AU2016101447A4 - Synergistic combination of a phyllanthus amarus extract and 5-fluorouracil for treatment of subjects with cancer - Google Patents
Synergistic combination of a phyllanthus amarus extract and 5-fluorouracil for treatment of subjects with cancer Download PDFInfo
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- AU2016101447A4 AU2016101447A4 AU2016101447A AU2016101447A AU2016101447A4 AU 2016101447 A4 AU2016101447 A4 AU 2016101447A4 AU 2016101447 A AU2016101447 A AU 2016101447A AU 2016101447 A AU2016101447 A AU 2016101447A AU 2016101447 A4 AU2016101447 A4 AU 2016101447A4
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- Prior art keywords
- fluorouracil
- phyllanthus amarus
- extract
- extraction solvent
- cancer
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Abstract
The present invention relates in a first aspect to a method for treating a subject suffering from cancer, in particular liver cancer. The method comprises administrating synergistic amounts of a Phyllanthus amarus extract and 5-fluorouracil or a prodrug 5 thereof to a subject, in particular a human. In another aspect of the present invention, a method of potentiating the activity of 5-fluorouracil in cancer cells is provided. In a further aspect, the present invention provides a kit comprising the Phyllanthus amarus extract and the 5-fluorouracil or a prodrug thereof. It has been found that the combination treatment with the Phyllanthus amarus extract 10 of the present invention and 5-fluorouracil or a prodrug thereof allows for synergistically potentiating the activity of 5-fluorouracil. Combined administration of Phyllanthus amarus and 5-fluorouracil or a prodrug thereof, thus, represents a highly promising treatment option for treating patients with cancer with enhanced anti-cancer activity of 5-fluorouracil while reducing or preventing resistances.
Description
SYNERGISTIC COMBINATION OF A PHYLLANTHUS AMARUS EXTRACT AND 5-FLUOROURACIL FOR TREATMENT OF SUBJECTS WITH CANCER
TECHNICAL FIELD
The present invention relates in a first aspect to a method for treating a subject suffering from cancer, in particular liver cancer. The method comprises administrating synergistic amounts of a Phyllanthus amarus extract and 5-fluorouracil or a prodrug thereof to a subject, in particular a human. In another aspect of the present invention, a method of potentiating the activity of 5-fluorouracil in cancer cells is provided. In a further aspect, the present invention provides a kit comprising the Phyllanthus amarus extract and the 5-fluorouracil or a prodrug thereof.
BACKGROUND OF THE INVENTION
Nucleoside analogues (NAs) have been developed almost 50 years ago for treatment of cancer or viral infections. NAs usually produce their major therapeutic effect through disturbing DNA and RNA synthesis, affecting key enzymes, cell signaling and intracellular metabolisms. This inevitably affects the natural ribonucleotides (RNs) and deoxyribonucleotides (dRNs) pool sizes, which play an important role in various cellular functions such as DNA replication, proliferation and cell cycle which requires an appropriate balance of the RNs and dRNs. Influencing intracellular RN and dRN pool sizes might represent a strategy for potentiating antitumor effects of NAs and for enhancing cancer-cell selectivity. 5-Fluorouracil (5-FU), also referenced as fluorouracil, is a fluoropyrimidine chemotherapeutic compound and has been widely used for treatment of many types of cancers such as colorectal, liver and breast cancer. 5-Fluorouracil and its metabolites inhibit cancer cell proliferation and cancer cell growth by means of various mechanisms. For example, 5-fluorouracil is converted to the active metabolite 5-fluoroxyuridine monophosphate (FUMP), which is metabolized to FUTP inhibiting RNA and protein synthesis by competing with uridine triphosphate. 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) as active metabolite of 5-fluorouracil is an irreversible inhibitor of the enzyme thymidylate synthase (TS) leading to a decrease of intracellular deoxythymidine triphosphate (dTTP) necessary for DNA synthesis and increase of deoxyuridine monophosphate (dUMP). Other metabolites of 5-fluorouracil are incorporated into DNA or RNA as false nucleotides with respective defects in RNA or DNA, wherein ribonucleotide reductase (RR) plays an important role in the metabolic pathways of 5-fluorouracil. Thymidylate synthase and ribonucleotide reductase are thereby considered as major determinants of the outcome of a 5-fluorouracil treatment. In sum, the intracellular RN and dRN pool sizes become unbalanced by 5-FU due to complex metabolic networks of RNs and dRNs. In turn, disturbing the balance of RN and dRN pools could have an impact on the activity of 5-fluorouracil.
There is an increasing interest in using herbal medicines combined with conventional chemotherapeutic compounds in order to improve chemotherapeutic activity. Several herbal medicines were found for being suitable adjuvants for enhancing the activity of cancer chemotherapies as well as for reducing side effects and resistances. This might also allow reducing the dose of the chemotherapeutic compound with a resulting improved efficacy-side effect profile.
Phyllanthus amarus Schum. & Thonn. (PHA) which is a plant of the family Euphorbiaceae that growths in tropical and subtropical areas has been used to treat various diseases including jaundice, dysentery disorders, scabies and in case of wounds (Konkimalla, V.B., Efferth, T, J Ethnopharmacol 2008,116:207-210). PHA has, further, been found to be useful for inhibiting DNA polymerase of HBV and related hepatitis viruses and to downregulate HBV mRNA transcription and translation (Blumberg, B.S. et al., Vaccine 1990,8 Suppl:S86-92, Ott, M. et al., Eur J Clin Invest 1997,27:908-915, Rajeshkumar, N.V. et al., J Ethnopharmacol 2002,81:17-22). A combined use with 5-fluorouracil was, however, neither known nor described so far -let alone any synergistic mechanism when administering both.
Consequently, there remains a strong need for methods and means allowing for an effective therapeutic treatment of cancer, in particular for means and methods for potentiating the activity of already known and therapeutically used chemotherapeutic compounds while ensuring an acceptable level of side effects.
SUMMARY OF THE INVENTION
The present invention relates in a first aspect to a method for treating a subject suffering from cancer. Said method comprises the step of administering synergistic amounts of (i) a Phyllanthus amarus extract, and (ii) 5-fluorouracil or a prodrug thereof to a subject, in particular a human. The cancer is in particular liver cancer. Preferred prodrugs of 5-fluorouracil include tegafur, floxuridine, doxifluridine or capecitabine, in particular capecitabine.
The Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof may be administered to the subject simultaneously or one of them is administered before or subsequent to the other, in particular in form of one single or in form of separate compositions including the Phyllanthus amarus extract, the 5-fluorouracil or prodrug thereof or both of them, i.e. the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof.
The Phyllanthus amarus extract is obtained or obtainable by an extraction comprising contacting a Phyllanthus amarus plant material with an extraction solvent which is in particular selected from water, an aliphatic alcohol or mixtures thereof. The Phyllanthus amarus plant material can include the whole plant or the aerial parts of the plant. The extraction solvent in particular comprises ethanol and most preferably essentially consists of about 75% (v/v) ethanol.
