Patent Application
20060110467
Cancer, a leading fatal disease, features an abnormal mass of malignant tissue resulting from excessive cell division. Cancer cells proliferate in defiance of normal restraints on cell growth, and invade and colonize territories normally reserved for other cells.
Modes of cancer therapy include chemotherapy, surgery, radiation, and combinations of these treatments. Chemotherapy typically involves use of one or more compounds that inhibit cancer cell growth. While many chemotherapeutic agents have been developed, there remains a need for more effective chemotherapy.
This invention is based on a surprising discovery that Lushanrubescensin A (LuA), Lushanrubescensin B (LuB), and Lushanrubescensin E (LuE) enhance efficacy of a chemotherapeutic agent (i.e., a medication used to treat various forms of cancer) in inhibiting the growth of cancer cells.
Thus, this invention relates to a method of treating cancer, e.g., esophagus carcinoma, lung carcinoma, gastric adenocarcinoma, breast carcinoma, lung carcinoma, liver carcinoma, prostate carcinoma, colon carcinoma, pancreatic carcinoma, leukemia, or melanoma. The method includes administering to a subject an effective amount of a chemotherapeutic agent and an effective amount of a diterpene compound of Formula I:
in which each of R1, R2, R3, R4 and R5 is, independently, H, OC(O)CH3, or OH.
Referring to Formula I, one subset of the diterpene compounds feature that each of R1, R2, R3, and R5 is OC(O)CH3; and R4 is OH. Another subset of the diterpene compounds feature each of R1, R2, and R3 is OC(O)CH3; and each of R4 and R5 is OH. Still another subset of the diterpene compounds feature that each of R1, R2 and R3, independently, is OC(O)CH3; R4 is H; and R5 is OH. Still another subset of the diterpene compounds feature a stereospecificity shown in Formula II below:
Set forth below are three exemplary diterpene compounds that can be used to practice this method:
Examples of chemotherapeutic agents used in this method include, but are not limited to, cisplatin, mitomycin C, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophyllotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, and cytokines (e.g., interleukin-2 and transforming growth factor β).
Also within the scope of this invention is a composition containing a diterpene compound of Formula I, a chemotherapeutic agent, and a pharmaceutically acceptable carrier for treating cancer, as well as use of a diterpene compound of Formula I for the manufacture of a medicament for treating cancer.
Details of several embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description, and also from the claims.
A diterpene compound of Formula I enhances the efficacy of a chemotherapeutic agent in treating cancer, when they are both administered to a subject in need thereof. As such, a lower dose of the chemotherapeutic agent is needed to achieve a desired therapeutic goal, thereby lessening side effects. Thus, an aspect of this invention relates to a method of treating cancer by administering to a subject in need thereof an effective amount of one or more of the above-described diterpene compounds and an effective amount of a chemotherapeutic agent. Examples of cancer include, but are not limited to, esophagus carcinoma, lung carcinoma, gastric adenocarcinoma, breast carcinoma, lung carcinoma, liver carcinoma, prostate carcinoma, colon carcinoma, pancreatic carcinoma, leukemia, or melanoma. The term “an effective amount” refers to the amount of the active agent that is required to confer the intended therapeutic effect in a subject. Effective amounts may vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other agents. The term “treating” refers to administering a diterpene compound and a chemotherapeutic agent to a subject that has cancer, or has a symptom of cancer, or has a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the cancer, the symptoms of the cancer, or the predisposition toward the cancer.
Some of the diterpene compounds used to practice this method are naturally occurring and can be isolated from plants. For example, LuA, LuB and LuE can be isolated from the leaves of Rabdosia rubescens Hara f. lushanensis Gao et Li. Others can be synthesized by methods well known in the art or prepared from the naturally-occurring compounds via simple transformations. The chemicals used in the isolation and synthesis of the diterpene compounds may include, for example, solvents, reagents, catalysts, and protecting group and deprotecting group reagents. The isolation and synthesis may also include steps to add or remove suitable protecting groups in order to ultimately obtain desired diterpene compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing applicable diterpene compounds are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.
Diterpene compounds used to practice the method of this invention may contain one or more asymmetric centers. Thus, they can occur as racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures. All such isomeric forms are contemplated.
