Use of compound in treatment of tumors

Abstract

Provided are a use of (1R,2R,4S)-3-elemene in treatment of tumors and a method for preparing (1R,2R,4S)-3-elemene. The (1R,2R,4S)-3-elemene is obtained by heating and converting H-germacrene A, and the H-germacrene A is obtained by means of fermentation of an engineered bacterium containing a nucleotide encoding an enzyme capable of synthesizing (-)-germacrene A.

Description

Use of a compound in treating tumors

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to and the benefit of Chinese Patent Application No. 202310770007.0, filed on June 27, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of medicine, and in particular to the use of the compound (+)-β-elemene in treating tumors and a preparation method thereof. Background Art

[0004] Cancer is currently a major threat to human health, with approximately 1.8 million patients diagnosed annually in my country, and the incidence rate is increasing year by year. Currently, cancer treatments primarily rely on surgery, chemotherapy, and radiotherapy. However, many chemotherapy drugs currently in clinical use still suffer from significant systemic side effects, drug resistance, and limited selectivity.

[0005] In recent years, researchers have tried to find natural products from natural plants, such as camptothecin, matrine, berberine, paclitaxel, elemene, etc., to overcome the problems of chemotherapy drugs in the treatment of cancer. In the 1970s, people found that zedoary oil extracted from zedoary turmeric had antiviral and anti-tumor effects. In the 1990s, elemene was isolated from zedoary oil and found to include α-elemene, β-elemene, γ-elemene, and δ-elemene. It has been reported that β-elemene has the following three structures (W.Chen.etc., Royal Society of Chemistry, 2022):

[0006] Among them, (-)-β-elemene (also known as (1S,2S,4R)-β-elemene) has been widely used in cancer treatment. (1S,2S,4S)-β-elemene has also been reported to be used in cancer treatment and has superior activity to (1S,2S,4R)-β-elemene. However, the synthesis of (1S,2S,4S)-β-elemene is extremely difficult (W. Chen, etc., Royal Society of Chemistry, 2022). Not only does it require at least eight reaction steps and has an extremely low yield of less than 10%, it is very difficult to commercialize.

[0007] Currently, there are few reports on whether (1R,2R,4S)-β-elemene ((+)-β-elemene) has pharmacological activity, and the extraction or synthesis of (1R,2R,4S)-β-elemene is difficult, making it difficult to adapt to the needs of industrial applications.

[0008] SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure provides use of (1R,2R,4S)-β-elemene (i.e., (+)-β-elemene) or a pharmaceutical composition comprising the compound (1R,2R,4S)-β-elemene in the preparation of a medicament for treating cancer.

[0010] On the other hand, the present disclosure provides a method for preparing (1R, 2R, 4S)-β-elemene, which comprises heating (-)-germane A to convert it into (1R, 2R, 4S)-β-elemene, wherein the (-)-germane A is obtained by fermenting an engineered bacterium containing a nucleotide encoding an enzyme capable of synthesizing (-)-germane A.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG1 shows a curve showing the changes in bioluminescent signals of orthotopic liver cancer tumors over time after (+)-β-elemene and (-)-β-elemene were administered to mice, respectively.

[0013] FIG2 shows the inhibition rates of (+)-β-elemene and (-)-β-elemene on liver cancer in situ tumors, respectively.

[0014] FIG3 shows a curve showing the time-dependent changes in the bioluminescent signal of an orthotopic gastric cancer tumor after administration of (+)-β-elemene to mice.

[0015] Detailed description

[0016] In one aspect, the present disclosure provides use of (1R,2R,4S)-β-elemene (i.e., (+)-β-elemene) or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene in the preparation of a medicament for treating cancer.

[0017] In another aspect, the present disclosure provides a method for treating cancer in an individual in need thereof, comprising administering to the individual (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene.

[0018] In yet another aspect, the present disclosure provides (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for use in treating cancer.

[0019] In yet another aspect, the present disclosure provides use of (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene in treating cancer.

[0020] In some embodiments, the cancer is selected from one or more of pancreatic cancer, leukemia, breast cancer, ovarian cancer, colorectal cancer, thyroid cancer, lung cancer, lymphoma, endometrial cancer, renal cell carcinoma, esophageal cancer, prostate cancer, gastric cancer, bladder cancer, brain tumor, liver cancer, cervical cancer, head and neck cancer, bone cancer, malignant melanoma, cancerous pleural effusion, malignant pleural mesothelioma and brain glioma.

[0021] In some embodiments, the cancer is selected from one or more of gastric cancer, brain glioma, pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer, and liver cancer.

[0022] In some embodiments, the cancer is selected from one or more of pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer, and liver cancer.

