators of multidrug resistance from Euphorbia dendroides L Bioorganic & Medicinal Chemistry (2003), 11(23), 5221-5227.
Corea, Gabriella; Fattorusso, Ernesto; Lanzotti, Virginia; Taglialatela-Scafati, Orazio; Appendino, Giovanni; Ballero, Mauro; Simon, Pierre-Noeel; Dumontet, Charles; Di Pietro, Attilio Jatrophane Diterpenes as P-Glycoprotein Inhibitors. First Insights of Structure-Activity Relationships and Discovery of a New, Powerful Lead Journal of Medicinal Chemistry (2003), 46(15), 3395-3402.
Fatope MO, Zeng L, Ohayaga JE, Shi G, McLaughlin JL 1996. Selectively cytotoxic diterpenes from Euphorbia poisonii. J Med Chem 39: 1005-1008.
Sevil Oksiiza, b, Faliha Giireka, Long-ze Line, Roberto R. Gilc, John M. Pezzutoc and Geoffrey A. Cordell, Aleppicatines A and B from Euphorbia aleppica, Phytochemistry Volume 42, Issue 2, May 1996, Pages 473-478
A. Evans, M. A. Osman, Carcinogenicity of bracken and shikimic acid, Nature 250, 348 - 349 (26 July 1974)
Starvic and Stolz, Food Cosmet. Toxicol. 14: 141, 1976
Hecker "Cocarcinogens from Euphorbiaceae and Thymeleaceae" in " Symposium on Pharmacognosy and Phytochemistry", 147-165, (Wagner et al., eds., Springer Verlag, 1970).
Gundidza and Kufa, Centr. Afr. J. Med. 38: 444-447, 1992.
Imai, S., M. Sugiura, et al. (1994). African Burkitt's lymphoma: A plant, Euphorbia tirucalli, reduces Epstein-Barr virus-specific cellular immunity. Anticancer Research 000004
8
14(3a): 933-936. Dep. Virol., Cancer Inst., Hokkaido Univ. Sch. Med., Sapporo 060, N15 W7, Japan
35. M. Pesic, J. Z. Markovic, D. Jankovic, S. Kanazir, I. D. Markovic, L. Rakic and S.
Ruzdijic, Induced Resistance in the Human Non Small Cell Lung Carcinoma (NCI- H460) Cell Line In Vitro by Anticancer Drugs, Journal of Chemotherapy, Vol. 18 (2006) - pp. 66-73
36. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107-1112.
DISCLOSURE OF THE INVENTION
Antineoplastic agents are ususally highly toxic, and there is a strong need to find one with high antineoplastic activity and low toxicity. A pharmaceutical composition of very low toxicity containing diterpene compounds with antineoplastic activities was found in the Euphorbiaceae family of plants, including weeds of Euphorbia species, and particularly in Euphorbia dendroides, and is preferred in the practice of the present invention. Reference herein to Euphorbia dendroides includes various varieties, strains, lines, hybrids or derivatives of this plant as well as its botanical or horticultural relatives. Furthermore, the present invention may be practiced using a whole Euphorbiaceae plant or parts thereof including sap or seeds or other reproductive material. Generally, for seeds or reproductive material to be used, a plant or plantlet is first required to be propagated.
Reference herein to an Euphorbiaceae plant, a Euphorbia species or Euphorbia dendroides further encompasses genetically modified plants. Genetically modified plants include transgenic plants or plants in which a trait has been removed or where an endogenous gene sequence has been down-regulated, up-regulated, mutated or otherwise altered including the alteration or introduction of genetic material which exhibits a regulatory effect on a particular gene. Consequently, a plant which exhibits a character not naturally present in an
Euphorbiaceae plant or a species of Euphorbia or in Euphorbia dendroides is nevertheless encompassed by the present invention and is included within the scope of the above- mentioned terms. Furthermore, the present invention contemplates hybrid plant cells or plants comprising hybrid plant cells formed by the fusion of two or more plant cells from different strains, species or genera and optionally regenerating a plant therefrom. Such hybrid plant cells are proposed to generate novel secondary metabolites having useful therapeutic properties.
The diterpenes are generally in extracts of the Euphorbiaceae plants. An extract may comprise, therefore, sap or liquid or semi-liquid material exuded from, or present in, leaves, stem, flowers, seeds and bark or between the bark and the stem. Most preferably, the extract is from sap. Furthermore, the extract may comprise liquid or semi-liquid material located in fractions extracted from sap, leaves, stems, flowers, bark or other plant material of the Euphorbiaceae plant. For example, plant material may be subject to physical manipulation to disrupt plant fibres and extracellular matrix material and inter- and intra-tissue extracted into a solvent including an aqueous environment. The fractions may include aqueous or alcohol extracts. Other extraction media are also contemplated including fractions prepared by BPLC or other fractionation systems. All such sources of the d iterpenes are encompassed by the present invention including diterpenes obtained by synthetic routes.
The chemical agents of the present invention may be in purified or isolated form meaning that the preparation is substantially devoid of other compounds or contaminating agents other than diluent, solvent or carrier or isofbrms of the agents. Furthermore, the term "chemical agent" includes preparations of two or more compounds either admixed together or co-purified from a particular source. The chemical agent may also be a chemical fraction, extract or other preparation including sap from the Euphorbiaceae plant. The chemical agents or extracts or fractions of the present invention may also be referred to as "drugs" or "actives" or "active ingredients". The term "agent" is not to imply a synthetic compound and may include a fraction obtainable from the sap of the Euphorbiaceae plant. The term "obtainable" also includes "obtained".
Consequently, reference herein to a "chemical agent" includes a purified form of one or more compounds or a chemical fraction or extract such as from the sap of an Euphorbiaceae plant and in particular a species of Euphorbia, and most preferably from Euphorbia dendroides or botanical or horticultural relatives or variants thereof.
Accordingly, one aspect of the present invention contemplates a method for the treatment or prophylaxis of cancer or a related condition, said method comprising the administration to said subject of a symptom-ameliorating effective amount of a chemical agent obtainable from a plant of the Euphorbiaceae family or a derivative or chemical analog thereof which chemical agent is a diterpene selected from compounds extracted from an Euphorbiaceae plant of which chemical agent or derivative or chemical analog is represented by any one of the general formulae I, II and I I I,
Figure imgf000011_0001
Formula I Formula II Formula III wherein R represents hydrogen atom, hydroxy group, aliphatic alkoxy group with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, or a radical represented by general formula RCOO-, wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group. Supstituents R2, R3, R4, R5, R6and R7 may be same or different and represent hydrogen atoms, aliphatic groups with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, or radicals represented by general formula RCO~, wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group.
R1 in the formulae I, II and III represents hydrogen atom, hydroxy group, aliphatic alkoxy group with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, containing preferably from 1 to 30 carbon atoms, or a radical represented by general formula RCOO-. Supstituents R2, R3, R4, R5, R6 and R7 in the formulae I, II and ΙΠ represent preferably linear or branched, saturated or unsaturated aliphatic groups containing preferably from 1 to 30 carbon atoms, or a radical represented by general formula RCO . The aliphatic group may be substituted with a halogen atom, a hydroxyl group, a carbonyl group, a carboxyl group, an amino group and an amide group.
Examples of carboxylic acids from which radicals RCOO- or RCO- are derived (wherein R represents an alkyl radical) include saturated aliphatic acids containing from 1 to 16 carbon atoms, such as acetic acid, propionoc acid, butiric acid, zso-butiric acid, 2,3-dimethyl butiric acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and unsaturated aliphatic acids containing from 3 to 16 carbon atoms, such as 2,4-decadienoic acid. Examples of carboxylic acids from which RCOO- or RCO- is derived (wherein R represents an aromatic radical), include aromatic carboxylic acids, such as benzoic acid, phtalic acid, salicylic acid, and antranilic acid. Examples of carboxilic acid from which RCOO- or RCO- is derived (wherein R represents an heteroaromatic radical), include furanocarboxylic acid, thiophenecarboxilic acid, pyridinecarboxilic acid, such as nicotinic acid and wo-nicotinic acid. The aromatic and heteroaromatic radicals may be substituted with halogen atoms, hydroxyl radicals, carbonyl radicals, carboxyl radicals, amino radicals, and amide radicals.