Examples of chemotherapeutic agents that can be used in this method are mentioned in the Summary section. Also see, e.g., Isselbacher et al., Harrison's Principles of Internal Medicine 13th, McGraw-Hill, 1994. All of them are commercially available or can be prepared by methods well-known in the art. A chemotherapeutic agent can be selected based on, for example, the type of neoplasm being treated, the expression of one or more markers by cancer, and the age and general health of the subject to be treated.
To practice this method, a diterpene compound and a chemotherapeutic agent can be applied at the same time or at different times. They can be administered orally, parenterally, by inhalation spray, or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
An oral composition can be any orally acceptable dosage form including, but not limited to, tablets, capsules, emulsions and aqueous suspensions, dispersions and solutions. Commonly used carriers for tablets include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added to tablets. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
A sterile injectable composition (e.g., aqueous or oleaginous suspension) can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or di-glycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.
An inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
A topical composition can be formulated in form of oil, cream, lotion, ointment and the like. Suitable carriers for the composition include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohols (greater than C12). The preferred carriers are those in which the active ingredient is soluble. Emulsifiers, stabilizers, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired. Additionally, transdermal penetration enhancers may be employed in these topical formulations. Examples of such enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762. Creams are preferably formulated from a mixture of mineral oil, self-emulsifying beeswax and water in which mixture the active ingredient, dissolved in a small amount of an oil, such as almond oil, is admixed. An example of such a cream is one which includes about 40 parts water, about 20 parts beeswax, about 40 parts mineral oil and about 1 part almond oil. Ointments may be formulated by mixing a solution of the active ingredient in a vegetable oil, such as almond oil, with warm soft paraffin and allowing the mixture to cool. An example of such an ointment is one which includes about 30% almond and about 70% white soft paraffin by weight.
A carrier in a pharmaceutical composition must be “acceptable” in the sense that it is compatible with active ingredients of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents, such as cyclodextrins (which form specific, more soluble complexes with one or more of active compounds of the extract), can be utilized as pharmaceutical excipients for delivery of the active ingredients. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.
Suitable in vitro assays can be used to preliminarily evaluate the efficacy of the combination of one or more of the above-described diterpene compounds and a chemotherapeutic agent in inhibiting growth of cancer cells. The combination can further be examined for its efficacy in treating cancer by in vivo assays. For example, the combination can be administered to an animal (e.g., a mouse model) having cancer and its therapeutic effects are then assessed. Based on the results, an appropriate dosage range and administration route can also be determined.
Without further elaboration, it is believed that the above description has adequately enabled the present invention. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All of the publications, including patents, cited herein are hereby incorporated by reference in their entirety.
Isolation and Identification of LuA, LuB, and LuE
Aerial herb of Rabdosia rubescens Hara f. lushanensis Gao et Li (200 g, debris) was immersed in 2000 ml of 95% ethanol at room temperature for three days and then filtered out. The herb debris was treated in the same manner twice. The ethanol in the combined filtrates was removed under vacuum, and the residue thus obtained was diluted in 200 ml of water and washed with 200 ml of petroleum ether. The petroleum ether layer was then removed and the aqueous layer was washed with 200 ml of ethyl acetate. The ethyl acetate layer was separated and concentrated to dryness. The crude extract was loaded onto a silica gel column (8 cm×3 cm) and eluted with a gradient solution system of petroleum ether/acetone (9:1, 500 ml→8:2, 500 ml→7:3, 500 ml). Three fractions of eluate (500 ml each) were collected. The three fractions contain 9:1, 8:2, and 7:3 petreolum ether/acetone, respectively. Strong UV absorbance at 254 nm was observed in each fraction. The first fraction, i.e., an eluate containing 9:1 petroleum ether/acetone, was concentrated and further purified using RP-18 chromatography with 50:50 methanol/water to provide pure LuA (37 mg, 0.02%). Similarly, the second fraction, i.e., an eluate containing 8:2 petreolum ether/acetone, and the third fraction, i.e., an eluate containing 7:3 petroleum ether/acetone, were concentrated and purified using RP-18 chromatography with 45:55 methanol/water and 40:60 methanol/water to provide pure LuB (59 mg, 0.03%) and LuE (81 mg, 0.04%), respectively.