[0023] In some embodiments, the cancer is selected from lung cancer, glioma, liver cancer, and gastric cancer. In some embodiments, the cancer is selected from gastric cancer and glioma. In some embodiments, the cancer is selected from gastric cancer. In some embodiments, the cancer is selected from liver cancer.

[0024] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Examples of the excipient include, but are not limited to, injection oil, carrier, filler, disintegrant, binder, lyoprotectant, pH regulator, preservative, antioxidant, wetting agent, and the like.

[0025] In some embodiments, the pharmaceutical composition can be prepared into various dosage forms, for example, solid dosage forms, semisolid dosage forms, liquid dosage forms and gaseous dosage forms, and the dosage forms can be, for example, liposomes, tablets, capsules, pills, mixtures, gels, ointments, granules, powders, creams, granules, pills, lozenges, solutions, suspensions, emulsions, pellets or injections, or one or more of these, or any other suitable dosage forms.

[0026] In some embodiments, the (1R,2R,4S)-β-elemene is obtained by converting (-)-germarene A by heating.

[0027] In some embodiments, the heating temperature is 100° C.-380° C. In some embodiments, the heating temperature is 100° C.-200° C. In some embodiments, the heating temperature is 150° C.-200° C. In some embodiments, the heating temperature is 150° C.

[0028] In some embodiments, the (-)-germarene A is obtained by a fermentation process.

[0029] In some embodiments, the fermentation method includes the following steps: culturing the engineered bacteria in a seed culture medium to obtain a seed liquid; then inoculating the seed liquid into a fermentation culture medium for fermentation culture, and recording the product of the fermentation culture as a fermentation system; during the fermentation culture process, adding the fermentation culture medium to the fermentation system.

[0030] In a further embodiment, the fermentation culture comprises yeast fermentation or E. coli fermentation. The yeast fermentation or E. coli fermentation can be performed using methods known in the art.

[0031] In some embodiments, the fermentation culture comprises a two-phase fermentation or a single-phase fermentation.

[0032] In some embodiments, the fermentation culture is carried out at a temperature of 20-40° C. In some embodiments, the fermentation culture is carried out at a temperature of 20-37° C. In some embodiments, the fermentation culture is carried out at a temperature of 37° C. In some embodiments, the fermentation culture is carried out at a temperature of 20-30° C. In some embodiments, the fermentation culture is carried out at a temperature of 25-30° C. In some embodiments, the fermentation culture is carried out at a temperature of 25° C.

[0033] In some embodiments, the fermentation culture is carried out at a rotation speed of 100-500 rpm. In some embodiments, the fermentation culture is carried out at a rotation speed of 200-300 rpm.

[0034] In some embodiments, the fermentation culture time is 1-240 hours. In some embodiments, the fermentation culture time is 40-120 hours.

[0035] In some embodiments, the seed culture medium and fermentation medium contain 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 ml / L calcium chloride mother liquor, 10 ml / L trace metal salt mother liquor, 3 ml / L vitamin mother liquor, 100 mL / L succinic acid mother liquor, and the balance is water.

[0036] In some embodiments, the engineered bacteria comprises one or more enzymes capable of synthesizing (-)-germarene A. In further embodiments, the one or more enzymes capable of synthesizing (-)-germarene A are selected from enzymes capable of synthesizing (-)-germarene A having an amino acid sequence that is at least 80% homologous to the amino acid sequence of SEQ ID NO. 2.

[0037] In a further embodiment, the engineered bacteria contains an expression cassette of a nucleotide sequence encoding an enzyme capable of synthesizing (-)-germarene A.

[0038] In some embodiments, the nucleotides encoding the enzyme capable of synthesizing (-)-germarene A are independently selected from nucleotide sequences that are at least 90% homologous to the nucleotide sequence of SEQ ID NO. 1.

[0039] In some embodiments, the enzyme capable of synthesizing (-)-germarene A is an enzyme capable of converting farnesyl pyrophosphate into germarene.

[0040] In some embodiments, the (-)-germarene A is separated and purified by extraction or chromatography. In some embodiments, the organic solvent used for extraction includes but is not limited to n-dodecane, isopropyl myristate, n-hexane, n-heptane, etc. In some embodiments, the organic solvent used for extraction is n-dodecane.

[0041] The inventors of the present application surprisingly discovered that (+)-β-elemene unexpectedly exhibited better tumor inhibitory effects in in vitro and / or in vivo models compared to (-)-β-elemene and / or (1S,2S,4S)-β-elemene, providing a new option for the treatment of tumors. The present disclosure also provides a method for the biosynthesis of (+)-β-elemene, wherein (-)-germaene A is obtained by fermentation with an engineered bacterium containing nucleotides encoding an enzyme capable of synthesizing (-)-germaene A, and then (-)-germaene A is thermally converted into (+)-β-elemene, thereby solving the problem of (+)-β-elemene being difficult to obtain and providing an important source for the clinical application and industrial production of (+)-β-elemene. Example

[0042] The embodiments of the present disclosure are further described in detail below with reference to specific examples, but the implementation of the present disclosure is not limited thereto. Equivalent substitutions, combinations, improvements or modifications made by those skilled in the art to the embodiments of the present disclosure based on the description of the present disclosure should all be included in the scope of protection of the present disclosure.

