USE OF EXTRACT FROM RABBIT SKIN INFLAMED BY VACCINIA VIRUS IN TREATMENT OF CANCER
20220354900 · 2022-11-10
Inventors
Cpc classification
A61K31/513
HUMAN NECESSITIES
A61K31/7068
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/7072
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K35/36
HUMAN NECESSITIES
A61K31/513
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/7068
HUMAN NECESSITIES
A61K31/7072
HUMAN NECESSITIES
Abstract
The present invention relates to the therapeutic use of extract from rabbit skin inflamed by vaccinia virus. More specifically, the present invention relates to the use of the extract for preventing or treating cancer, or the use of the extract for stimulating secretion of cytokine by somatic cells in a patient. In addition, the present invention also relates to a drug combination comprising the extract as the first anticancer agent and a second anticancer agent as well as its use for preventing or treating cancer.
Claims
1. Use of extract from rabbit skin inflamed by vaccinia virus in the preparation of a medicament for preventing or treating cancer in a patient.
2. The use of claim 1, wherein the cancer is selected from the group consisting of liver cancer, lung cancer, cervical cancer, cholangiocarcinoma, bladder cancer, brain cancer, bone cancer, breast cancer, head and neck cancer, colorectal cancer, large intestine cancer, gastric cancer, esophageal cancer, bile cancer, hepatocellular carcinoma, kidney cancer, multiple myeloma, nasopharyngeal carcinoma, oral cancer, pancreatic cancer, ovarian cancer, ureteral cancer, pituitary adenoma, urinary tract cancer, prostate cancer, small cell lung cancer, squamous cell carcinoma, endometrial cancer, leukemia, lymphoma, neuroblastoma, retinoblastoma, Ewing sarcoma, soft tissue sarcoma, glioma, skin cancer or melanoma.
3. The use of extract from rabbit skin inflamed by vaccinia virus in the preparation of a medicament for stimulating secretion of cytokine by somatic cells in a patient.
4. The use of claim 3, wherein the cytokine comprises IL or IFN, preferably selected from IL-2, IFN-γ, IL-4 or IL-12.
5. The use of claim 3 or 4, wherein the somatic cells are immune cells, such as lymphocytes.
6. Use of the extract from rabbit skin inflamed by vaccinia virus in the preparation of a medicament for promoting the apoptosis of tumor cells or activating the apoptosis signal pathway of tumor cells in a patient.
7. The use of claim 6, wherein promoting the apoptosis of tumor cells or activating the apoptosis signal pathway of tumor cells is achieved by increasing the expression level of cytochrome C, Caspase-3, cleaved Caspase-3, Caspase-9, p53 and/or Bax, or by reducing the expression level of Bcl2.
8. Use of extract from rabbit skin inflamed by vaccinia virus in the preparation of a medicament for inducing G2/M cell cycle arrest of tumor cells in a patient.
9. The use of claim 8, wherein inducing G2/M cell cycle arrest of tumor cells is achieved by reducing the expression level of cyclin A2, cyclin B1 and/or CDK1.
10. The use of any one of claims 1-9, wherein the extract from rabbit skin inflamed by vaccinia virus as an anticancer agent in combination with another anticancer agent is for the preparation of a medicament.
11. A drug combination for the treatment of cancer, the drug combination comprising extract from rabbit skin inflamed by vaccinia virus as a first anticancer agent and a second anticancer agent.
12. Use of the extract from rabbit skin inflamed by vaccinia virus as the first anticancer agent and a second anticancer agent in the preparation of a medicament for preventing or treating cancer in a patient.
13. The drug combination of claim 11 or the use of claim 7, wherein the second anticancer agent is a cytotoxic agent; preferably an agent that acts on the chemical structure of DNA or an agent that affects nucleic acid synthesis.
14. The drug combination or use of claim 13, wherein the agent that acts on the chemical structure of DNA is an alkylating agent or a pharmaceutically acceptable salt thereof, preferably nitrogen mustards.
15. The drug combination or use of claim 14, wherein the nitrogen mustards comprises fatty nitrogen mustards, aromatic nitrogen mustards, amino acid nitrogen mustards, steroidal nitrogen mustards or heterocyclic nitrogen mustards or a pharmaceutically acceptable salt thereof.
16. The drug combination or use of claim 15, wherein the heterocyclic nitrogen mustard comprises cyclophosphamide, ifosfamide, 4H-peroxocyclophosphamide, defosfamide, mafosfamide, perfosfamide, trifosfamide or a pharmaceutically acceptable salt thereof, preferably cyclophosphamide.
17. The drug combination or use of claim 13, wherein the agent that affects nucleic acid synthesis is a thymidylate synthase inhibitor or a pharmaceutically acceptable salt thereof, preferably a pyrimidine antagonist.