A compound represented by the general formula I, wherein R!and R7 are hydrogen atoms, R2 is propanoyl radical, R3and R5 are acetyl radicals, R4 is /so-butanoyl radical and R6 is nicotinyl radical (compound 1), and a compound represented by the general formula I wherein R!and R7 are hydrogen atoms, R3and R5 are acetyl radicals, R2 and R4 are iso- butanoyl radicals and R6 is nicotinyl radical (compound 2), are prepared by extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, followed by purifying the extracts according to known procedures.
A compound represented by the general formula Π, wherein R1 is acetoxy radical, R2, R3, R5 and R7are acetyl radicals, R4 is /so-butanoyl radical, R6 is nicotinyl radical (compound 6), is prepared by extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, and then purifying the extracts. A compound represented by the general formula 111, wherein R1 is hydroxy radical, R2 hydrogen atom, R3 benzoyl radical and R4 z'so-butanoyl radical, (compound 7), is prepared extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, and then purifying the extracts according to known procedures.
These isolated compounds may be used as starting materials for the preparation of other compounds represented by the general formulae I, II and ΙΠ, respectively. For example, compound 1 is hydrolyzed to give the compound of formula I wherein R2 is hydrogen atom, and then the obtained hydroxyl compound is converted to ether compound of the present invention in accordance with Williamson Synthesis described below, that is the action of sodium alkoxides and then alkylhalides R'X, wherein R' has the same meaning as R2 and X represents a halogen atom, (Fig. 1). Further, compound 1 after hydrolysis (formula I wherein R2 is a hydrogen atom) may react with acid anhydrides, i.e., (R"CO)20 in the presence of anhidrous pyridine to give ester compounds of the present invention (Fig 2).
ISOLATION
To the aerial parts of Euphorbia dendroides (1 kg) collected on the territory of west Balkans, 60% (v/v) of aqueous ethanol (3 L) was added and the mixture left for ten days at room temperature with stirring. The extract was lyophilized at 40° C to give the concentrated extract (lOOg). The concentrated extract was mixed with hexane (350 ml) and placed in an ultra sound bath for 45 minutes. The soluble part was decanted and the extraction repeated under same conditions. The combined extracts were concentrated under reduced pressure at 40° C to give the concentrated extract (4.65 g). The crude extract was subjected to dry flash chromatography on silica gel using toluene/ethyl acetate in different proportions as eluent. The fraction 1, eluated with 5% - 20 % ethyl acetate hadn't shown biological activity and consisted mostly of fatty acid esters, according to proton NMR spectrum. Fractions 2-6 were eluated with higher percent of ethyl acetate in toluene and exibited biological activity. These fractions were purified by preparative thin layer chromatography (TLC) on silica gel plates 20 cm x 20 cm, with the layer thicknes of 0.5 mm, 1 mm or 2 mm, the choice of the layer thickness depended on complexity of the separating mixture. Final purification was performed by HPLC to obtain diterpene compounds 1-7.
Fraction 2 (0.26 g, eluated with 30 % ethyl acetate in toluene) was subjected to preparative TLC on silica gel, layer thicknes 2 mm, developing system hexane-acetone = 7:3 (the plates were developed 3 times) to give subtraction 2-1 (77.7 mg). Final purification by HPLC1 afforded pure compounds 4 (4.5 mg), 5 (4.8 mg) and 7 (5 mg).
Fraction 3 (0.25 g, eluated with the second portion of 30 % ethyl acetate in toluene) after preparative TLC on silica gel, layer thickness 1 mm, developing system hexane-acetone = 7:3 (the plates were developed 5 times) gave two subfractions. Subtraction 3-1 (81 mg) was purified by HPLC1 to give 1 (8 mg) and 2 (16 mg). Subtraction 3-2 (63 mg) after purification by HPLC yelded additional amount of 2 ( 8.6 mg).
1 Performed on Zorbax XDB-C18 column, with the mobile phase a mixture of 0.20 % formic acid in MilliQ water and acetonitrile (gradient mode). P T/RS2010/000004
13
Preparative TLC of fraction ^ (0.16 g, eluated with 40 % ethyl acetate in toluene) on silica gel, layer thickness 2 mm, developing system hexane-acetone = 7:3 (the plates were developed 3 times) afforded two subfractions. Subtraction 4-1 (50 mg), after HPLC purification afforded 1 (17 mg) and 3 (2 mg). Subtraction 4-2 (51 mg) upon HPLC yelded aditional 1 (16 mg) and 2 (8 mg).
Fraction 5 (0.09 g, eluated with 50 %- 100 % ethyl acetate in toluene) was subjected to preparative TLC on silica gel, layer thickness 0.5 mm, developing system hexane-acetone = 65:35 (the plate was developed 4 times) to afford three subfractions. Subfractions 5-1 (12.4 mg) and 5-2 (23 mg), after purification by HPLC, both yielded compound 6 (2 mg and 4.4 mg, respectively). Purification of subfraction 5-3 (21 mg) by HPLC afforded additional amount of 1 (2.2 mg).
Preparative TLC of fraction 6 (0.12 g, eluated with 10 % methanol in ethyl acetate) on silica gel plate, layer thickness 0.5 mm, developing system hexane-acetone = 65:35 (the plate was developed 4 times) afforded subfraction 6-1 (30 mg), purified by HPLC, to give additional amount of compound 6 (2.5 mg).
The MS, UV and IR data for compounds 1-7 are given in Table 1.
Table 1
Figure imgf000015_0001
Figure imgf000015_0002
1H NMR and 13C NMR data for compounds 1 -7 are given in Tables 2-4. 04
15
Table 2
Figure imgf000016_0001
Table 3
Figure imgf000017_0001
Table 4
Figure imgf000018_0001
Accordingly, the structure of compounds 1 to 7 were identified from the data given above, and interpretation of two dimensional NMR spectroscopy (COSY, HSQC, HMBC, NOESY).
Table 5
Structures of the compounds 1 to 7
Figure imgf000019_0001
Formula I Formula II Formula III
Figure imgf000019_0002
Pr-propanoyl, Ac- acetyl, iBu- iso-butanoyl, Nic- nicotinyl. and Bz- benzoyl
THE PROCEDURE FOR DETERMINATION OF BIOLOGICAL ACTIVITY
Each compound was tested against four cultured, cancer cell lines of human origin, three of which (NCI-H460, DLD-1 and U-87 MG) were from ATCC (American Type Culture Collection). NCI-H460/R cell line with multidrug resistant phenotype was originally selected from NCI-H460 cells and cultured in a medium containing ΙΟΟηΜ doxorubicin (DOX) (reference 35). NCI-H460 (non-small cell lung carcinoma), NCI-H460/R (multidrug resistant non-small cell lung carcinoma) and DLD-1 (colorectal adenocarcinoma) cells were 10 000004
19
maintained in RPMI 1640 supplemented with 10% FBS (Fetal Bovine Serum), 2mM L- glutamine, 4.5 g/1 glucose, 10.000 U/ml penicillin, 10 mg/ml streptomycin, 25 μ¾ ι 1 amphotericin B solution at 37° C in a humidified 5 % C02 atmosphere. U-87 MG (glioblastoma) cells were maintained in Eagle Minimum Essential Medium supplemented with 10% FBS, 2mM L-glutamine, 10.000 U/ml penicillin and 10 mg/ml streptomycin at 37° C in a humidified 5 % C02 atmosphere. All cell lines were sub cultured at 72 h intervals using 0.25 % trypsin/EDTA and seeded into a fresh medium at the following densities: 8.000 cells/cm2 for NCI-H460, 16.000 cells/cm2 for NCI-H460/R, 16.000 cells/cm2 for DLD-1 and 16.000 cells/cm2 for U-87 MG. Paclitaxel (PTX) is used as positive control for cross- resistance.
Additional cancer cell line against which compounds were tested in certain series was K-562 (chronic myelogenous leukemia). K-562 cells were maintained in RPMI 1640 with 10% FCS (Fetal Calf Serum).This cell line was sub cultured at 72 h intervals using 0.1 % trypsin/EDTA and seeded into a fresh medium at 20.000 cells/cm2. Human normal lymphocytes were used in order to determine the level of toxicity on healthy cells. Cisplatin (CPt) is used as positive control for toxicity.
Cells grown in 25 cm2 tissue flasks were trypsinized, seeded into flat-bottomed 96-well tissue culture plates in 150 μΐ medium and incubated overnight. The working solution of ImM for each compound was in 25 % ethanol. Further dilutions were made in a cell culture medium. Tested concentrations in final volume of culture medium (200 μΐ) were 0.1 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ, 10 μΜ, 25 μΜ and 50 μΜ. The compounds were added to the cells in 50 μΐ of all samples under continuous culture for 72 hours. Inhibition of growth relative to untreated control was determined by the SRB (Sulforhodamine B) assay and MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. These assays test the relative ability of a compound to inhibit cell growth, but not survival of the cells. However, inhibition of growth may reflect cell death (necrosis or apoptosis) and/or cytostasis.