Analytical data for LuA:
m.p.=188˜190° C.; [α]D13 =−62.1° (c=0.99, pyridine);
U (EtOH) λmax: 239 nm;
IR (KBr): 3440, 1730, 1703, 1645, 1248, 1225 cm−1;
1H-NMR (C5D5N, 400 MHz) δ: 5.56 (1H, ddd, J=12.5, 4.0, 2.6 Hz), 5.30 (1H, d, J=2.6 Hz), 2.64 (1H, br), 5.54 (1H, dd, J=3.4, 1.7 Hz), 4.02 (1H, d, J=3.4 Hz), 2.40 (1H, br), 5.36 (1H, d, J=4.3 Hz), 3.00 (1H, br), 2.55 (1H, d, J=12.5 Hz), 1.48 (1H, dd, J=12.5, 3.6 Hz), 6.05 (1H, br), 5.35 (1H, br), 1.04 (3H, s), 1.12 (3H, s), 1.49 (3H, s);
13C-NMR (C5D5N, 100 MHz) δ: 212.7 (s), 170.6 (s), 170.4 (s), 169.8 (s), 169.0 (s), 150.2 (s), 114.6 (t), 77.6 (d), 73.1 (d), 71.0 (d), 68.3 (d), 67.6 (d), 55.0 (d), 50.0 (s), 42.0 (d), 40.8 (t), 39.8 (s), 38.3 (s), 38.3 (t), 37.3 (d) 34.5 (t), 28.0 (q), 23.2 (q), 21.2 (q), 20.9 (q), 20.6 (q), 20.6 (q), 20.6 (q);
EIMS m/z: 535[M+1]+, 474, 414, 354, 312, 294, 279.
Analytical data for LuB:
m.p.=219˜221° C.; [α]D19=−90° (c=0.1, methanol);
IR (KBr): 3440, 1740, 1710, 1646, 1265˜1220 cm−1;
1H-NMR (C5D5N, 400 MHz) δ: 5.54 (1H, ddd, J=12.0, 4.2, 2.8 Hz), 5.20 (1H, d, J=2.8 Hz), 2.58 (1H, br), 5.45 (1H, dd, J=3.2, 1.4 Hz), 4.00 (1H, d, J=3.2 Hz), 2.55 (1H, br), 4.34 (1H, d, J=4.3 Hz), 3.03 (1H, br), 2.58 (1H, d, J=12.6 Hz), 1.46 (1H, dd, J=12.6, 3.4 Hz), 6.02 (1H, br), 5.36 (1H, br), 1.00 (3H, s), 1.13 (3H, s), 1.51 (3H, s);
13C-NMR (C5D5N, 100 MHz) δ: 213.6 (s), 170.6 (s), 170.6 (s), 169.9 (s), 151.0 (s), 112.9 (t), 77.8 (d), 73.7 (d), 71.5 (d), 67.9 (d), 65.0 (d), 59.1 (d), 49.9 (s), 42.1 (d), 40.8 (t), 40.8 (t), 39.4 (s), 38.3 (s), 38.1 (d), 35.2 (t), 28.0 (q), 23.3 (q), 21.3 (q), 21.3 (q), 21.0 (q), 20.7 (q);
EIMS m/z: 492[M]+, 432, 372, 312, 297, 279, 252, 240.
Analytical data for LuE:
m.p.=215˜217° C.; [α]D19=−77.5° (c=0.1, methanol);
UV (EtOH) λmax: 238 nm;
IR (KBr): 3550˜3370, 1740˜1720, 1710, 1650, 1265˜1225 cm−1;
1H-NMR (C5D5N, 400 MHz) δ: 5.74 (1H, d, J=3.0 Hz), 5.35 (1H, m), 4.68˜4.46 (1H, m), 3.08 (1H, br), 6.03 (1H, br), 5.29 (1H, br), 1.13 (3H, s), 1.10 (3H, s), 1.55 (3H, s);
13C-NMR (C5D5N, 100 MHz) δ: 208.0 (s), 170.8 (s), 169.9 (s), 150.8 (s), 111.4 (t), 81.4 (d), 69.0 (d), 65.0 (d), 63.8 (d), 63.6 (d), 49.2 (d), 48.8 (s), 44.5 (t), 41.3 (t), 39.7 (s), 38.5 (s), 38.5 (d), 37.9 (d) 37.9 (t), 28.2 (q), 22.9 (q), 21.5 (q), 20.9 (q), 20.2 (q);
EIMS m/z: 434[M]+, 416, 392, 374, 356, 314, 299, 296, 281, 253, 235.