In particular, the Phyllanthus amarus extract is obtained by an extraction comprising: a) contacting Phyllanthus amarus plant material with an extraction solvent which comprises and more preferably essentially consists of 75 Vol.-% ethanol for obtaining a crude extract such as by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 8 h and subjecting the mixture to ultrasonication for at least 45 min; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step b) for obtaining a residue and preferably drying the residue such as by evaporating the extraction solvent in a rotary evaporator and drying the residue by means of vacuum drying at at least 40°C.
The present invention provides in a second aspect a method of potentiating the activity of 5-fluorouracil in cancer cells comprising contacting the cancer cells with: (i) a Phyllanthus amarus extract; and (ii) 5-fluorouracil.
The cancer cells are in particular from a human liver cancer. The cancer cells may be contacted with the Phyllanthus amarus extract and the 5-fluorouracil simultaneously or the cancer cells are contacted with one of them before or subsequent to the other. In particular, the cancer cells are contacted with both simultaneously.
The Phyllanthus amarus extract is in particular obtained or obtainable by an extraction as described above.
Contacting the cancer cells in particular means administering the Phyllanthus amarus extract and 5-fluorouracil or a prodrug thereof to a subject, in particular a human.
In still another aspect, the present invention provides a kit comprising synergistic amounts of: (i) a Phyllanthus amarus extract; and (ii) 5-fluorouracil or a prodrug thereof.
The Phyllanthus amarus extract is, in particular, obtained or obtainable by an extraction as described above.
The Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof may be contained in the kit in solid, semisolid or liquid form. The kit can further comprise at least one pharmaceutically tolerable excipient such as one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a coloring agent, a surfactant and a preservative.
Further in accordance with the present invention is a pharmaceutical composition comprising the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof and an excipient such as selected from a pharmaceutically acceptable carrier, salt, buffer, water, or a combination thereof, in particular comprising: (i) the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof, and (ii) one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a coloring agent, a surfactant and a preservative.
Accordingly, the present invention provides a novel and highly advantageous approach for treating cancer from various origins including the administration of (i) the Phyllanthus amarus extract and (ii) the 5-fluorouracil or a prodrug thereof as synergistic combination. It has been found that the Phyllanthus amarus extract obtained or obtainable as described above is especially suitable for inhibiting the proliferation and growth of cancer cells when used in combination with 5-fluorouracil.
In particular, the inventors unexpectedly found that although Phyllanthus amarus alone has no significant impact on the levels of RNs and dRNs, Phyllanthus amarus when combined with 5-fluorouracil enhances the anticancer activity of 5-fluorouracil through affecting the ribonucleotide and deoxyribonucleotide pool sizes which may follow from the induced cell cycle arrest or the affected regulation of key enzyme steps in intracellular RN and dRN metabolism. The inventors in particular found that high amounts and long term treatment with Phyllanthus amarus exerted a cytostatic effect on HepG2 cells. At the same time, comparatively low amounts of Phyllanthus amarus synergistically enhanced the antitumor activity of 5-fluorouracil via its effects on RN and dRN pools.
Combined administration of Phyllanthus amarus and 5-fluorouracil or a prodrug thereof, thus, represents a highly promising treatment option for treating patients with cancer with enhanced anti-cancer activity of 5-fluorouracil and improved response rates to 5-fluorouracil while reducing or preventing resistances. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows MTT results measured in a HepG2 cell line in the presence of a Phyllanthus amarus (PHA) extract of the present invention, 5-fluorouracil (5-FU) or a combination of the Phyllanthus amarus extract of the present invention and 5-fluorouracil. Fig. 1A is a diagram showing the MTT results measured in a HepG2 cell line in the presence of a Phyllanthus amarus extract of the present invention after 24, 48 or 72 h. Fig. 1B is a diagram showing the MTT results measured in a HepG2 cell line in the presence of 5-fluorouracil or a combination of 5-fluorouracil and the Phyllanthus amarus extract of the present invention after 24 or 48 h. (*P<0.05, **P<0.01, compared with the control group).
Fig. 2 shows score plots of PCA models with the statistical parameters as follows: Fig. 2A (RN pool sizes): R2X=0.998, Q2=0.993, Fig. 2B (dRN pool sizes): R2X=0.978, Q2=0.915; Scores plots of OPLS-DA models with the statistical parameters as follows: Fig. 2C (RN pool sizes): R2X=0.995, R2Y=0.984, Q2=0.954, Fig. 2D (dRN pool sizes): R2X=0.987, R2Y=0.985, Q2=0.965; VIP plot Fig. 2E (RNs pool sizes) and Fig. 2F (dRN pool sizes) (N: Normal control group, F: 5-FU-treated group, P: PHA-treated group, P-F: combination group with 5-FU and PHA)
Fig. 3 shows the levels of ribonucleotides in HepG2 cells after treatment with the Phyllanthus amarus extract (PHA) of the present invention, 5-fluorouracil (5-FU) or a combination of the Phyllanthus amarus extract of the present invention and 5-fluorouracil for 24 h and a control group without treatment. Fig. 3A is a diagram showing the levels of nucleoside triphosphates in each group. Fig. 3B is a diagram showing the levels of nucleoside diphosphates in each group. Fig. 3C is a diagram showing the levels of nucleoside monophosphates in each group. All group data are represented as mean ± SD of three independent experiments (*P<0.05, **P<0.01, compared with the control group; #P<0.05, ##P<0.01, compared with the 5-FU group). (N: Normal control group, F: 5-FU-treated group, P: PHA-treated group, P-F: combination group with 5-FU and PHA)
Fig. 4 is a diagram showing the levels of dUMP and dTTP after treatment with the Phyllanthus amarus extract of the present invention, 5-fluorouracil or a combination of the Phyllanthus amarus extract of the present invention and 5-fluorouracil for 24 h and a control group without treatment. All group data are represented as mean ± SD. of three independent experiments (*P<0.05, **P<0.01, compared with the control group; *P<0.05, ##P<0.01, compared with the 5-FU group). (N: Normal control group, F: 5-FU-treated group, P: PHA-treated group, P-F: combination group with 5-FU and PHA).
Fig. 5 shows Western Blots obtained with HepG2 cells. Fig. 5A is a Western Blot showing the expression of thymidylate synthase (TS) and β-tubulin as control after treatment with the Phyllanthus amarus extract of the present invention, 5-fluorouracil or a combination of the Phyllanthus amarus extract of the present invention and 5-fluorouracil for 24 h and a control group without treatment. Fig. 5B is a Western Blot showing the expression of ribonucleotide reductase subunits M1 (RRM1), M2 (RRM2), P53-controlled ribonucleotide reductase (P53R2) and β-tubulin as control after treatment with the Phyllanthus amarus extract of the present invention, 5-fluorouracil or a combination of the Phyllanthus amarus extract of the present invention and 5-fluorouracil for 24 h and a control group without treatment.