SRB assay (reference 36): The cellular proteins were stained with sulforhodamine B (SRB) assay that was performed with some changes. Briefly, the cells in 96-well plates were fixed in 50 % trichloroacetic acid (50 μΐ/well) for 1 h at 4° C, rinsed four times in tap water and stained with 0.4 % (w/v) sulforhodamine B in 1% acetic acid (50 μΐ/well) for 30 min at room temperature. The cells were then rinsed three times in 1% acetic acid (250 μΐ) to remove the unbound stain. The protein-bound stain was extracted with 200 μΐ 10 mM Tris base (pH 10.5) per well. The optical density was read at 540 nm, with correction at 670 nm (LKB 5060-006 Micro Plate Reader, Vienna, Austria). Values are average of quanta plicate determinations. Growth inhibition (I) was determined according to the following equitation:
I (%) = (1 - (A treated sample / untreated control) X 100.
IC30 and IC50 values were defined as the concentration of the drug that inhibited cell growth by 30% and 50%, respectively. The inhibitory concentrations for the compounds were calculated by linear regression analysis using Excel software.
RESULTS
Results of the in vitro cytotoxicity testing, specifically IC30 and IC5o values for the various cancer cell lines, are given in Table 1. Cross-resistance profile of tested compounds compared with PTX is shown in Table 2.
Table 1. Growth inhibition effect of tested compounds on various cancer cell lines
Co NCI-H460 NCI-H460/R DLD-1 U-87 MG K-562 mp IC30 IC50 IC30 IC50 IC30 IC50 IC30 IC50 IC30 IC50 ou
nd
1 >50μΜ >50μΜ >50μ >50μ 6.1 Μ 46μΜ >50μΜ >50μΜ 1.4μΜ 5.1μΜ
2 2.5μΜ 5.6μΜ 25μΜ 46μΜ 1.9μΜ 18μΜ 5.6μΜ >50μΜ >50μΜ >50μΜ
3 4.8μΜ Ι Ι . ΙμΜ 12μΜ 23μΜ 12μΜ 25μΜ 35μΜ >50μΜ 4.5μ 13μΜ
4 Ι ΙμΜ 20μ 14μΜ 28μΜ 14μΜ 38μΜ 10μΜ 46μΜ 6.2μΜ 14μΜ
5 8.3μΜ 22μΜ 15μΜ 50μΜ 14μΜ 39μΜ 10μΜ >50μΜ 5.7μΜ 15μΜ
6 5μΜ 16μΜ 50μΜ >50μΜ 37μ >50μΜ 24μΜ >50μΜ 3.1μΜ 6.7μΜ
7 6.3μΜ 13μΜ 16μΜ 24μΜ 13μΜ 24μΜ 35μΜ >50μΜ 4.6μΜ Π μΜ
An IC50 value above 50 μΜ indicates that there was insufficient cytotoxicity of the compound to achieve a 50% inhibition of cell growth at 50 μΜ.
Table 2. Relative cross-resistance to tested compounds
Resistance
Compound Factor
Paclitaxel 185.7
Vinblastine 34,6
Etoposide 21,2
Epirubicin 8,9 Doxorubicin 54,5
2 8.2
3 2.1
4 1.4
5 2.3
7 1.8
Resistance factor is relation between ICso values of resistant and sensitive non-small cell carcinoma cell lines (Resistance factor = ICso ofNCI-H460/R/IC50 ofNCI-H460)
Figure imgf000022_0001
Figure 1. Effect of 1, 2, 3, 4, 5, 6, 7 and CPt on viability of normal human lymphocytes measured by MTT. The healthy lymphocytes were preserved after 72h treatment with 1, 2, 3, 4, 5, 6 and 7 (at tested concentrations the percent of viable lymphocytes was about 90-95%). CPt induced significant decrease in lymphocyte counts (57% of lymphocytes survived at 5 μΜ and only 35% at 50 μΜ). 04
22
1 is active against colorectal adenocarcinoma and chronic myelogenous leukemia (Table 1). 1 shows great potential for treatment of leukemia, but not for solid tumors. This cytotoxic effect is followed by low toxicity of 1 observed on healthy lymphocytes (Figure 1). IC50 for 1 was above 50 μΜ for sensitive and resistant non-small cell lung carcinoma cell lines, and therefore resistance factor was not shown in Table 2.
2 is active against non-small cell lung carcinoma, colorectal adenocarcinoma and glioblastoma (Table 1). 2 shows great potential for treatment of non-small cell lung carcinoma and other solid tumors, but not for leukemia. This cytotoxic effect is followed by low toxicity of 2 observed on healthy lymphocytes (Figure 1). 2 is also active against resistant non-small cell lung carcinoma, with cross-resistance below the one obtained with PTX (Table 2).
4 is active against non-small cell lung carcinoma, colorectal adenocarcinoma, glioblastoma and leukemia (Table 1). 4 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).
7 is active against non-small cell lung carcinoma, colorectal adenocarcinoma and leukemia (Table 1). 7 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).
5 is active against non-small cell lung carcinoma, colorectal adenocarcinoma, glioblastoma and leukemia (Table 1). 5 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).
6 is active against non-small cell lung carcinoma and chronic myelogenous leukemia (Table 1). 6 shows great potential for treatment of leukemia, but not for solid tumors. This cytotoxic effect is followed by low toxicity of 6 observed on healthy lymphocytes (Figure 1). IC50 for 6 was above 50 μΜ for resistant non-small cell lung carcinoma cell line, and therefore resistance factor was not shown in Table 2.
3 is active against against non-small cell lung carcinoma, both nonresistant and resistant cell lines, colorectal adenocarcinoma as well as chronic myelogenous leukemia (Table 1). Resistance factor was shown in Table 2. The best effect on non-small cell lung carcinoma (NCI-H460) is achieved by 2, 7, 6 and 3 (Table 1).
The best effect on colorectal adenocarcinoma (DLD-1) is achieved by 2, 7, 3 and 1 (Table 1). The best effect on glioblastoma (U-87 MG) is achieved by 4, 2 and 5 (Table 1). The best effect on chronic myelogenous leukemia (K-562) is achieved by 1, 6 and 3 (Table 1). The lowest cross-resistance is obtained with 4, 7, 5, and 3 (Table 2).
2 and 3 demonstrate great potential for treatment of solid tumors, while 1, 6 and 3 show strong anti-cancer effect toward lymphoid malignances. All seven compounds do not exert toxic effect on healthy human cells. CPt, which is already in use as first line chemotherapeutic, significantly lowers the number of healthy human lymphocytes at 5 μΜ (the concentration effective against cancerous cells). 2, 4, 7, 5 and 3 are prospective drugs for treatment of resistant cancer cells. The cross-resistance to these compounds is enormously lower than the one obtained with PTX, which is also first line chemotherapeutic.
Raising the dose of paclitaxel, vinblastine, etoposide, epirubicin or doxorubicin, as shown in Table 2 by any amount increases their toxicity by the same amount, and therefore increases negative side effects to the human organism. In order to eliminate resistant cancer cells in vitro or in human organism, substances 2, 4, 7, 5 and 3 have in vitro shown results under 50 μΜ while, at the same time, it showed no toxic effects on healthy human cells.
BEST MODE FOR CARRYING OUT THE INVENTION
The novel substances may be used as dietary supplements or pharmaceutical compositions. The dietary composition may be in form of food such as dairy products (yoghurts), in form of fortified food such as cereal bars and bakery items such as cakes and cookies, in form of dietary supplements such as tablets, pills, granules, dragees, capsules, and effervescent formulations, in form of non-alcoholic drinks such as soft drinks, sport drinks, fruit juices, lemonades, near-water drinks, teas and milk based drinks, in form of liquid food such as soups and dairy products (muesli drinks), in form of alcoholic drinks such as brandy, wme or beer, or in any other beverage.
The pharmaceutical compositions may be administered in various forms, such as tablets, powders, granules, capsules, injections, suppositories, ointments and cataplasms. The pharmaceutical composition containing the active compounds of the present invention may be formulated by using conventional carriers and additives such as vehicles and resolvents, bases, diluents, fillers, adjuvants, such as solvent adjuvants, emulsifying agents, dispersers, disintegrants, solubilizers, viscosity-increasing agents, and lubricants, and additives such as antioxidans, preservatives, flavoring agents and sweetening agents.
INDUSTRIAL APPLICABILITY
Euphorbia dendroides can be planted industrially, it gives lots of juice from which the basic diterpenes may be extracted. It is a long living plant, and only the aerial parts of it are used, so it need not be destroyed when taking parts of it for the process needed. The chemical processes needed to extract the diterpenes which are presented by this invention are standard, whereas the processes needed to transform them are tested during the preparation of this application, and are fully feasible in ammounts needed for mass production of the compounds.