18. The drug combination or use of claim 17, wherein the pyrimidine antagonist is selected from the group consisting of 5-fluorouracil, capecitabine, tegafur, tegadifur, carmofur, uracil-tegafur, furtulon, floxuridine, doxifluridine or a pharmaceutically acceptable salt thereof, preferably 5-fluorouracil.
19. The drug combination or use of any one of claims 11-18, wherein the cancer is selected from liver cancer, lung cancer, cervical cancer, cholangiocarcinoma, bladder cancer, brain cancer, bone cancer, breast cancer, head and neck cancer, colorectal cancer, large intestine cancer, gastric cancer, esophageal cancer, bile cancer, hepatocellular carcinoma, kidney cancer, multiple myeloma, nasopharyngeal carcinoma, oral cancer, pancreatic cancer, ovarian cancer, ureteral cancer, pituitary adenoma, urinary tract cancer, prostate cancer, small cell lung cancer, squamous cell carcinoma, endometrial cancer, leukemia, lymphoma, neuroblastoma, retinoblastoma, Ewing sarcoma, soft tissue sarcoma, glioma, skin cancer or melanoma.
20. The drug combination or use according to any one of claims 1-19, wherein the extract from rabbit skin inflamed by vaccinia virus is Lepalvir.
21. The drug combination or use of any one of claims 1-20, wherein the extract from rabbit skin inflamed by vaccinia virus is formulated into an oral formulation or an injection, preferably an intramuscular injection, intraperitoneal injection, IP injection, subcutaneous injection or intravenous injection.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0065] Unless otherwise specified, all scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, and equivalents can be used. All publications and other references mentioned herein are incorporated by reference in their entirety. The following examples are provided to further illustrate the present invention. The following examples are not intended to limit the scope of the present invention for any reason.
EXAMPLES
Example 1—In Vitro Anti-Tumor Activity of Lepalvir
[0066] 1.1. The Inhibitory Effect of Lepalvir on Tumor Cell Proliferation
[0067] Human liver cancer cells HepG2, highly metastatic liver cancer cells LM3, cervical cancer cells HeLa, alveolar epithelial cells A549 and large cell lung cancer cells H460 in logarithmic growth phase were digested with 0.25% trypsin to prepare a cell suspension. The cell concentration was adjusted to 5×10.sup.4/ml. The cells were inoculated in a 96-well cell culture plate (100 μl per well), and incubated overnight in a constant temperature (37° C.) incubator under 5% CO.sub.2. The supernatant was discarded, and pre-prepared complete culture medium containing different concentrations of Lepalvir was added at 5 working concentrations (the final concentration of Lepalvir was 1.63, 0.815, 0.326, 0.163 and 0.0815 U/ml). For the control group (no drug solution added) and the cell-free blank group, 10% FBS-containing DMEM medium or RPMI-1640 medium was added. There were 3 replicate wells for each concentration, and the total volume of each well was 100 μl. After incubation in a constant temperature cell incubator for 24 hours, 10 μl of CCK-8 reagent was added to each well. After further incubation for 1 hour, the absorbance (OD) for each well was measured at 450 nm in an enzyme-linked immunoassay, and the cell proliferation inhibition rate for each group was calculated. Cell inhibition rate (%)=1−[(experimental group OD value-blank group OD value)/(control group OD value-blank group OD value)]×100%.
[0068] HepG2, LM3, HeLa, A549 and H460 cells were treated with different concentrations of Lepalvir (the final concentration was 1.63, 0.815, 0.326, 0.163 and 0.0815 U/ml), and the cell proliferation activity was detected using the CCK-8 method. The results were shown in Table 1. Lepalvir had a significant inhibitory effect on the proliferation of the above five types of tumor cells at a higher concentration, especially on HepG2, LM3 and H460 cells. When the concentration of Lepalvir reached 1.63 U/ml, the inhibition rate on HepG2, LM3, and H460 cells reached 58.95%, 55.08%, and 57.28%, respectively, and the inhibition rate on HeLa and A549 cells was slightly lower, being 48.18% and 45.80% respectively. When the concentration of Lepalvir was 0.8151.63 U/ml, compared with the inhibition rate of HeLa and A549 cells, the difference in inhibition rate of HepG2 and LM3 cells was statistically significant (P<0.05).