Fig. 6 is a diagram showing the cell cycle effects of 5-fluorouracil and the Phyllanthus amarus extract of the present invention in HepG2 cells after 24 h. All data were expressed as mean ± SD values by three independent experiments. P value of less than 0.05 (*P< 0.05, **P< 0.01, versus the control group; *P< 0.05, mP< 0.01, versus the 5-FU group) were considered significant. (N: Normal control group, F: 5-FU-treated group, P: PHA-treated group, P-F: combination group with 5-FU and PHA).
DESCRIPTION OF THE EMBODIMENTS
The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and for representing preferred embodiments thereof. The technical terms used in the present patent application have the meaning as commonly understood by a respective skilled person unless specifically defined otherwise.
As used herein, “comprising” means including the following elements or components but not excluding others. “Essentially consisting of” means that the material or compound consists of the respective element or components along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. “Consisting of” means that the material solely consists of, i.e. is formed by the respective element or component. As used herein, the forms “a,” “an,” and “the,” are intended to include the singular and plural forms unless the context clearly indicates otherwise.
The present invention relates in a first aspect to a method for treating a subject suffering from cancer. Said method of treating cancer comprises the step of administering synergistic amounts of a Phyllanthus amarus extract and 5-fluorouracil or a prodrug thereof to a subject.
As used herein the term “Phyllanthus amarus extract” refers to an active preparation derived from Phyllanthus amarus plant material which is optionally further processed and/or purified, i.e. a preparation comprising one or more active components contained in the Phyllanthus amarus plant material. By "active" it is meant that the Phyllanthus amarus extract and the one or more components comprised therein, respectively, are capable of producing a desired synergistic effect when combined with 5-fluorouracil ora prodrug thereof for treating certain disease, which is according to the present invention cancer. More specifically, the desired effect is a synergistic inhibition or suppression of the proliferation and/or growth of the cancer cells, a synergistic reduction of cancerous cells and/or a synergistic amelioration of symptoms related to the cancer, in particular a synergistic reduction of the growth and proliferation of the cancer cells.
An extract is generally obtained by an "extraction", which will be understood by those skilled in the art as treating plant materials with an extraction solvent to dissolve the active components and separate them from unwanted plant material. The Phyllanthus amarus extract can be in liquid form, in particular a decoction, solution, infusion or tincture or in solid form, in particular a powder or granules. The term Phyllanthus amarus extract also covers liquid or solid preparations comprising the one or more active components and parts of Phyllanthus amarus plant material more or less broken into pieces and optionally the extraction solvent. Likewise, the term Phyllanthus amarus extract covers embodiments in which the preparation is liquid comprising the one or more active components and a solvent, which can in embodiments be the extraction solvent or which is a different or further solvent like water, ethanol or the like such as added after at least partially removing the extraction solvent.
The Phyllanthus amarus extract comprises components such as selected from one or more of alkaloids such as pyrrolizidine alkaloids, flavonoids, lignans such as dibenzylbutane-type lignans, tannins such as ellagitannins, polyphenols, triterpenes, sterols and/or higher molecular polysaccharides. In particular, the Phyllanthus amarus extract comprises one or more of phyllanthin, hypophyllanthin, phyllanthusiin D, amarin, amarulone and amarinic acid, in particular it comprises phyllanthin as major component with an amount of between about 0.03 % by weight and about 0.1 % by weight.
The term “prodrugs” of 5-fluorouracil includes any compound that, following oral or parenteral administration to the subject, is metabolized to 5-fluorouracil or to 5-fluorouracil among others. Preferred prodrugs include tegafur, capecitabine, galocitabine, floxuridine and doxifluridine or polymeric prodrugs of 5-fluorouracil such as conjugates of 5-fluorouracil to polymers like polysaccharides or amino sugar or amino polysaccharide derivatives. More preferably, the prodrug is selected from tegafur, floxuridine, doxifluridine or capecitabine, in particular it is capecitabine.
The administration of the Phyllanthus amarus extract and of the 5-fluorouracil or prodrug thereof to the subject can be carried out independently or simultaneously in particular by oral administration or parenteral administration such as intravenous or intra-arterial injection or infusion. The Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof may be administered to the subject simultaneously or one of them is administered before or subsequent to the other. The administration of the Phyllanthus amarus extract and of the 5-fluorouracil or prodrug thereof may be carried out independently or simultaneously once or several times a day, a week or a month.
The Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof may be administered in combination with: a) at least one chemotherapeutic compound, which chemotherapeutic compound is a compound selected from the group consisting of a topoisomerase-ll inhibitor, an anthracycline, a coordination complex of platinum, a taxane, a protein kinase inhibitor, a vinca alkaloid or derivative thereof, a topoisomerase-l inhibitor and a further nucleotide analog or precursor analog; b) radiotherapy, and/or c) immunotherapy.
The term “synergistic amounts" as used herein refers to amounts of the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof which have after administration a “synergistic effect”. The exact nature of the effect varies depending on the specific disorder which is treated. When the disorder is cancer as according to the present invention, the effect is an inhibition or suppression of the proliferation and/or growth of the cancer cells, a reduction of cancerous cells and/or the amelioration of symptoms related to the cancer. A synergistic effect is, thus, an effect greater than the expected additive effect of Phyllanthus amarus extract and 5-fluorouracil or prodrug thereof. In other words, “synergistic” means according to the present invention that the combination of Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof achieves improved effects, namely improved inhibition or suppression of the proliferation and/or growth of the cancer cells, an improved reduction of cancerous cells and/or an improved amelioration of symptoms related to the cancer relative to the additive effects when the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof are administered independently. The synergistic amounts may depend on the species and body weight of the subject. The skilled person is able to determine and select such synergistic amounts such as by means of a cytotoxicity assay for determining cell viability and IC50 like an MTT assay and a combination index analysis (Cl) as known in the art (Chou, T.C. et al., J Natl Cancer Inst 1994,86:1517-1524, Chou, T.C. et al., Adv Enzyme Regul 1984,22:27-55). A combination index (Cl) of Cl = 1 indicates that the compounds have additive effects, Cl < 1 indicates more than additive effects (“synergism”) and Cl > 1 indicates less than additive effects (“antagonism”). Preferably, the synergistic amounts of the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof are such that a Cl of less than 0.8, more preferably of less than 0.7 and in particular of about 0.66 is obtained.