Claims

CLAIMS What is claimed is:
1. A diterpene compound represented by the formulae I, II and ΠΙ:
Figure imgf000026_0001
Formula I Formula Π Formula III wherein R represents hydrogen atom, hydroxy group, aliphatic alkoxy group with linear or branched, saturated or unsaturated , substituted or unsubstituted structure, or a radical represented by general formula RCOO-, wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group. Supstituents R2, R3, R4, R5, R6and R7 may be same or different and represent hydrogen atoms, aliphatic groups with linear or branched, saturated or unsaturatd, substituted or unsubstituted structure, or radicals represented by general formula formula RCO-, wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group.
2. A compound according to claim 1, wherein said compound is represented by formula IV:
Figure imgf000026_0002
IV wherein Pr represents propanoyl radical, Ac represents acetyl radical, -Bu represents iso- butanoyl radical, and Nic represents nicotinyl radical.
3. A compound according to claim 1, wherein said compound is represented by formula V:
Figure imgf000027_0001
wherein i-Bu represents ώο-butanoyl radical, Ac represents acetyl radical and Nic represents nicotinyl radical.
4. A compound according to claim 1, wherein said compound is represented by formula VI:
Figure imgf000027_0002
VI
wherein Pr represents propanoyl radical, i-Bu represents «ο-butanoyl radical, Ac represents acetyl radical and Nic represents nicotinyl radical.
5. A compound according to claim 1, wherein said compound is represented by formula VII:
Figure imgf000028_0001
VII
wherein i-Bu represents wo-butanoyl radical, Ac represents acetyl radical and Bz represents benzoyl radical.
6. A compound according to claim 1, wherein said compound is represented by formula VIII:
Figure imgf000028_0002
VIII wherein Pr represents propanoyl radical, Ac represents acetyl radical, i-Bu represents iso- butanoyl radical and Bz represents benzoyl radical.
7. A compound according to claim 1, wherein said compound is represented by formula IX:
Figure imgf000028_0003
IX
wherein Ac represents acetyl radical, i-Bu represents wo-butanoyl radical, and Nic represents nicotinyl radical.
8. A compound according to claim 1, wherein said compound is represented by formula X:
Figure imgf000029_0001
X wherein i-Bu represents wo-butanoyl radical and Bz represents benzoyl radical.
9. A diterpene compound according to claim 1 represented by formula I, wherein R1, R2, R3 and R4 may be same or different and represent hydrogen atoms, linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, substituted or unsubstituted aromatic or heteroaromatic group; and R5 radical represented by RCO-, wherein R denotes a substituted or unsubstituted heteroaromatic group.
10. A diterpene compound according to claim 1, wherein compound is represented by formula Π, wherein Ac represents acetyl radical, i-Bu represents wo-butanoyl radical, Pr represents propanoyl radical, and Nic represents nicotinyl radical.
11. A diterpene compound according to claim 1 represented by formula III, wherein Ac represents acetyl radical, i-Bu represents ώο-butanoyl radical, and Nic represents nicotinyl radical.
12. A dietary or pharmaceutical composition comprising an extract of Euphorbia dendroides containing compounds of formula I, II or ΠΙ according to claims 1 to 11.
13. A dietary or pharmaceutical composition comprising an extract of any plant containing compounds of formula I, II or III according to claims 1 to 11. An extract may comprise sap or liquid or semi-liquid material exuded from, or present in, leaves, stem, flowers, seeds and bark or between the bark and the stem. Furthermore, the extract may comprise liquid or semi-liquid material located in fractions extracted from sap, leaves, stems, flowers, bark or other plant material, even after being subjected to physical manipulation in order to disrupt plant fibres and extracellular matrix material and inter- and intra-tissue extracted into a solvent including an aqueous environment. The fractions may include aqueous or alcohol extracts or other extraction media including fractions prepared by BPLC or other fractionation systems.
14. A dietary or pharmaceutical composition comprising diterpene compounds of formula I, II or III according to claims 1 to 11 including diterpenes obtained by synthetic routes.
15. A dietary or pharmaceutical composition comprising diterpene compounds of formula I, Π or ΙΠ according to claims 1 to 1 1 obtained from extracts from genetically modified plants. Genetically modified plants include transgenic plants or plants in which a trait has been removed or where an endogenous gene sequence has been down-regulated, up- regulated, mutated or otherwise altered including the alteration or introduction of genetic material which exhibits a regulatory effect on a particular gene. Consequently, a plant which exhibits a character not naturally present in an Etiphorbiaceae plant or a species of Euphorbia or in Euphorbia dendroides is nevertheless encompassed by the present invention, including hybrid plant cells or plants comprising hybrid plant cells formed by the fusion of two or more plant cells from different strains, species or genera and optionally regenerating a plant therefrom.
16. A dietary or pharmaceutical composition comprising diterpene compounds of formula I, II or III according to claims 1 to 1 1 obtained from extracts from genetically modified organisms of any kind, other than plants, including anymals, viruses, bacteria, and fungi.
17. A method of treating cancer sensitive to the compounds in claims 1 to 1 1 , comprising administering to a patient an effective amount of a pharmaceutical composition.
18. The dietary composition according to any one of claims 12 to 16 being in form of food such as dairy products (yoghurts), in form of fortified food such as cereal bars and bakery items such as cakes and cookies, in form of dietary supplements such as tablets, pills, granules, dragees, capsules, and effervescent formulations, in form of non-alcoholic drinks such as soft drinks, sport drinks, fruit juices, lemonades, near-water drinks, teas and milk based drinks, in form of liquid food such as soups and dairy products (muesli drinks), in form of alcoholic drinks such as brandy, wine or beer, or in any other beverage.
19. Use of the dietary composition according to any one of claims 12 to 16 or the pharmaceutical composition according to any one of claims 12 to 16 as antineoplastic agent.
20. Extract of Euphorbia dendroides as defined in claim 12 for use as medicament.
21. The extract of Euphorbia dendroides as defined in claim 12 for use as medicament for the treatment of malignant deseases.
22. The extract of Euphorbia dendroides as defined in claim 12 for use according to claim 21 for the treatment of leukemia.