Biological Assay
An in vitro assay was conducted to evaluate the efficacy of combinations of cisplatin and LuA, cisplatin and LuB, and cisplatin and LuE in inhibiting proliferation of cancer cells.
Human tumor cell lines, esophagus carcinoma (Eca-109, TE-1), gastric adenocarcinoma (AGS, BGC-823), breast carcinoma (MCF-7), lung carcinoma (A549), and lung adenocarcinoma (SPC-A-1), all purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences, were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum (FBS) in an incubator at 37° C. under 5% CO2. Cells of 70˜80% confluence were trypsinized, resuspended in IMDM medium containing 10% FBS at 1×105 cells/ml, and seeded in 96-well plates (100 μl in each well). The plates were incubated at 37° C. under 5% CO2 overnight.
To wells was added a DMSO solution containing LuA, LuB, or LuE, or a PBS solution containing cisplatin (Qilu Pharmaceutical Ltd.), or a combination of the just-mentioned DMSO solution and PBS solution. For wells which contained only LuA, LuB, LuE, or cisplatin, efficacy at six final concentrations, 100, 30, 10, 3, 1, and 0.3 μg/ml, was studied. For wells which contained both LuA and cisplatin, both LuB and cisplatin, or both LuE and cisplatin, efficacy at as many as eighteen final concentrations (LuA/LuB/LuE; cisplatin), 50:50, 15:15, 5:5, 1.5:1.5, 0.5:0.5, 0.15:0.15, 75:25, 22.5:7.5, 7.5:2.5, 2.25:0.75, 0.75:0.25, 0.225:0.075, 25:75, 7.5:22.5, 2.5:7.5, 0.75:2.25, 0.25:0.75, and 0.075:0.225 μg/ml, was studied. Wells, to which only DMSO was added, were used as the control; and wells, to which no LuA, LuB, LuE, cisplatin, and DMSO were added, were used as the background. The plates were then incubated at 37° C. under 5% CO2 for 48 hrs.
The plates were spun at 1000 rpm for 15 minutes, and then the supernatant was removed by vacuum carefully. After washed with 150 μl of PBS, the cells were placed in 100 μl of fresh growth medium.
3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to determine the concentration of living cells. 10 μl of 5 mg/ml MTT was added to all wells except for the background wells. After being incubated for additional 3-4 hrs, the plates were spun at 1000 rpm for 15 minutes and the supernatants were carefully removed by vacuum.
150 μl of DMSO was added to each well. The plates were placed on a shaker at 150 rpm for 15 minutes to dissolve the precipitate in the wells. Absorbance was measured at 492 nm using a microplate reader.
Inhibition ratios for cells treated with LuA, LuB, LuE, cisplatin, or various combinations described above were calculated according to the following equation:
A software program, XLfit (ID Business Solutions), was used to calculate the concentrations required to reach 10, 20, . . . 90% inhibition (i.e., IC10, IC20, . . . IC90) on each tumor cell line. Compared with LuA, LuB, LuE, or cisplatin alone, each combination had unexpectedly lower IC10, IC20, . . . IC90 values for cell lines, Eca-109, TE-1, AGS, BGC-823, MCF-7, A549, and SPC-A-1. The results show that the combination was more effective in inhibiting these cancer cells than LuA, LuB, LuE, or cisplatin alone.
Combination Indexes (CIs) were calculated according to the method described in Bertino J. et al. Chemotherapy: Synergism and Antagonism, Encyclopedia of Cancer, 1996, Academic Press, Inc. A CI represents the combination effect, such as synergism, antagonism, or addition of two or more drugs. When it is lower than 1, the combination effect is synergistic; when it is equal to 1, the combination effect is additional; and when it is higher than 1, the combination effect is antagonistic. The calculated CI values indicated that most of the above-mentioned combinations unexpectedly exhibited synergetic effect in inhibiting growth of cancer cells. More specifically, for cell lines BGC-823, SPC-A-1, AGS, and TE-1, the combination of LuA and cisplatin showed synergetic effect. For cell lines BGC-823, AGS, and TE-1, the combination of LuB and cisplatin showed synergetic effect. For cell lines BGC-823, Eca-109, and AGS, the combination of LuE and cisplatin showed synergetic effect.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
Pursuant to 35 USC § 119(e), this application claims priority to U.S. Provisional Application Ser. No. 60/626,171, filed Nov. 9, 2004, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60626171 | Nov 2004 | US |