For example, the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof may be administered in concentrations of at least about 25 pg/ml, in particular at least about 50 pg/ml or at least about 100 pg/ml of the Phyllanthus amarus extract, in particular about 50 pg/ml, and at least about 50 pM of 5-fluorouracil, in particular 50 pM, to the subject for obtaining the synergistic effects preferably for at least 24 h, more preferably at least 48 h and in particular at least 72 h. In humans, the amount of 5-fluorouracil can be, for example, between at least about 250 mg up to 1000 mg per intravenous injection or intravenous or intra-arterial infusion, in particular between about 5 mg/kg bodyweight to 15 mg/kg bodyweight.
The subject can be a human or animal, in particular the subject is a mammal, preferably a human. The subject is, thus, preferably a human having cancer.
The Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof may be administered in form of one single or in form of separate compositions including the Phyllanthus amarus extract, the 5-fluorouracil or prodrug thereof or both of them and at least one excipient such as selected from a pharmaceutically acceptable carrier, salt, buffer, water, or a combination thereof. Preferably, the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof are administered in form of one single or in form of separate pharmaceutical compositions comprising: • the Phyllanthus amarus extract, the 5-fluorouracil or prodrug thereof or both of the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof, and • at least one pharmaceutically tolerable excipient such as one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a coloring agent, a surfactant and a preservative.
The skilled person is able to select suitable pharmaceutically tolerable excipients depending on the form of the pharmaceutical composition and is aware of methods for manufacturing pharmaceutical compositions as well as able to select a suitable method for preparing the pharmaceutical composition depending on the kind of pharmaceutically tolerable excipients and the form of the pharmaceutical composition.
The pharmaceutical composition(s) can be present in solid, semisolid or liquid form to be administered by an oral or parenteral, rectal, topical, or transdermal route to a subject, preferably a human.
The Phyllanthus amarus extract is obtained or obtainable, in particular obtained, by an extraction comprising contacting Phyllanthus amarus plant material with an extraction solvent which comprises water, an aliphatic alcohol or mixtures thereof. In particular, the extraction solvent is selected from water, an aliphatic alcohol or mixtures thereof.
Preferably, the alcohol is an aliphatic alcohol, which means herein an aliphatic hydrocarbon, preferably a branched or straight chain alkane, wherein at least one hydrogen atom of the aliphatic hydrocarbon is substituted with a hydroxyl group, preferably one hydrogen atom is substituted with a hydroxyl group referenced as monohydric aliphatic alcohol. More preferably, the aliphatic alcohol is a monohydric aliphatic alcohol, still more preferably a monohydric alcohol with 1 to 3 carbon atoms, further preferably with 1 to 2 carbon atoms. I.e. the aliphatic alcohol is more preferably selected from methanol, ethanol, propanol or /sopropanol or mixtures thereof and further preferably from methanol, ethanol or mixtures thereof. More preferably, the aliphatic alcohol is methanol or ethanol. The extraction solvent most preferably essentially consists of water, ethanol, methanol or mixtures thereof, preferably the extraction solvent comprises ethanol and most preferably essentially consists of ethanol such as 75% (v/v) ethanol.
The Phyllanthus amarus plant material can include the whole plant including underground parts and aerial parts or only the aerial parts of the plant. Preferably, the Phyllanthus amarus plant material comprises and further preferably essentially consists of the whole plant of Phyllanthus amarus.
The Phyllanthus amarus plant material is preferably at least one of: a) dried, and/or b) cut, shredded, milled and/or pulverized before the extraction.
In particular, the Phyllanthus amarus plant material is dried and cut, shredded, milled and/or pulverized before the extraction. Drying is usually carried out with air at room temperature, i.e. at 25+/-2°C, or alternatively at temperatures of 50°C to 100°C for 30 min to 1 h.
In particular, the amount of Phyllanthus amarus plant material in relation to the total amount of the extraction solvent used for the extraction is preferably between 5 mg/ml and 100 mg/ml, further preferred between 20 mg/ml and 100 mg/ml, in particular about 50 mg/ml Phyllanthus amarus plant material relative to the total amount of extraction solvent extracting the Phyllanthus amarus plant material.
In more preferred embodiments, the Phyllanthus amarus extract is obtained or obtainable, in particular obtained, by an extraction comprising: a) contacting the Phyllanthus amarus plant material with an extraction solvent which comprises ethanol for obtaining a crude extract; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step (ii) for obtaining a residue and optionally drying the residue.
The extraction solvent in step a) preferably essentially consists of about 75 Vol.-% ethanol. Step a) is preferably carried out by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 5 h and subjecting the mixture to sonication for at least 30 min.
More preferably, step a) is carried out by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 8 h at a temperature between 20°C and 30°C, more preferably at about 25±2°C and subjecting the mixture to ultrasonication for at least 45 min, more preferably for about 1 h at a temperature between 20°C and 30°C, more preferably at about 25±2°C.
Sonication means applying sound energy to agitate particles in the mixture. The application of ultrasonic frequencies (more than about 20 kHz) is herein referenced as ultrasonication.
Step c) is preferably carried out by evaporating the extraction solvent at least partially and in particular completely in a rotary evaporator and drying the residue by means of vacuum drying at at least 40°C. The temperature for vacuum drying is preferably at least 50°C, more preferably about 60°C. The expression “partially removed” as used herein means that at least 50 % by weight of the extraction solvent in the filtrate is removed, in particular at least 80 % by weight and further preferred at least 90% by weight of the extraction solvent is removed from the filtrate. Completely removing the extraction solvent means removing more than 95% by weight of the extraction solvent from the filtrate.
In most preferred embodiments, the extraction comprises and in particular consists of the steps: a) immersing the Phyllanthus amarus plant material which has been dried and powdered in the extraction solvent which essentially consists of 75 Vol.-% ethanol for at least 8 h at about 25±2°C and subjecting the mixture to ultrasonication for at least about 1 h at a temperature of about 25±2°C for obtaining a crude extract; the amount of Phyllanthus amarus plant material in relation to the total amount of the extraction solvent used for the extraction is about 50 mg/ml Phyllanthus amarus plant material relative to the total amount of extraction solvent; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step b) in a rotary evaporator and drying the residue by means of vacuum drying at about 60°C.
The cancer is preferably liver cancer. In particular, the growth and proliferation of the cancer cells is synergistically inhibited by administering the Phyllanthus amarus extract and 5-fluorouracil or a prodrug thereof to the subject.
In preferred embodiments, the method of the present invention comprises administering synergistic amounts of a Phyllanthus amarus extract and 5-fluorouracil to the subject.
The Phyllanthus amarus extract of the present invention is effective for potentiating the activity of 5-fluorouracil, i.e. for increasing the effectiveness of 5-fluorouracil in particular for inhibiting the proliferation and/or the growth of the cancer cells. “Potentiating the activity” as used herein means any measurable and in particular statistically significant increase in the activity of 5-fluorouracil, namely the effects after contacting cancer cells with 5-fluorouracil, in particular an increase of at least 5%, preferably of at least 10% and more preferably of at least 20% in the activity, i.e. in the effects of 5-fluorouracil on the cancer.