23. The extract of Euphorbia dendroides as defined in claim 12 for use according to claim 21 for the treatmennt of cancer including acute lymphoblastic leukemia (adult), acute aymphoblastic leukemia (childhood), acute myeloid leukemia (adult), acute myeloid leukemia (childhood), adrenocortical carcinoma, adrenocortical carcinoma (childhood), AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (childhood, cerebellar), astrocytoma (childhood cerebral), basal cell carcinoma (nonmelanoma), bile duct cancer (extrahepatic), bladder cancer, bladder cancer (childhood), bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain stem glioma (childhood), brain tumor (adult), central nervous system embryonal tumors (childhood), cerebellar astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), ependymoblastoma (childhood), ependymoma (childhood), medulloblastoma (childhood), medulloepithelioma (childhood), pineal parenchymal tumors of intermediate differentiation (childhood), supratentorial primitive neuroectodermal tumors and pineoblastoma (childhood), visual pathway and hypothalamic glioma (childhood), brain and spinal cord tumors (childhood), breast cancer, breast cancer and pregnancy, breast cancer (childhood), breast cancer (male), bronchial tumors (childhood), Burkitt lymphoma, carcinoid tumor (childhood), carcinoid tumor (gastrointestinal), carcinoma of head and neck, central nervous system embryonal tumors (childhood), central nervous system lymphoma (primary), cerebellar astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), cervical cancer, cervical cancer (childhood), childhood cancers, chordoma (childhood), chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, embryonal tumors (central nervous system, childhood), endometrial cancer, ependymoblastoma (childhood), ependymoma (childhood), esophageal cancer, esophageal cancer (childhood), Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastric (stomach) cancer (childhood), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gastrointestinal stromal cell tumor (childhood), germ cell tumor (extracranial, childhood), germ cell tumor (extragonadal), germ cell tumor (ovarian), gestational trophoblastic tumor, glioma (adult), glioma (childhood) brain stem glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer (adult, primary), hepatocellular (liver) cancer (childhood, primary), Hodgkin lymphoma (adult), hodgkin lymphoma (childhood), hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposi sarcoma, kidney (renal cell) cancer, kidney cancer, laryngeal cancer, laryngeal cancer (childhood), leukemia (acute lymphoblastic, adult), leukemia (acute lymphoblastic, childhood), leukemia (acute myeloid, adult), leukemia (acute myeloid, childhood), leukemia (chronic lymphocytic), leukemia (chronic myelogenous), hairy cell, lip and oral cavity cancer, non-small cell lung cancer, small cell lung cancer, non-Hodgkin adult lymphoma, non-Hodgkin childhood lymphoma, macroglobulinemia (Waldenstrom), melanoma, Merkel cell carcinoma, mesothelioma (adult malignant), mesothelioma (childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplasia syndromes, myelodysplasia / myeloproliferative diseases, myelogenous leukemia, chronic myeloid leukemia (adult acute), myeloid leukemia (childhood acute), myeloma (multiple), myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer (childhood), neuroblastoma, oral cancer (childhood), oral cavity cancer, lip and oropharyngeal cancer, ovarian cancer (childhood), ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (childhood), papillomatosis (childhood), parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter transitional cell cancer, respiratory tract carcinoma involving the NUT gene on chromosome 15, rhabdomyosarcoma (childhood), salivary gland cancer, salivary gland cancer (childhood), soft tissue sarcoma (adult), soft tissue sarcoma (childhood), uterine sarcoma, small intestine cancer, squamous neck cancer with occult primary (metastatic), supratentorial primitive neuroectodermal tumors (childhood), testicular cancer, throat cancer, thymoma and thymic carcinoma, thymoma and thymic carcinoma (childhood), thyroid cancer, thyroid cancer (childhood), trophoblastic tumor (gestational, unknown primary site), carcinoma of adult (unknown primary site), cancer of childhood, unusual cancers of childhood, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, vaginal cancer (childhood), vulvar cancer, Wilms tumor, women's cancers)
24. A method for the treatment of malignant diseases in animals including humans, said method comprising administering an effective dose of the extract of Euphorbia dendroides as defined in claim 12 to animals including humans which are in need thereof.
25. The method according to claim 24, wherein the animal is a human, a pet animal or a farm animal.
26. The use of compounds defined in claims 12 to 16 in the manufacture of a medicament for the prophylaxis and/or treatment of malignant diseases.
27. The use of compounds defined in claims 12 to 16 administered in various forms, such as tablets, powders, granules, capsules, injections, suppositories, ointments and cataplasms. The pharmaceutical composition containing the active compounds of the present invention may be formulated by using conventional carriers and additives such as vehicles and resolvents, bases, diluents, fillers, adjuvants, such as solvent adjuvants, emulsifying agents, dispersers, disintegrants, solubilizers, viscosity-increasing agents, and lubricants, and additives such as antioxidants, preservatives, flavoring agents and sweetening agents.
28. The use of compounds defined in claims 12 to 16, used alone, together, or in any combination with other known or novel dietaiy or pharmaceutical composition.
29. The use of compounds defined in claims 12 to 16, used together with radiation therapy of any type of cancer, reducing the risk of inducing leukemia as a side effect of the therapy.
30. The use of compounds defined in claims 12 to 16, used together with chemotherapy of any type of cancer, reducing the risk of inducing leukemia as a side effect of the therapy.
31. The use of compounds defined in claims 12 to 16, used preventively to reduce the risk of leukemia for any human or animal exposed to any type of radiation.
32. The use of compounds defined in claims 12 to 16, used preventively to reduce the risk of leukemia for any human or animal exposed to benzene.
33. The use of compounds defined in claims 12 to 16, used preventively to reduce the risk of leukemia for any human with Down syndrome and other inherited diseases.
34. The use of compounds defined in claims 12 to 16, used preventively to reduce the risk of leukemia for any human with myelodysplasia syndrome and other blood disorders.
35. The use of compounds defined in claims 12 to 16, used preventively to reduce the risk of leukemia for any human with human T-cell leukemia virus type I (HTLV-I).
36. The use of compounds defined in claims 12 to 16 in the manufacture of a medicament for the prophylaxis and/or treatment of malignant diseases.
37. A method of treating cancer sensitive to the compounds in claims 1 to 11, comprising administering to a patient an effective amount of a pharmaceutical composition containing any amount of said compounds.
38. A method for treating deseases by stymulating the immunity of the organism as a whole or any of its parts by compounds in claims 1 to 11, comprising administering to a patient an effective amount of a pharmaceutical composition containing any amount of said compounds.
39. A pharmaceutical composition comprising an immune system stimulator composition, said immune system stimulator composition comprising a biologically effective amount of herb, or extracts thereof, of Euphorbia dendroides or any of the compounds in claims 1 to 1 1.
PCT/RS2010/000004 2010-05-06 2010-05-06 Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same WO2011139172A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
RS20120482A RS58784B1 (en) 2010-05-06 2010-05-06 Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same
PCT/RS2010/000004 WO2011139172A1 (en) 2010-05-06 2010-05-06 Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/RS2010/000004 WO2011139172A1 (en) 2010-05-06 2010-05-06 Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same