[0043] Unless otherwise indicated, the experimental methods used below are routine methods well known to those skilled in the art. For example, they can be performed using the standard procedures described in the following works: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001; Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA, 1995; and Juan S. Bonifacino et al., Current Protocols in Cell Biology, John Wiley and Sons, Inc.

[0044] Identification of (-)-germarene A: Based on the disclosure in Alfred J. Weinheimer et al., Tetrahedron Letters No. 7, pp. 497-500, 1970.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] The equipment and reagents used in the present disclosure are as follows: microplate reader (Spark JC437), balance (SECURA225D-1CN JC395), centrifuge (MODEL0406-1 GY067), inverted microscope (CKX53SF JC406), cell counter (Countstar Rigel S2 JC422), CO2 incubator (371YL110), clean bench (ESCO, SW-CJ-2FD, FZ084), anhydrous ethanol (Sigma WXBC939V), CCK8 (Beyotime 061223231013), PBS (Procell WHB823U071), double antibody (Gibco 2441832), DMEM medium (Gibco 2537564), 1640 medium (Gibco 2732541), FBS (Sciencell 38890), trypsin (Gibco 2764663).

[0047] The cell lines used in the present invention are: human in situ pancreatic adenocarcinoma cell line (BXPC-3), human blast crisis chronic myeloid leukemia cell line (LAMA-84), human lung squamous cell carcinoma cell line (NCI-H226), human colorectal cancer cell line (HCT116), human pancreatic cancer cell line (PANC-1), human ovarian cancer cell line (A2780), human chronic myeloid leukemia cell line (K562), human non-small cell lung cancer cell line (A549), human breast cancer cell line (MDA-MB-231), human esophageal cancer cell line (Eca-109), human prostate adenocarcinoma cell line (PC-3), human liver cancer cell line (HepG-2), human gastric cancer cell line (MKN-28), and human glioma cell line (U251).

[0048] The (+)-β-elemene used in Examples 1 to 7 can be obtained by the preparation method described in Example 8.

[0049] Example 1

[0050] (1) Prepare cell suspension (BXPC-3, density: 8×10 4 / mL), 100 μL of cell suspension was added to each well of a 96-well cell culture plate;

[0051] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h. The drug was diluted with 1640 medium to the desired working concentration, and 100 μL of drug-containing medium was added to each well (final drug concentration: 150 μM). A vehicle control group (1640 medium without drug was added to the vehicle control group) and a blank control group (1640 medium was added to the blank control group without cells or drug) were also established.

[0052] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h;

[0053] (4) Discard the supernatant and add 100 μL of 10% CCK8 detection reagent solution (prepared by adding 0.8 mL of CCK8 to 7.2 mL of serum-free 1640 culture medium and mixing well) to each well. Continue culturing in the incubator for 2.5 h. Observe that the bottom of the 96-well plate containing cells turns yellow.

[0054] At a detection wavelength of 450 nM, the OD value of each well was read using a microplate reader, where the tumor cell growth inhibition rate IR (%) = (1-(OD compound - OD blank control) / (OD vehicle control - OD blank control)) * 100%. The calculated inhibition factor of (+)-β-elemene relative to (-)-β-elemene was 6.77 times, calculated using the following formula: inhibition factor = ((+)-β-elemene inhibition rate - (-)-β-elemene inhibition rate) / (-)-β-elemene inhibition rate.

[0055] Example 2

[0056] (1) Prepare cell suspension (LAMA-84, density: 3×10 5 / mL), 100 μL of cell suspension was added to each well of a 96-well cell culture plate;

[0057] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h. The drug was diluted with 1640 medium to twice the required working solution concentration, and 100 μL of drug-containing medium was added to each well (final drug concentration: 150 μM). A vehicle control group (medium without drug) and a blank control group (medium only was added without cells or drugs) were also established.

[0058] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h. 20 μL of 10% CCK8 detection reagent (7.2 mL of serum-free 1640 medium plus 0.8 mL of CCK8) was added to each well and cultured in the incubator for 2.5 h. The bottom of the 96-well plate containing cells turned yellow.

[0059] At a detection wavelength of 450 nM, the OD value of each well was read using a microplate reader, where the tumor cell growth inhibition rate IR (%) = (1-(OD compound - OD blank control) / (OD vehicle control - OD blank control)) * 100%. The calculated inhibition rate of (+)-β-elemene relative to (-)-β-elemene was 0.54 times. The calculation method of the enhancement factor was as described in Example 1.