For example, potentiating the activity of 5-fluorouracil can be an increase with regard to the effects on cell viability and/or cell cycle arrest by 5-fluorouracil, in particular the cell viability after contacting cancer cells with Phyllanthus amarus extract and 5-fluorouracil preferably for at least about 24 h and most preferably for at least about 48 h is at least 10, more preferably at least 12 and in particular at least 15, and further preferably at least 18 percentage points decreased compared to the percentage of cell viability in cancer cells which have been contacted with the same amount of 5-fluorouracil, but not with the Phyllanthus amarus extract. The cell viability is preferably measured with a MTT assay.
Alternatively, potentiating the activity of 5-fluorouracil or its prodrug can be an increase in the dUMP levels after contacting cancer cells with 5-fluorouracil and the
Phyllanthus amarus extract compared to the same amount of 5-fluorouracil alone, such as a statistically significant increase of dUMP, in particular an increase of at least 20% and further preferred of at least 50% under the combination compared to 5-fluorouracil alone. It is assumed that the accumulation of dUMP and the depletion of dTTP is the key mechanism of action of 5-fluorouracil by the inhibition of thymidylate synthase (TS). Additionally or alternatively, potentiating the activity of 5-fluorouracil or its prodrug means a statistically significant decrease of the dTTP levels after contacting cancer cells with 5-fluorouracil and the Phyllanthus amarus extract compared to the same amount of 5-fluorouracil alone, preferably a decrease of at least 10%, more preferably of at least 15%. The levels of dUMP and dTTP can be determined as previously described (Guo, J.R. etal., Scientific Reports 2015,5).
Thus, the present invention refers in another aspect to a method of potentiating the activity of 5-fluorouracil in cancer cells comprising contacting the cancer cells with: (i) a Phyllanthus amarus extract; and (ii) 5-fluorouracil.
The 5-fluorouracil can be derived from a prodrug thereof.
The cancer cells can be from a human or an animal, in particular from a mammal, preferably from a human.
The cancer cells may be contacted with the Phyllanthus amarus extract and the 5-fluorouracil simultaneously or the cancer cells are contacted with one of them before or subsequent to the other. More preferably, the cancer cells are contacted with both simultaneously.
For example, the cancer cells are contacted with the Phyllanthus amarus extract and the 5-fluorouracil in amounts of at least about 50 pg/ml of the Phyllanthus amarus extract and at least about 50 μΜ of the 5-fluorouracil preferably for at least 24 h, more preferably at least 48 h and in particular at least 72 h.
The step of contacting the cancer cells with the Phyllanthus amarus extract and the 5-fluorouracil may be carried out by applying at least one incubation solution comprising the Phyllanthus amarus extract and/or the 5-fluorouracil to said cancer cells which incubation solution may further comprise suitable excipients such as solvents, buffers or a suitable growth medium.
Contacting the cancer cells can include adding an incubation solution to the cancer cells or administering the Phyllanthus amarus extract and 5-fluorouracil or a prodrug thereof to a subject. The prodrug will, thus, be metabolized to 5-fluorouracil. The administration can, in particular, be carried out by oral administration or parenteral administration such as intravenous or intra-arterial administration to the subject.
The cancer cells are preferably from a human liver cancer and contacted with the Phyllanthus amarus extract and 5-fluorouracil.
The Phyllanthus amarus extract for contacting the cancer cells is obtained or obtainable, in particular obtained, by an extraction as described above. More preferably, the Phyllanthus amarus extract for contacting the cancer cells is obtained or obtainable, in particular obtained, by an extraction comprising: a) contacting Phyllanthus amarus plant material with an extraction solvent which comprises ethanol for obtaining a crude extract; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step b) for obtaining a residue and optionally drying the residue.
In further preferred embodiments, the extraction solvent in step a) essentially consists of ethanol with about 75 Vol.-% and step a) is carried out by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 5 h, in particular for at least 8 h, and subjecting the mixture to sonication, in particular ultrasonication, for at least 30 min, in particular for about 1 h, and step c) is carried out by evaporating the extraction solvent in a rotary evaporator, wherein the residue is dried by means of vacuum drying at at least 40°C, in particular at about 60°C. The amount of Phyllanthus amarus plant material in relation to the total amount of the extraction solvent used for the extraction is about 50 mg/ml Phyllanthus amarus plant material relative to the total amount of extraction solvent.
The present invention in another aspect refers to a kit comprising synergistic amounts of: (i) a Phyllanthus amarus extract; and (ii) 5-fluorouracil or a prodrug thereof.
The kit may comprise excipients, in particular a pharmaceutically tolerable carrier, salt, buffer, water, or a combination thereof. The kit in particular comprises at least one pharmaceutically tolerable excipient selected from one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a coloring agent, a surfactant and a preservative.
The kit can further comprise an instruction leaflet. Still further, the kit may comprise at least one container.
The Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof may be contained in the kit in solid, semisolid or liquid form.
The Phyllanthus amarus extract contained in the kit is obtained or obtainable, in particular obtained, by an extraction as described above. More preferably, the Phyllanthus amarus extract contained in the kit is obtained or obtainable, in particular obtained, by an extraction comprising: a) contacting Phyllanthus amarus plant material with an extraction solvent which comprises ethanol for obtaining a crude extract; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step b) for obtaining a residue and optionally drying the residue.
In further preferred embodiments, the extraction solvent in step a) essentially consists of ethanol with about 75 Vol.-% and step a) is carried out by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 5 h, in particular for at least 8 h, and subjecting the mixture to sonication, in particular ultrasonication, for at least 30 min, in particular for about 1 h, and step c) is carried out by evaporating the extraction solvent in a rotary evaporator, wherein the residue is dried by means of vacuum drying at at least 40°C, in particular at about 60°C. The amount of Phyllanthus amarus plant material in relation to the total amount of the extraction solvent used for the extraction is about 50 mg/ml Phyllanthus amarus plant material relative to the total amount of extraction solvent.
Further in accordance with the present invention is a pharmaceutical composition comprising the Phyllanthus amarus extract and the 5-fluorouracil or a prodrug thereof and an excipient such as selected from a pharmaceutically tolerable carrier, salt, buffer, water, or a combination thereof, in particular essentially consisting of: (i) the Phyllanthus amarus extract and the 5-fluorouracil or prodrug thereof, and (ii) one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a coloring agent, a surfactant and a preservative.
The skilled person is able to select suitable pharmaceutically tolerable excipients depending on the form of the pharmaceutical composition and is aware of methods for manufacturing pharmaceutical compositions as well as able to select a suitable method for preparing the pharmaceutical composition depending on the kind of pharmaceutically tolerable excipients and the form of the pharmaceutical composition.