Publications (1)

Publication Number Publication Date
WO2011139172A1 true WO2011139172A1 (en) 2011-11-10

Family

ID=43741940

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RS2010/000004 WO2011139172A1 (en) 2010-05-06 2010-05-06 Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same

Country Status (2)

Country Link
RS (1) RS58784B1 (en)
WO (1) WO2011139172A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104165961A (en) * 2014-08-29 2014-11-26 四川汇宇制药有限公司 Detection method of related substances in docetaxel raw material
CN108640890A (en) * 2018-04-10 2018-10-12 山东省分析测试中心 A method of isolating and purifying frankincense Chinese and Western pine alkane type macrocyclic diterpene compounds
CN113710681A (en) * 2019-01-30 2021-11-26 罗特哈姆斯泰德研究有限公司 Dehydroepiloside cyclic dimers and derivatives thereof isolated from Salix species for cancer therapy

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330094A1 (en) 1988-02-25 1989-08-30 Eszter geb. Szénási Tamás Use of the plant Euphorbia hirta L. and the extracts as well as their active ingredients
DE4102054A1 (en) 1991-01-24 1992-07-30 Geb Szenasi Tamas Euphorbia hirta L. to increase immunity - e.g. against influenza, winter colds and AIDS and as antifungal agent to treat open wounds
HU208790B (en) 1991-12-05 1994-01-28 Tamas Process for producing pharmaceutical compositions against arc and aids, comprising euphorbia hirta l. plant or its parts or their extracts
JPH08245505A (en) * 1994-09-09 1996-09-24 Tosoh Corp Diterpene derivative, its production and antitumor agent containing the same as active ingredient
US6333348B1 (en) 1999-04-09 2001-12-25 Aventis Pharma S.A. Use of docetaxel for treating cancers
US20050003031A1 (en) * 1997-08-19 2005-01-06 Aylward James Harrison Anti-cancer compounds
US7378445B2 (en) 2000-08-07 2008-05-27 Peplin Research Pty. Ltd. Treatment of prostate cancer

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330094A1 (en) 1988-02-25 1989-08-30 Eszter geb. Szénási Tamás Use of the plant Euphorbia hirta L. and the extracts as well as their active ingredients
DE4102054A1 (en) 1991-01-24 1992-07-30 Geb Szenasi Tamas Euphorbia hirta L. to increase immunity - e.g. against influenza, winter colds and AIDS and as antifungal agent to treat open wounds
HU208790B (en) 1991-12-05 1994-01-28 Tamas Process for producing pharmaceutical compositions against arc and aids, comprising euphorbia hirta l. plant or its parts or their extracts
JPH08245505A (en) * 1994-09-09 1996-09-24 Tosoh Corp Diterpene derivative, its production and antitumor agent containing the same as active ingredient
US20050003031A1 (en) * 1997-08-19 2005-01-06 Aylward James Harrison Anti-cancer compounds
US6333348B1 (en) 1999-04-09 2001-12-25 Aventis Pharma S.A. Use of docetaxel for treating cancers
US7378445B2 (en) 2000-08-07 2008-05-27 Peplin Research Pty. Ltd. Treatment of prostate cancer