[0060] Example 3

[0061] (1) Prepare cell suspension (NCI-H226, density: 8×10 4 / mL), 100 μL of cell suspension was added to each well of a 96-well cell culture plate;

[0062] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h. The drug was diluted with 1640 medium to the desired working concentration, and 100 μL of drug-containing medium was added to each well (final drug concentration: 150 μM). A vehicle control group (medium without drug) and a blank control group (medium only, without cells or drug) were also established.

[0063] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h;

[0064] (4) Discard the supernatant and add 100 μL of 10% CCK8 detection reagent solution (7.2 mL of serum-free 1640 culture medium plus 0.8 mL of CCK8 and mix well) to each well. Continue to culture in the incubator for 2 h. Observe that the bottom of the 96-well plate containing cells turns yellow.

[0065] At a drug concentration of 150 μM and a detection wavelength of 450 nM, the OD value of each well was read using a microplate reader to calculate the inhibition rate. Tumor cell growth inhibition rate IR (%) = (1-(OD compound - OD blank control) / (OD vehicle control - OD blank control)) * 100%. The calculated inhibition rate of (+)-β-elemene relative to (-)-β-elemene was 3.85 times higher. The calculation method of the enhancement factor is as described in Example 1.

[0066] Example 4

[0067] (1) Preparation of cell suspension: HCT116 density: 4×10 4 / mL, A549 density: 4.5×10 4 / mL, MDA-MB-231 density: 5×10 4 / mL, Eca-109 density: 4.5×10 4 PC-3 density: 6×10 4 cells / mL, HepG-2 density: 3.5×10 4 cells / mL, PANC-1 density: 6×10 4 / mL, A2780 density: 8×10 4 / mL, K562 density: 1×10 5 100 μL of cell suspension was added to each well of a 96-well cell culture plate.

[0068] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h. The drug was diluted with 1640 medium to the desired working concentration, and 100 μL of drug-containing medium was added to each well. The final drug concentrations in each group of wells were 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM, respectively. Each group had three replicate wells with the same final drug concentration. A vehicle control group (medium without drug) and a blank control group (medium only, without cells or drug) were also established.

[0069] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 72 h.

[0070] (4) Discard the supernatant and add 100 μL of 10% CCK8 detection reagent solution (7.2 mL of serum-free 1640 medium and 0.8 mL of CCK8 mixed) to each well. Continue incubating in the incubator for 2.5 hours. Observe that the bottom of the 96-well plate containing cells turns yellow. Under the detection wavelength of 450 nM, the OD value of each well is read using a microplate reader, and the inhibition rate is calculated, where the tumor cell growth inhibition rate IR (%) = (1-(OD compound-OD blank control) / (OD solvent control-OD blank control)) * 100%. Based on the inhibition rate of the drug at each concentration group, the IC50 value of the drug is calculated using GraphPad Prism 10 software, that is, the drug concentration value corresponding to the inhibition rate of 50%. The experimental results are shown in Table 1.

[0071] Table 1 Inhibitory effects of different drugs in different cell lines

[0072] Example 5

[0073] (1) Prepare cell suspension (MKN-28 density: 8×10 4 / mL, U251 density: 3.5×10 4 100 μL of cell suspension was added to each well of a 96-well cell culture plate.

[0074] (2) The 96-well MKN-28 cell culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24 h. The drug was diluted with 1640 medium to the desired working concentration, and 100 μL of drug-containing medium was added to each well. The final drug concentrations in each group of wells were 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM, respectively. Each group had three replicate wells with the same final drug concentration. A vehicle control group (medium without drug was added to the vehicle control group) and a blank control group (no cells or drug were added to the blank control group, only 1640 medium was added). 96-well U251 cell culture plates were incubated in a 37°C, 5% CO2, and saturated humidity incubator for 24 hours. Drugs were diluted with 1640 medium to the desired working concentration, and 100 μL of drug-containing medium was added to each well. The final drug concentrations in each group of wells were 1.96 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM, respectively. Each group had three replicate wells with the same final drug concentration. A vehicle control group (drug-free medium was added to the vehicle control group) and a blank control group (no cells or drug were added to the blank control group, containing only 1640 medium) were also established.

[0075] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 72 h.

[0076] (4) Discard the supernatant and add 100 μL of 10% CCK8 detection reagent solution (7.2 mL of serum-free 1640 medium and 0.8 mL of CCK8 mixed) to each well. Continue incubating in the incubator for 2.5 hours. Observe that the bottom of the 96-well plate containing cells turns yellow. Under the detection wavelength of 450 nM, the OD value of each well is read using a microplate reader, and the inhibition rate is calculated, where the tumor cell growth inhibition rate IR (%) = (1-(OD compound-OD blank control) / (OD solvent control-OD blank control)) * 100%. Based on the inhibition rate of the drug at each concentration, the IC50 value of the drug is calculated using GraphPad Prism10 software, that is, the drug concentration value corresponding to the inhibition rate of 50%. The experimental results are shown in Table 2.