The pharmaceutical composition can be present in solid, semisolid or liquid form to be administered by an oral, parenteral, rectal, topical, or transdermal or inhalative route to a subject, preferably a human.
Further in accordance with the present invention is the use of the Phyllanthus amarus extract as described above for treatment of cancer in combination with 5-fluorouracil or a prodrug thereof as well as the use of the Phyllanthus amarus extract as described above for preparing a medicament for use in the treatment of cancer in combination with 5-fluorouracil or a prodrug thereof.
EXAMPLES
Chemicals and reagents LC-MS grade methanol, acetonitrile and acetic acid were purchased from Anaqua Chemical Supply Co., Houston, TX, USA. Hexylamine (HA), diethylamine (DEA), trioctylamine, 1,1,2-trichlorotrifluoroethane, stable isotope labeled adenosine-13C1015N5-triphosphate (ATP13C15N), dimethyl sulfoxide (DMSO), trypsin-EDTA solution and 3-[(4,5)-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich Chemical Co., St. Louis, MO, USA. Ultra-pure water was obtained from a Milli-Q Gradient Water System (Millipore Corp., Bedford, MA, USA). For culturing cells, phosphate buffered saline, pH=7.8 (PBS), Dulbecco's Modified Eagle Medium (DMEM), penicillin-streptomycin solution and fetal bovine serum (FBS) were obtained from Gibco Invitrogen Corp., Carlsbad, CA, USA. Human hepatocellular cancer HepG2 cell line was supplied by American Type Culture Collection (ATCC), Rockville, MD, USA. LC/MS/MS Assay LC/MS/MS Assay was performed on a Thermo Fisher TSQ LC-MS/MS system consisting of an Accela Autosampler, an Accela pump and a Quantum Access triple quadrupole mass spectrometer (Thermo Fisher Scientific Co., San Jose, CA, USA). Data acquisition was performed with the Xcalibur software version 2.0.7, and data processing using the Thermo LCquan 2.5.6 data analysis program (Thermo Fischer). The chromatographic separation was achieved using an XTerra-MS C18 column (150 mm X2.1 mm i.d., 3.5 pm, Waters Corp., Milford, MA, USA). The two eluents were: (A) 5 mM HA-0.5% DEA in water, pH adjusted to 10 with acetic acid; and (B) 50% acetonitrile in water. The mobile phase consisted of a linear gradient of A and B: 0-15 min, 100-80% A (v/v); 15-35 min, 80-70% A; 35-45 min, 70-45% A; 45-46 min, 45-0% A; 46-50 min, 0-0% A; 51-70 min, 100-100% A. The liquid flow-rate was set at 0.3 mL/min, and the column temperature was maintained at 35°C. For all dRNs, the following optimized parameters were obtained. The sheath gas pressure reached 40 psi. The ion spray voltage was set at 3000 V for negative mode and 4000 V for positive mode at a temperature of 350°C and auxiliary gas pressure of 15 psi. Quantification was performed using multiple reactions monitoring (MRM) as previously published (Guo, J.R. et al., Scientific Reports 2015, 5).
Cell Culture
Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 pg/mL streptomycin in a 37°C humidified incubator with a 5 % CO2 atmosphere. HepG2 cells were seeded in 100 mm by 20 mm dishes (Corning Inc, Corning, NY, USA). After overnight culture, cells were divided into different groups as follows: Control group (N), cells incubated with medium alone; 5-FU group (F), cells exposed to 50.0 pM of 5-fluorouracil (5-FU) for 24 h; Phyllanthus amarus (PHA) group (P), cells exposed to 50.0 pg/mL of PHA extract for 24 h; combination group (P-F), cells exposed to 50.0 pM of 5-FU combined with 50.0 pg/mL of PHA extract for 24 h; An extra dish of a cell line was incubated for cell counting on the day of cell harvest for normalization of nucleotide pools, and the viability assessed by trypan blue exclusion assay.
Preparation of cell pellets
Monolayer HepG2 cells were washed with ice-cold PBS once and were trypsinized with 0.25% trypsin-EDTA. Cells from two or three dishes were then re-suspended with 12 ml_ ice-cold PBS. After centrifugation at 1,000 rpm for 5 min, the cell pellet was washed with 1 ml_ ice-cold PBS again and spun down at 1,000 rpm for 5 min. The cell pellet was incubated with 150 μΙ_ of 15% trichloroacetic acid (TCA) containing 7.5 pl_ of 20.0 μΜ ATP13C15N as internal standard and placed on ice for 10 min. After centrifugation at 13,500 rpm for 15 min in the cold room, the acidic supernatant was separated and neutralized twice with 80 μΙ_ mixture of trioctylamine and 1,1,2-trichlorotrifluoroethane (45:55 v/v). Samples were stored at -80°C until analysis within two days. EXAMPLE 1
Preparation of a Phyllanthus amarus (PHA) extract of the present invention
Phyllanthus amarus was collected from Guangxi province and identified by Professor Burning Liu at Guangxi Key Laboratory of Traditional Chinese Medicine Quality standards, China. The dried powder of Phyllanthus amarus (about 5 g) was accurately weighed into a 250 mL flask and immersed in 100 mL of 75% ethanol (v/v) overnight. The mixture was ultrasonically extracted at room temperature for 1 h before the extract was filtered. The filtrate was condensed by a rotary evaporator followed with vacuum drying at 60°C. The fingerprint was also recorded by a previously published method (data not shown) (Guo, J. et al., Anal Bioanal Chem 2015,407:1389-1401). EXAMPLE 2 MTT assay
The inhibitory effect of different groups of HepG2 cells was determined by the cytotoxic MTT assay. HepG2 cells were seeded in 96 well plates (LabServ, Thermo Fisher Scientific Co., Beijing, China) at 1*104 cells/well. After incubation, they were treated with indicated concentrations of 5-FU, PHA or a combination for 24, 48 and 72 h. MTT solution (final concentration of 0.5 mg/ml in medium) was added to each well and incubated further for 4 h. The medium was removed, and 100 pLof DMSO was added to each well to dissolve the purple crystals of formazan. Absorbance was measured at 570 nm with a microplate UV/VIS spectrophotometer (Infinite M200 PRO, Tecan Austria GmbH 5082, Grodig, Austria); reference wavelength was 650 nm. The cell number was determined using a hemocytometer. IC50 (half maximal (50%) inhibitory concentration) values of 5-FU were calculated by GraphPad Prism software (GraphPad Software, Inc. CA, USA). Cell viability (%) = OD treated / OD control (untreated) * 100. Drug interaction was evaluated by the combination index (Cl) methods, whereas Cl<1, Cl=1, and Cl>1 indicates synergism, additive effect and antagonism, respectively (Chou, T.C. et al., J Natl Cancer Inst 1994,86:1517-1524, Chou, T.C. et al., Adv Enzyme Regul 1984,22:27-55).