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
"Anonymous. Oncology Tools: Approved Claims for microtubule inhibitors", US FOOD AND DRUG ADMINISTRATION, 17 September 2006 (2006-09-17)
"Anonymous. Taxotere Docetaxel concentrate for infusion", MEDSAFE, 25 September 2006 (2006-09-25)
A. EVANS; M. A. OSMAN: "Carcinogenicity of bracken and shikimic acid", NATURE, vol. 250, 26 July 1974 (1974-07-26), pages 348 - 349
ANONYMOUS. DOCETAXEL: CLINICAL PHARMACOLOGY. RXLIST., 29 June 2006 (2006-06-29)
ANONYMOUS. DRUGDEX EVALUATIONS: DOCETAXEL. THOMSON MICROMEDEX, 26 September 2006 (2006-09-26)
ANONYMOUS. NEW ZEALAND PHARMACEUTICAL SCHEDULE. WELLINGTON: PHARMAC, 2006, pages 133
ANONYMOUS. TAXOTERE.COM FOR HEALTHCARE PROFESSIONALS: ABOUT. SANOFI-AVENTIS U.S. LLC., July 2005 (2005-07-01)
ANONYMOUS. TAXOTERE.COM FOR HEALTHCARE PROFESSIONALS: EFFICACY AND SAFETY. SANOFI-AVENTIS U.S. LLC., 24 September 2006 (2006-09-24)
ANONYMOUS. TAXOTERE.COM FOR HEALTHCARE PROFESSIONALS: PHARMACOKINETICS. SANOFI-AVENTIS U.S. LLC., 23 September 2006 (2006-09-23)
BAKER SD; ZHAO M; LEE CKK; VERWEIJ J; ZABELINA Y; BRAHMER JR ET AL.: "Comparative pharmacokinetics of weekly and every-three-weeks docetaxel", CLIN CANCER RES., vol. 10, no. 6, 2004, pages 1976 - 83
BARILE; ELISA; COREA; GABRIELLA; LANZOTTI; VIRGINIA: "Diterpenes from Euphorbia as potential leads for drug design", NATURAL PRODUCT COMMUNICATIONS, vol. 3, no. 6, 2008, pages 1003 - 1020
CLARKE SJ; RIVORY LP.: "Clinical pharmacokinetics of docetaxel", CLIN PHARMACOKINET, vol. 36, no. 2, 1999, pages 99 - 114
COREA ET AL: "Jatrophane Diterpenes as Modulators of Multidrug Resistance. Advances of Structure-Activity Relationships and Discovery of the Potent lead Pepluanin A", J.MED.CHEM., vol. 47, no. 4, 2004, pages 988 - 992, XP002629805 *
COREA, G.; DI PIETRO, A.; DUMONTET, C.; FATTORUSSO, E.; LANZOTTI, V: "Jatrophane diterpenes from Euphorbia spp. as modulators of multidrug resistance in cancer therapy", PHYTOCHEMISTRY REVIEWS, vol. 8, no. 2, 2009, pages 431 - 447
COREA, G.DI PIETRO, A.DUMONTET, C.FATTORUSSO, E.LANZOTTI, V: "Jatrophane diterpenes from Euphorbia spp. as modulators of multidrug resistance in cancer therapy", PHYTOCHEMISTRY REVIEWS, vol. 8, no. 2, 20 September 2001 (2001-09-20), pages 431 - 447, XP019686452 *
COREA, GABRIELLA; FATTORUSSO, ERNESTO; LANZOTTI, VIRGINIA; TAGLIALATELA-SCAFATI, ORAZIO; APPENDINO, GIOVANNI; BALLERO, MAURO; SIMO: "Jatrophane Diterpenes as P-Glycoprotein Inhibitors. First Insights of Structure-Activity Relationships and Discovery of a New", POWERFUL LEAD JOURNAL OF MEDICINAL CHEMISTRY, vol. 46, no. 15, 2003, pages 3395 - 3402
COREA, GABRIELLA; FATTORUSSO, ERNESTO; LANZOTTI, VIRGINIA; TAGLIALATELA-SCAFATI, ORAZIO; APPENDINO, GIOVANNI; BALLERO, MAURO; SIMO: "Modified jatrophane diterpenes as modulators of multidrug resistance from Euphorbia dendroides L", BIOORGANIC & MEDICINAL CHEMISTRY, vol. !, no. 23, 2003, pages 5221 - 5227
COREA, GABRIELLAFATTORUSSO, ERNESTOLANZOTTI, VIRGINIATAGLIALATELA-SCAFATI, ORAZIOAPPENDINO, GIOVANNIBALLERO, MAUROSIMON, PIERRE-NO: "Jatrophane Diterpenes as P-Glycoprotein Inhibitors. First Insights of Structure-Activity Relationships and Discovery of a New", POWERFUL LEAD JOURNAL OF MEDICINAL CHEMISTRY, vol. 46, no. 15, 2003, pages 3395 - 3402, XP002629804 *
DATABASE WPI Derwent World Patents Index; AN 1996-488256, XP002629811 *
DEP. VIROL.
EISENHAUER EA; VERMORKEN JB.: "The taxoids: Comparative clinical pharmacology and therapeutic potential", DRUGS, vol. 55, no. 1, 1998, pages 5 - 30
FATOPE MO; ZENG L; OHAYAGA JE; SHI G; MCLAUGHLIN JL: "Selectively cytotoxic diterpenes from Euphorbia poisonii", J MED CHEM, vol. 39, 1996, pages 1005 - 1008
FATOPE MOZENG LOHAYAGA JESHI GMCLAUGHLIN JL: "Selectively cytotoxic diterpenes from Euphorbia poisonii", J MED CHEM, vol. 39, 1996, pages 1005 - 1008, XP002374791 *
G. COREA ET AL: "Modified Jatrophane Diterpenes as Modulators of Multidrug Resistance from Euphorbia Dendroides L.", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 11, 2003, pages 5221 - 5227, XP002629806 *
GOYLE S; MARAVEYAS A.: "Chemotherapy for colorectal cancer", DIG SURG., vol. 22, no. 6, 2005, pages 401 - 14
GUITTON J; COHEN S; TRANCHAND B; VIGNAL B; DROZ JP; GUILLAUMONT M ET AL.: "Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry", RAPID COMMUN MASS SPECTROM, vol. 19, no. 17, 2005, pages 2419 - 26
GUNDIDZA; KUFA, CENTR. AFR. J. MED., vol. 38, 1992, pages 444 - 447
H. HELMBOLDT ET AL: "Enantioselective synthesis of the C-14 to C-5 cyclopentane segment of jatrophane diterpenes", TEDRAHEDRON LETTERS, vol. 45, 2004, pages 289 - 292, XP004478886 *
IMAI, S.; M. SUGIURA ET AL.: "African Burkitt's lymphoma: A plant, Euphorbia tirucalli, reduces Epstein-Barr virus-specific cellular immunity", ANTICANCER RESEARCH, vol. 14, no. 3A, 1994, pages 933 - 936
LUIS M. BEDOYA ET AL: "SJ23B, a jatrophane diterpene activates classical PKCs and displays strong activity against HIV in vitro", BIOCHEMICAL PHARMACOLOGY, vol. 77, 2009, pages 965 - 978, XP025990021 *
LYSENG-WILLIAMSON KA; FENTON C.: "Docetaxel: a review of its use in metastatic breast cancer", DRUGS, vol. 65, no. 17, 2005, pages 2513 - 31
M. PESIC; J. Z. MARKOVIC; D. JANKOVIC; S. KANAZIR; 1. D. MARKOVIC; L. RAKIC; S. RUZDIJIC: "Induced Resistance in the Human Non Small Cell Lung Carcinoma (NCI-H460) Cell Line In Vitro by Anticancer Drugs", JOURNAL OF CHEMOTHERAPY, vol. 18, 2006, pages 66 - 73
MATHERS, COLIN D; CYNTHIA BOSCHI-PINTO; ALAN D LOPEZ; CHRISTOPHER JL MURRAY: "Cancer incidence, mortality and survival by site for 14 regions of the world", GLOBAL PROGRAMME ON EVIDENCE FOR HEALTH POLICY DISCUSSION PAPER NO. 13, 2001
PIERRE POTIER, CHEMIST, 1998
SEVIL OKSIIZA, B; FALIHA GIIREKA; LONG-ZE LINC; ROBERTO R. GILC; JOHN M. PEZZUTOC; GEOFFREY A. CORDELL: "Aleppicatines A and B from Euphorbia aleppica", PHYTOCHEMISTRY, vol. 42, no. 2, May 1996 (1996-05-01), pages 473 - 478
SKEHAN, P.; STORENG, R.; SCUDIERO, D.; MONKS, A.; MCMAHON, J.; VISTICA, D.; WARREN, J. T.; BOKESCH, H.; KENNEY, S.; BOYD, M. R.: "New colorimetric cytotoxicity assay for anticancer-drug screening", J. NATL. CANCER INST., vol. 82, 1990, pages 1107 - 1112
SNYDER JP; NETTLES JH; CORNETT B; DOWNING KH; NOGALES E.: "The binding conformation of Taxol in b-tubulin: A model based on electron crystallographic density", PNAS, vol. 98, no. 9, 2001, pages 5312 - 16
STARVIC; STOLZ, FOOD COSMET. TOXICOL., vol. 14, 1976, pages 141
TANNOCK IF; DE WIT R; BERRY WR; HORTI J; PLUZANSKA A; CHI KN ET AL.: "Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer", N ENGL J MED., vol. 351, no. 15, 2004, pages 1502 - 12
URIEN S; BARRE J; MORIN C; PACCALY A; MONTAY G; TILLEMENT JP.: "Docetaxel serum protein binding with high affinity to alpha I-acid glycoprotein", INVEST NEW DRUGS, vol. L4, no. 2, 1996, pages 147 - 51
YVON AC; WADSWORTH P; JORDAN MA.: "Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells", THE AMERICAN SOCIETY FOR CELL BIOLOGY, vol. 10, 1999, pages 947 - 959