[0077] Table 2 Inhibitory effects of different drugs in different cell lines

[0078] Example 6

[0079] 1. Experimental Materials

[0080] 1.1 Animals: BALB / c female nude mice, 6-8 weeks old, weighing 17-23 g, 18 mice, supplied by Shanghai Bikeway Biotechnology Co., Ltd. 1.2 Test drugs:

[0081] 1) Test sample: (+)-β-elemene (2% ( W / V )) or (-)-β-elemene (2% ( W / V )) and soybean oil (4.8% ( W / V )) were mixed as the oil phase, albumin solution (6% ( W / V )) is used as the aqueous phase and subjected to shear high-pressure homogenization treatment to obtain a stable aqueous dispersion system with an average particle size of the dispersed phase ≤1μm.

[0082] 2) Reference substance: 0.9% sodium chloride injection (Shijiazhuang Siyao Co., Ltd., national standard: H13023200)

[0083] 2. Experimental process

[0084] 1) Cell Culture: Human hepatocellular carcinoma Hep3B-Luc (Hep3B, ATCC-HB-8064) cells were cultured as monolayers in DMEM medium supplemented with 10% FBS and 1% antibiotic-antimycolic acid at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number was reached, cells were harvested, counted, and plated.

[0085] 2) Tumor cell inoculation: Anesthetized mice were placed in supine position. The abdominal skin was disinfected with 75% ethanol to maintain the sterility of the surgical field. A transverse incision was made in the abdominal wall to fully expose the left lobe of the liver. 20 μl of tumor cell inoculation containing approximately 3×10 6 A cell suspension of Hep3B-Luc cells (15% Matrigel) was injected into the left lobe of the liver. The injection site was checked for leakage. The left lobe of the liver was returned to the abdominal cavity and sutured. The mouse was observed until it fully regained consciousness.

[0086] 3) Experimental method: After inoculation, the mice were intraperitoneally injected with 15 mg / mL of fluorescein solution at 10 μL / g body weight (the fluorescein injection dose for each animal was 150 mg / kg). Ten minutes later, the mice were pre-anesthetized with a mixture of oxygen and isoflurane. After the mice were deeply anesthetized, they were moved to the imaging chamber of IVIS (Lumia II) to begin bioluminescence detection, and the bioluminescence signals in the animals and the generated image information were detected and recorded. Six days after tumor inoculation, group dosing began when the average fluorescence value reached 2.29E+07, with each group containing 6 mice. The day of grouping was defined as Day 0, and dosing began on Day 0 (i.e., treatment began on the day of grouping). Dosing started on Day 0 and ended on Day 28. The dosage, route of administration, and frequency of administration for the three groups are as described in Table 3. Tumor growth inhibition rate TGI% was calculated as [(1-(average bioluminescence signal of a treatment group at the end of drug administration-average bioluminescence signal of the treatment group at the beginning of drug administration)) / (average bioluminescence signal of the vehicle control group at the end of treatment-average bioluminescence signal of the vehicle control group at the beginning of treatment)] x 100%.

[0087] Table 3 Route and frequency of administration

[0088] Note: iv: intravenous injection; QD: once a day;

[0089] 3. Experimental results

[0090] Figure 1 shows the changes in bioluminescence signals over time in each group of mice after drug administration. The tumor growth inhibition rate (TGI%) of each group was calculated based on the bioluminescence signals, and the results are shown in Figure 2.

[0091] Example 7

[0092] 1. Experimental Materials

[0093] 1.1 Animals: BALB / c female nude mice, 6-8 weeks old, weighing 19-23 g, 12 mice were supplied by Beijing Ankai Yibo Biotechnology Co., Ltd.

[0094] 1.2 Test drug:

[0095] 1) Test sample: (+)-β-elemene (2% ( W / V )) and soybean lecithin (2% ( W / V )) added to soybean oil (5% ( W / V )) and heated to form an oil phase; the oil phase was added to a mixture containing sodium oleate (0.1% ( W / V )) and an appropriate amount of glycerol in an aqueous solution, and after shearing and high-pressure homogenization treatment, a stable aqueous dispersion system with an average particle size of the dispersed phase ≤1μm is obtained.

[0096] 2) Reference substance: 0.9% sodium chloride injection (Shijiazhuang Siyao Co., Ltd., national standard: H13023201)

[0097] 2. Experimental process

[0098] 1) Cell Culture: Human gastric cancer NCI-N87-Luc (NCI-N87, ATCC-CRL-5822) cells were cultured as monolayers in RPMI 1640 medium supplemented with 10% FBS and 2 μg / mL puromycin at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number was reached, cells were harvested, counted, and plated.