Cells were exposed to PHA at various concentrations over different incubation periods (24, 48 and 72 h). High amounts and longtime treatment with PHA was able to inhibit proliferation of HepG2 cells (Fig. 1A). The IC50 value of PHA on HepG2 cells after 72 h was 114.0±24.6 pg/mL. Furthermore, lower amounts of PHA (50.0 pg/mL) proved to enhance antitumor activity of 5-FU (Fig. 1B) although PHA alone had no strong inhibitory effect on HepG2 cells in said amount (more than 88% of control values). 5-FU combined with 50.0 pg/mL PHA for 48 h decreased cell viability of HepG2 cells from 71.26±3.93 % to 54.66±2.56 % (P<0.01). At the same time, the value of Cl was 0.66, indicating a synergistic action between 5-FU and PHA. These observation and comparison illustrates the promising and high potential of PHA to potentiate the chemotherapy with 5-FU in liver cancer. EXAMPLE 3
Cell cycle analysis and Western Blot and multivariate statistical analysis
The cell cycle analysis was carried out as previously described (Guo, J.R. et al., Biol Res 2015,48:40). In brief, cells were seeded at 4.5x105 cells/well in 6-well culture plates in duplicate, and incubated with indicated concentrations of 5-FU, PHA or a combination thereof for 24 h. They were then harvested and fixed in 70% (v/v) cold ethanol overnight at 4°C. The fixed cells were collected by centrifugation and re-suspended in PBS and incubated with 5 mg/mL propidium iodide (Sigma-Aldrich) and 10 mg/mL RNase A (Sigma-Aldrich) at room temperature for 30 min in the dark. The cells were then analyzed on a flow cytometer (Muse™ cell analyzer, Merck Millipore, Darmstadt, Germany). Finally, the percentages of cells in different phases (G0/G1, S and G2/M) were calculated using Modfit software (Verity Software House, USA).
The cells were treated with 5-FU, PHA and a combination of them for 24 h, respectively. At the end of the incubation time, the cells of every group were harvested and lysed in RIPA buffer (Cell Signaling Technologies Inc. Beverly, MA, USA). Bradford reagent (Bio-Rad, Hercules, CA, USA) was used to determine the protein concentration. Then, the cell lysates were mixed with 5xSDS-loading buffer (4:1, v/v) and heated at 100°C with locked cap for 5 min. The cell lysates (40 pg) were subjected to 10% SDS-PAGE. After electrophoresis, the cell extracts from SDS-PAGE were transferred to nitrocellulose membrane. Then, the membranes were incubated with rabbit RRM1 (D12F12) antibody (Cell Signaling Technology, Inc. USA), RRM2 [EPR11820] antibody (abeam®, Cambridge, UK), p53R2 [EPR8816] antibody (abeam®, Cambridge, UK), thymidylate synthase (D5B3) antibody (Cell Signaling Technology, Inc. USA) and β-tubulin antibody (Santa CruzBiotechnology, CA, USA) overnight at 4°C. Furthermore, the membranes were incubated with HRP-conjugated antibodies for one hour. Visualization of the protein bands was carried out by using the enhanced chemiluminescence reagents (Invitrogen, Paisley, Scotland, UK). The bands were analyzed by using the Image J 1.46r software (National Institutes of
Health, Bethesda, MD, USA).
After exposure to 5-FU, PHA or a combination thereof for 24 h, cell cycle arrest analysis in HepG2 cells showed that the PHA extract alone for 24 h just induced the cells arrest in G2/M phase. However, there were no significant differences in other phases (Fig. 6). At the same time, the 5-FU group showed a remarkable accumulation in the G0/G1 and S phases by a decreased distribution in the G2/M phase. Compared with the 5-FU group, a combined treatment with 5-FU and PHA extract induced a higher proportion of arrest in the S phase and decreased distribution in the G0/G1 phase in HepG2 cells. This proved that PHA could increase the role of cell cycle arrest at S phase induced by 5-FU in HepG2 cells.
Human ribonucleotide reductase (RR) is composed of three known subunits, RRM1 (large subunit), RRM2 (small subunit) and encoded P53-controlled ribonucleotide reductase (P53R2) that are differentially regulated during the cell cycle. R1 protein concentrations are relatively constant throughout the cell cycle. M2 protein is low outside S phase (Mah, V. et al., PLoS One 2015,10:e0127600, Zhou, B. et al., Cytogenet Cell Genet 2001,95:52-59). Compared with the control group, the expression of RRM2 was increased after 5-FU treatment with or without PHA, which was caused by cell cycle arrest at S phase (Fig. 5B). Thus high levels of deoxyadenosine diphosphate (dADP), deoxycytidine diphosphate (dCDP), deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) were associated with high level of RRM2. As already noted, dTTP stimulates the formation of deoxyguanosine diphosphate (dGDP) and hence of deoxyguanosine triphosphate (dGTP). The subsequent decrease in the levels of dGDP and dGTP in 5-FU and the combination groups may be, at least in part, caused by a decrease of dTTP due to the inhibition of thymidylate synthase (TS) by 5-FU. Because the appropriate balance of the four deoxyribonucleotides is required for the synthesis of DNA, PHA enhances antitumor activity of 5-FU via affecting the RN and dRN levels disturbed by 5-FU through effects on key enzymes and/or cell cycle arrest.
In order to understand and visualize the grouping trends, unsupervised principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using SIMCA-P version 14.0 software (Umetrics, Sweden). As can be seen from Fig. 2A an 2B, a clear group separation of sample points suggested that the exposure to 5-FU with or without PHA made a significant difference in the profiles of RN and dRN pools although PHA alone had no impact on intracellular RN and dRN pool sizes. Further, it can be seen from the OPLS-DA model in Fig. 2C and 2D that the 5-FU group and the combination group seem easily classified into two groups with satisfactory discriminating ability, which suggested PHA was able to stimulate the disturbance of RN and dRN levels induced by 5-FU. Finally, six RNs and dRNs including adenosine-triphosphate (ATP) , cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), dUMP and dTTP were screened out using VIP>1.0 and P<0.05 (Fig. 2E and 2F).