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104165961A (en) * 2014-08-29 2014-11-26 四川汇宇制药有限公司 Detection method of related substances in docetaxel raw material
CN108640890A (en) * 2018-04-10 2018-10-12 山东省分析测试中心 A method of isolating and purifying frankincense Chinese and Western pine alkane type macrocyclic diterpene compounds
CN113710681A (en) * 2019-01-30 2021-11-26 罗特哈姆斯泰德研究有限公司 Dehydroepiloside cyclic dimers and derivatives thereof isolated from Salix species for cancer therapy

Also Published As

Publication number Publication date
RS20120482A1 (en) 2013-06-28
RS58784B1 (en) 2019-06-28

Similar Documents

Publication Publication Date Title
Huang et al. A naturally occurring mixture of tocotrienols inhibits the growth of human prostate tumor, associated with epigenetic modifications of cyclin-dependent kinase inhibitors p21 and p27
US9820963B2 (en) Composition containing lignan compound as active ingredient for preventing or treating cancer
WO2011139172A1 (en) Diterpene compounds with antineoplastic activities and pharmaceutical compositions comprising same
KR100821683B1 (en) A composition containing icariside ii for preventing and treating diseases associated with abnormal angiogenesis
KR101673265B1 (en) A pharmaceutical composition for preventing or treating IL-6-mediated disease, comprising extract, fraction, or compounds derived from Ampelopsis brevipedunculata
KR101533733B1 (en) Composition for preventing or treating cancer comprising alnus japonica, descurainia sophia, and peucedanum praeruptorum dunn mixed extracts or fraction thereof
KR101300798B1 (en) Composition comprising Ligularia fischeri extract for protecting nerve cells
WO2013161821A1 (en) Cgrp responsiveness promoter
KR20170115764A (en) Combination therapy for treating neurodegenerative diseases using combination of isoliquiritigenin and flupirtine or mirtazapine
KR100965305B1 (en) Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27
KR101418164B1 (en) A pharmaceutical composition comprising extract of UV-induced rice for preventing or treating a colon cancer
KR100542587B1 (en) Composition containing an extract of Zingiber cassumunar or the compound isolated therefrom for preventing and treating cancer disease
KR101512095B1 (en) Composition for preventing or treating renal disease containing extract of amorpha fruticosa
KR100965304B1 (en) Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27
KR101349113B1 (en) Pharmacological composition for dementia prevention or treatment comprising specific substance extracted from Hericium erinacium and preparation method thereof
KR101244402B1 (en) A composition for enhancing the radiotherapy of cancer
KR101021975B1 (en) A composition for enhancing the radiotherapy of cancer
KR20130009084A (en) A pharmaceutical composition for preventing gastric cancer containing denbinobin as active ingredient
KR101703827B1 (en) Novel Curcumin Derivatives and Uses Thereof
KR100502835B1 (en) Compositions comprising extract of ganoderma lucidum, oleamide and its structural analogue as an effective component for preventing or treating dementia
KR102336045B1 (en) Novel compound and composition comprising the same for preventing or treating cancer
KR102156701B1 (en) A composition for inhibiting cancer metastasis comprising Oenothera odorata extracts
KR102018085B1 (en) Pharmaceutical Composition for Treatment and Inhibiting Metastasis of Brain Tumor Comprising Acteoside
KR101434500B1 (en) Composition for preventing and treating prostate cancer comprising anthocyanin extracted from black soybean
KR101404257B1 (en) Composition for Preventing and Treating Inflammatory or Immune diseases Comprising Octaphlorethol A

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10731832

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: P-2012/0482

Country of ref document: RS

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 21.03.2013)

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 21.03.13).

122 Ep: pct application non-entry in european phase

Ref document number: 10731832

Country of ref document: EP

Kind code of ref document: A1