[0099] 2) Tumor cell inoculation: Anesthetized mice were placed in a supine position. The abdominal skin was disinfected with 75% ethanol to keep the surgical area sterile. A longitudinal incision was made in the abdomen slightly to the left of the midline to expose the mouse stomach. 50 μl of the solution containing approximately 5 × 10 6 A cell suspension of NCI-N87-Luc cells (15% Matrigel) was injected subserosa into the gastric glandular area. A sterile cotton swab soaked in normal saline was used to press the injection site for approximately 1 minute. The injection site was checked for leakage. The stomach was returned to the abdominal cavity, and the abdominal wall and skin were sutured in layers. The mouse was observed until it fully regained consciousness.

[0100] 3) Experimental method: After inoculation, the mice were intraperitoneally injected with 15 mg / mL of fluorescein solution at 5 μL / g body weight (the fluorescein injection dose for each animal was 75 mg / kg). Ten minutes later, the mice were pre-anesthetized with a mixture of oxygen and isoflurane. After the mice were deeply anesthetized, they were moved to the imaging chamber of IVIS (Lumia III) to begin bioluminescence detection, and the bioluminescence signals in the animals and the generated image information were detected and recorded. 9 days after tumor inoculation, group dosing began when the average fluorescence value reached 26E+07, with each group containing 6 mice. The day of grouping was defined as Day 0, and dosing began on Day 1 (i.e., treatment began on the first day after grouping). Dosing started on Day 1 and ended on Day 28. The dosage, route of administration, and frequency of administration for one group are as described in Table 4. Tumor growth inhibition rate TGI% was calculated as [(1-(average bioluminescence signal of a treatment group at the end of drug administration - average bioluminescence signal of the treatment group at the beginning of drug administration)) / (average bioluminescence signal of the vehicle control group at the end of treatment - average bioluminescence signal of the vehicle control group at the beginning of treatment)] x 100%. The calculated TGI% value was greater than 40%.

[0101] Table 4 Administration route and frequency

[0102] Note: po: oral; QD: once a day; BID: twice a day; QW: once a week

[0103] 3. Experimental results

[0104] FIG3 shows the temporal changes in bioluminescence signals after drug administration in mice.

[0105] Example 8 Preparation of (+)-β-elemene

[0106] 1. Preparation of (-)-germaene

[0107] 1.1 Plasmid Preparation

[0108] The nucleotide sequence encoding MmoTPS was synthesized according to the nucleotide sequence in Table 5 by conventional methods known to those skilled in the art. In order to construct an expression vector for the fusion protein, GGGGS was selected as a connecting peptide (Qi Liu. et al, 2023), and the ERG20 gene fragment (SEQ ID NO: 3), SSA1 promoter fragment (SEQ ID NO. 7) and ADH1 terminator fragment (SEQ ID NO. 8) were obtained from Saccharomyces cerevisiae by conventional methods. The ERG20 gene fragment and the synthesized MmoTPS gene fragment were used as templates to obtain the fusion protein MmoTPS-GGGGS-ERG20 gene fragment by OE-PCR (amino acid sequence and nucleotide sequence are shown in Table 6). The full-length gold seamless cloning kit was used. The MmoTPS-GGGGS-ERG20 gene fragment, SSA1 promoter fragment, and ADH1 terminator fragment were ligated with the yeast expression vector pRS425 (Miaoling Biotechnology, China) using the ELISA-Basic Seamless Cloning and Assembly Kit (Quanshijin, China). The ligation product was transformed into Trans 5α competent cells (Quanshijin, China), plated on LB-Amp resistant plates, and cultured at 37°C for 16 h. Single clones were picked and cultured overnight at 37°C. The plasmids were extracted and verified by sequencing.

[0109] Table 5 Enzyme related information

[0110] Table 6 Fusion protein sequences

[0111] 1.2 Construction of Saccharomyces cerevisiae strain CEN124

[0112] In order to realize the synthesis of germarene A in Saccharomyces cerevisiae, CEN.PK2-1D (Euroscarf, Germany) was selected for transformation. Donor DNA was constructed by conventional methods and gene editing was performed as shown in Table 7 (Amanda Reider Apel et al, 2016). By knocking out LPP1 and GAL80 genes, EfmvaE (GEN BANK: WP_002382276) and IDI1 genes (GEN BANK: NP_015208) were inserted, URA3 (GEN BANK: NP_010893), ERG12 (GEN BANK: NP_013935), ERG8 (GEN BANK: NP_013947) and ERG19 genes (GEN BANK: NP_014441) were inserted at the neutral site YPRC15C, and HIS3 (GEN BANK: NP_01622b) was inserted at the neutral site. BANK:WP_020863086), ERG13 (GEN BANK:NP_013580), and EfmvaE genes were cloned, the TRP gene (GEN BANK:NP_010290) was backfilled in situ, the ERG9 promoter was replaced with HXT1 (GEN BANK:CP092951) and then with the original ERG9 promoter (GEN BANK:CP026287), and finally the Saccharomyces cerevisiae engineered strain CEN124 was obtained.