When the cells were exposed to the extract of PHA alone, there were no remarkably changes in the levels of the RNs, except for the slight decrease of ATP, GTP, guanosine monophosphate (GMP) and the small increase of cytidine diphosphate (CDP). The effects of 5-FU on RN and dRN pool sizes in cells upon exposure to 5-FU for different durations have already been reported (Guo, J.R. et al., Scientific Reports 2015,5). The levels of four nucleoside triphosphates exhibited similar increased after treatment of 5-FU with or without PHA (Fig. 3Ato 3C and table 1). The effects of 5-FU on ribonucleotides were possibly related with its cytostatic effect. However, it was interesting that ATP, CTP, GTP and UTP levels in combination group significantly decreased compared with 5-FU alone group. At the meantime, there was a significant increase in adenosine monophosphate (AMP), cytidine monophosphate (CMP) and GMP levels after combination with PHA. Nucleoside monophosphate kinase (NMPK) can phosphorylate monophosphate metabolites into diphosphate metabolites, which can then be further transferred to triphosphate metabolites by nucleoside diphosphate kinase (NDPK) (Van Rompay A.R., et al., Pharmacol Ther 2000,87:189-198). Given that there was no effect of PHA on the level of nucleoside diphosphates, NMPK and NDPK are assumed to be a potential target for PHA.
Table 1: Levels of RNs in HepG2 cells in each group (pmol/106cell)
All data were expressed as mean ± SD values by three independent experiments. P value of less than 0.05 (*P< 0.05, **P< 0.01, versus the control group; #P<0.05, ##P<0.01, compared with the 5-FU group) are considered significant.
Table 2 summarizes the levels of dRNs in each group. It is assumed that the accumulation of dUMP pools and the depletion of dTTP pools is the key mechanism of action of 5-FU by the inhibition of the enzyme thymidylate synthase (TS) (Kakito, H. et al., Cancer Invest 1993,11:530-533).
Furthermore, the level of dUMP in the 5-FU and PHA combined treatment group was significantly higher than that in the 5-FU group, while the dTTP pools were reduced significantly (Fig. 4). This suggests that PHA can enhance the effect of 5-FU through regulation of RN and dRN pool sizes.
The TS protein level was evaluated by Western Blot in a HepG2 cell line. HepG2 cells were incubated for 24 h with 50.0 μΜ 5-FU, 50.0 pg/mL PHA and the combination of these two, respectively. As seen in Fig. 5A, the level of TS protein remains unchanged after exposure to the PHA extract. The treatment of 5-FU induced the formation of two distinct bands of TS, one at 35 kDa and the other at approximately 38 kDa. The band at 35 kDa represents the normal TS. The second band of TS with higher molecular mass represents the conjugate formed by FdUMP, TS and CH2-THF (5, 10-methylene-tetrahydrofolate) (Chu, E. et al., Mol Pharmacol 1993,43:527-533). However, there was no inhibitory effect on this enzyme after combination with the PHA extract. Since deoxycytidine triphosphate (dCMP) is a precursor of dUMP (Maley, G.F., Maley, F., J Biol Chem 1964,239:168-176), the increase of dUMP in the combination group might be caused by an increase of dCMP.
Table 2: Levels of dRNs in HepG2 cells in each group (pmol/106cell)
All data were expressed as mean ± SD values by three independent experiments. P value of less than 0.05 (*P< 0.05, **P< 0.01, versus the control group; #P<0.05, ##P<0.01, compared with the 5-FU group) are considered significant; *UDL, under detected limit of assay.
Claims (20)
1. A method for treating a subject suffering from cancer comprising the step of administering synergistic amounts of (i) a Phyllanthus amarus extract, and (ii) 5-fluorouracil or a prodrug thereof to a subject.
2. The method of claim 1, wherein the Phyllanthus amarus extract is obtained by an extraction comprising contacting Phyllanthus amarus plant material with an extraction solvent which comprises water, an aliphatic alcohol or mixtures thereof.
3. The method of claim 2, wherein the extraction solvent comprises ethanol.
4. The method of claim 2, wherein the Phyllanthus amarus extract is obtained by an extraction comprising a) contacting Phyllanthus amarus plant material with an extraction solvent which comprises ethanol for obtaining a crude extract; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step b) for obtaining a residue and optionally drying the residue.
5. The method of claim 4, wherein the extraction solvent in step a) essentially consists of about 75 Vol.-% ethanol and wherein step a) is carried out by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 5 h and subjecting the mixture to sonication for at least 30 min.
6. The method of claim 4, wherein step c) is carried out by evaporating the extraction solvent in a rotary evaporator and wherein the residue is dried by means of vacuum drying at at least 40°C.
7. The method of claim 1, wherein the cancer is liver cancer.
8. The method of claim 1, wherein the subject is a human.
9. The method of claim 1, wherein the growth and proliferation of the cancer cells is synergistically inhibited by administering the Phyllanthus amarus extract and 5-fluorouracil or a prodrug thereof to the subject.
10. The method of claim 1 comprising administering synergistic amounts of a Phyllanthus amarus extract and 5-fluorouracil to the subject.
11. A method of potentiating the activity of 5-fluorouracil in cancer cells comprising contacting the cancer cells with (i) a Phyllanthus amarus extract; and (ii) 5-fluorouracil.
12. The method of claim 11, wherein the cancer cells are simultaneously contacted with the Phyllanthus amarus extract and with the 5-fluorouracil.
13. The method of claim 11, wherein the Phyllanthus amarus extract is obtained by an extraction comprising a) contacting Phyllanthus amarus plant material with an extraction solvent which comprises ethanol for obtaining a crude extract; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step b) for obtaining a residue and optionally drying the residue.
14. The method of claim 13, wherein the extraction solvent in step a) essentially consists of about 75 Vol.-% ethanol and wherein step a) is carried out by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 5 h and subjecting the mixture to sonication for at least 30 min and wherein step c) is carried out by evaporating the extraction solvent in a rotary evaporator and wherein the residue is dried by means of vacuum drying at at least 40°C.
15. The method of claim 11, wherein the cancer cells are from a human liver cancer and the cancer cells are contacted with the Phyllanthus amarus extract and 5-fluorouracil.
16. The method of claim 11, wherein contacting the cancer cells means administering the Phyllanthus amarus extract and 5-fluorouracil or a prodrug thereof to a subject.
17. A kit comprising synergistic amounts of (i) a Phyllanthus amarus extract; and (ii) 5-fluorouracil or a prodrug thereof.
18. The kit of claim 17, wherein the Phyllanthus amarus extract is obtained by an extraction comprising a) contacting Phyllanthus amarus plant material with an extraction solvent which comprises ethanol for obtaining a crude extract; b) filtering the crude extract for obtaining a filtrate; c) at least partially removing the extraction solvent from the filtrate of step b) for obtaining a residue and optionally drying the residue.
19. The kit of claim 17, wherein the extraction solvent in step a) essentially consists of about 75 Vol.-% ethanol and wherein step a) is carried out by immersing the Phyllanthus amarus plant material in the extraction solvent for at least 5 h and subjecting the mixture to sonication for at least 30 min and wherein step c) is carried out by evaporating the extraction solvent in a rotary evaporator and wherein the residue is dried by means of vacuum drying at at least 40°C.
20. The kit of claim 17 further comprising at least one pharmaceutically tolerable excipient such as one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a coloring agent, a surfactant and a preservative.
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