[0113] Table 7

[0114] 1.3 Preparation of competent CEN124 yeast

[0115] The CEN124 monoclone was picked and transferred to 3 mL SD-Trp His Ura (Panjinuo, China) liquid medium and cultured at 30°C for 24 h. Then, the clone was transferred to 50 mL YPD liquid medium at a 5% transfer volume and cultured until the OD 600 =0.6-0.8, the bacteria were collected and CEN124 competent cells were prepared using ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA) according to the kit operation procedure.

[0116] 1.4 Construction of engineered bacteria

[0117] According to the ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA) kit operating procedures, the plasmid (see Table 8) was transformed into the above-mentioned CEN124 competent cells, coated on SD-Leu plates, and cultured at 30°C for 48 hours to obtain the corresponding engineered bacteria (see Table 8).

[0118] Table 8 Engineering bacteria and corresponding plasmids

[0119] Preparation of 1.5(-)-Germaene A

[0120] 1.5.1 Culture medium preparation

[0121] Calcium chloride mother liquor: prepared according to Example 3 of the prior art CN108060092B.

[0122] Trace metal salt mother liquor: prepared according to Example 3 of the prior art CN108060092B.

[0123] Vitamin mother solution: 0.2g / L biotin, 0.8g / L sodium p-aminobenzoate, 4g / L niacin, 4g / L calcium pantothenate, 4g / L pyridoxine hydrochloride, 4g / L thiamine hydrochloride, 100g / L inositol.

[0124] Succinic acid mother liquor: 60 g / L succinic acid, adjust pH to 5.05 with NaOH.

[0125] F2 fermentation medium: 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 ml / L calcium chloride mother liquor, 10 ml / L trace metal salt mother liquor, 3 ml / L vitamin mother liquor, 100 mL / L succinic acid mother liquor.

[0126] 1.5.2 Fermentation of engineered Saccharomyces cerevisiae

[0127] Individual clones of the engineered bacteria obtained in 1.4 were transferred to 3 mL of SD-Leu (Panjinuo, China) liquid medium and cultured with shaking at 30°C, 250 rpm for 24 hours. A 5% transfer volume was then added to each 24-hour culture broth in a 100 mL Erlenmeyer flask containing 20 mL of F2 fermentation medium and cultured at 30°C, 250 rpm for 40 hours. 750 μL of the 40-hour fermentation broth was placed in a 2 mL centrifuge tube, 300 μL of n-dodecane was added, and the cells were ground with glass beads for 30 minutes. The supernatant n-dodecane solution was removed and diluted 10-fold with ethanol for yield analysis. The yield of (-)-germarene A in yeast was 362.25 mg / L.

[0128] 2. Preparation of (+)-β-elemene

[0129] To the fermentation broth containing (-)-germane A obtained above, n-dodecane was added at a volume ratio of 1:2 (V n-dodecane:V fermentation broth), and the mixture was stirred and extracted for 16 hours. The organic phase was then heated at 150°C for 2 hours to obtain a conversion solution. The conversion solution was then separated and purified by silver nitrate-silica gel and silica gel column chromatography, respectively, to obtain (+)-β-elemene (yield ≥ 95%). 1 H NMR(CDCl3,400MH z):1.03(3H,s,H-15), 1.48(3H,m,H-2,3a), 1.64(1H,m,H-5a), 1.56(2 H,m,H-3b,5b), 1.74(3H,s,H-11), 1.77(3H,s,H-14), 1.96(1H,m,H-6), 2.04(1H,m,H-4), 4.61(1H,s,H-10a), 4.73(1H,s,H-10b), 4.74(1H,brs,H-13a), 4.85(1H,brs,H-1 3b), 4.92 (1H, d, J = 11.2 Hz, H-8a), 4.93 (1H, d, J = 17.6 Hz, H-8b), 5.85 (1H, dd, J = 17.6, 11.2 Hz, H-7).

[0130] 13 C NMR (CDCl3, 100MHz): 16.6 (C-15), 21.1 (C-14), 24.8 (C-11), 26.8 (C-5), 32.9 (C-3), 39.8 (C-1), 39.9 (C-2) , 45.7(C-6), 52.7(C-4), 108.2(C-13), 109.8(C-8), 112.1(C-10), 147.7(C-9), 150.3(C-12), 150.4(C-7).

[0131] The following is an informal sequence listing

[0132] SEQ ID NO.1 (nucleotide sequence of MmoTPS)

[0133] SEQ ID NO.2 (amino acid sequence of MmoTPS)

[0134] SEQ ID NO.3 (nucleotide sequence of ERG20)

[0135] SEQ ID NO.4 (amino acid sequence of MmoTPS-GGGGS-ERG20 fusion protein)

[0136] SEQ ID NO.5 (nucleotide sequence of MmoTPS-GGGGS-ERG20 fusion protein)

[0137] SEQ ID NO.6 (pRS425-LEU-P SSA1 -MmoTPS-GGGGS-ERG20-T ADH1 nucleotide sequence of the plasmid)

[0138] SSA1 promoter fragment (SEQ ID NO.7)

[0139] ADH1 terminator fragment (SEQ ID NO.8)

Claims

1. Use of (1R, 2R, 4S)-β-elemene or a pharmaceutical composition comprising (1R, 2R, 4S)-β-elemene in the preparation of a medicament for treating cancer, wherein the (1R, 2R, 4S)-β-elemene has the following structure:

2. A method for treating cancer in an individual in need thereof, comprising administering to the individual (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene.

3. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer.

4. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to claim 1, the method according to claim 2, or according to claim 3, wherein the cancer is selected from pancreatic cancer, leukemia, breast cancer, ovarian cancer, colorectal cancer, thyroid cancer, lung cancer, lymphoma, endometrial cancer, renal cell carcinoma, esophageal cancer, prostate cancer, gastric cancer, bladder cancer, brain tumor, liver cancer, cervical cancer, head and neck cancer, bone cancer, malignant melanoma, carcinoma One or more of pleural effusion, malignant pleural mesothelioma and brain glioma; or, the cancer is selected from one or more of gastric cancer, brain glioma, pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer and liver cancer; or, the cancer is selected from one or more of pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer and liver cancer; or, the cancer is selected from one or more of lung cancer, liver cancer, gastric cancer and brain glioma; or, the cancer is selected from liver cancer or gastric cancer.

5. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to claim 1 or 4, or the method according to claim 2 or 4, or according to claim 3 or 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, such as an injection oil, a carrier, a filler, a disintegrant, a binder, a lyoprotectant, a pH regulator, a preservative, an antioxidant and a wetting agent.

6. The use according to claim 1, 4 or 5, the method according to claim 2, 4 or 5, or the (1R, 2R, 4S)-β-elemene or the pharmaceutical composition comprising (1R, 2R, 4S)-β-elemene for treating cancer according to claim 3, 4 or 5, wherein the pharmaceutical composition can be formulated into solid dosage forms, semisolid dosage forms, liquid dosage forms and gaseous dosage forms, such as one or more of liposomes, tablets, capsules, pills, mixtures, gels, ointments, granules, powders, creams, granules, pills, lozenges, solutions, suspensions, emulsions, pellets or injections.

7. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to the use according to any one of claims 1 and 4-6, the method according to any one of claims 2 and 4-6, or the pharmaceutical composition comprising (1R,2R,4S)-β-elemene according to any one of claims 3 and 4-6, wherein the (1R,2R,4S)-β-elemene is obtained by heating and converting (-)-germaene A, and the (-)-germaene A is obtained by fermenting an engineered bacterium containing a nucleotide encoding an enzyme capable of synthesizing (-)-germaene A.

8. The use, method or (1R,2R,4S)-β-elemene or pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to claim 7, wherein the fermentation comprises the following steps: culturing the engineered bacteria in a seed culture medium to obtain a seed solution; then inoculating the seed solution into a fermentation culture medium for fermentation culture, and recording the product of the fermentation culture as a fermentation system; during the fermentation culture, adding the fermentation culture medium to the fermentation system.

9. The use, method or (1R,2R,4S)-β-elemene or pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to claim 7 or 8, wherein the enzyme capable of synthesizing (-)-germaene A has an amino acid sequence of SEQ ID NO: 2 or at least 80% homologous to SEQ ID NO:

2.

10. The use, method or pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to claim 7 or 8, wherein the nucleotide encoding the enzyme capable of synthesizing (-)-germaene A has SEQ ID NO: 1 or a nucleotide sequence having at least 90% homology with SEQ ID NO:

1.

11. The use, method or (1R,2R,4S)-β-elemene or pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to any one of claims 7 to 10, wherein the seed culture medium and the fermentation culture medium contain 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 ml / L calcium chloride mother liquor, 10 ml / L trace metal salt mother liquor, 3 ml / L vitamin mother liquor, 100 mL / L succinic acid mother liquor, and the balance is water.

12. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to any one of claims 7 to 11, wherein the (-)-germarene A is heated at 100°C-380°C or 150°C.

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