TABLE-US-00001 TABLE 1 Inhibition rate of Lepalvir on the proliferation of various tumor cells Drug concentration Inhibition rate (%) (U/ml) HepG2 cells LM3 cells H460 cells A549 cells HeLa cells 0.0815 2.14 ± 0.30 6.77 ± 1.98 3.53 ± 1.98 4.04 ± 3.00 1.58 ± 0.72 0.163 3.88 ± 1.74 7.25 ± 1.54 3.59 ± 0.87 4.08 ± 1.89 2.02 ± 1.41 0.326 9.78 ± 1.24 9.28 ± 1.13 4.48 ± 2.43 6.04 ± 2.08 4.00 ± 2.32 0.815 22.44 ± 0.28*.sup.Δ 22.20 ± 1.71*.sup.Δ 15.82 ± 2.58 20.07 ± 1.02 14.29 ± 2.81 1.63 58.95 ± 2.56**.sup.## 55.08 ± 0.64**.sup.## 57.28 ± 0.42**.sup.## 45.80 ± 1.14 48.18 ± 2.10 Note: *P < 0.05, **P < 0.01, compared with Hela group; .sup.##P < 0.01, compared with A549 group; .sup.ΔP < 0.05, compared with H460 group;
[0069] 1.2 The Effect of Lepalvir on the Secretion of Cytokines by Mouse Splenic Lymphocytes
[0070] The prepared spleen lymphocyte suspension was obtained. The cell density was adjusted to 5×10.sup.6/ml. The cells were inoculated in a 96-well cell culture plate, 100 μl cell fluid per well, and then 50 μl ConA or LPS was added (the final concentration of ConA in the splenic lymphocyte suspension for determining IL-2, IFN-γ was 2 mg/L, the final concentration of ConA in the splenic lymphocyte suspension for determining IL-4 was 10 mg/L, and the final concentration of LPS in the splenic lymphocyte suspension for determining IL-12 was 10 mg/L). Then the pre-prepared 50 μl different concentrations of Lepalvir was added (the final concentration was 0.815, 0.163 and 0.0815 U/ml). ConA, LPS control groups were established. 3 multiple wells were included for each treatment, and the total volume of each well was 200 μl. After culturing in a 5% CO.sub.2, 37° C. constant temperature incubator for 24 hours, the cell suspension was centrifuged at 300×g at room temperature for 5 minutes. The supernatant of each group was then carefully aspirated for further use.
[0071] ELISA was used to detect the effects of different concentrations of Lepalvir (the final concentration was 0.815, 0.163 and 0.0815 U/ml) on the secretion of cytokines IL-2, IFN-γ, IL-4 and IL-12 from the collected splenic lymphocyte supernatant. The experimental results showed that the secretion level of the four cytokines increased after different concentrations of Lepalvir was given to splenic lymphocytes. Compared with the control group, Lepalvir 0.0815 U/ml had no significant effect on the secretion level of IL-2, while the IL-2 secretion level of Lepalvir 0.163 and 0.815 U/ml groups was significantly increased, with the effect of Lepalvir 0.815 U/ml group more pronounced (P<0.01,
[0072] 1.3 The Effect of Lepalvir Stimulated Lymphocyte Supernatant on the Proliferation of HepG2 Cells
[0073] The prepared spleen lymphocyte suspension was obtained. The cell density was adjusted to 5×10.sup.6/ml (containing ConA at a final concentration of 5 mg/L). The cells were inoculated in a 96-well cell culture plate, 100 μl cell fluid per well. One half of the wells was added with 100 μl Lepalvir (the final concentration of Lepalvir was 0.163 U/ml), and the other half was added with the same volume of RPMI-1640 culture medium containing 10% FBS, with a total volume of 200 μl per well. After culturing in a 5% CO.sub.2, 37° C. constant temperature incubator for 24 hours, the cell suspension was centrifuged at 300×g at room temperature for 5 minutes. The supernatant was then carefully aspirated for further use.
[0074] The HepG2 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a cell suspension. The cell density was adjusted to 5×10.sup.4/ml. The cells were seeded in a 96-well cell culture plate with a volume of 100 μl per well, and then cultured in a 5% CO.sub.2, 37° C. constant temperature incubator overnight. The supernatant was discarded, and the supernatant of splenic lymphocytes stimulated by Lepalvir was added. According to the experimental design, the following groups were included: experimental group, lymphocyte supernatant treatment group, Lepalvir single-use group, and blank control group. The experimental group was added with different proportions of lymphocyte supernatant which Lepalvir will act on, and the supernatant ratio by volume was 25%, 50%, 75%, 100% respectively. For the lymphocyte supernatant group without the effect of Lepalvir, the balance was DMEM medium containing 10% FBS. For the blank control group, the same volume of DMEM culture medium containing 10% FBS was added. For the Lepalvir single-use group, the final concentration of the drug was 0.163 U/ml. For each treatment, 3 replicate wells were set up, each with a volume of 100 μl. After culturing in a 5% CO.sub.2, 37° C. constant temperature incubator for 24 hours, the cells were determined for proliferation activity.
[0075] For the cells treated above to be tested, 10 μl of CCK-8 reagent was added to each well. After further incubation for 1.5 hour, the absorbance (OD) for each well was measured at 450 nm in an enzyme-linked immunoassay, and the cell proliferation inhibition rate for each group was calculated.
[0076] The lymphocyte supernatant contains a variety of immunologically active factors. Based on the above-mentioned ELISA test results, the splenic lymphocyte supernatant stimulated using 0.163 U/ml Lepalvir had an effect on the HepG2 cells. The results were shown in
[0077] 1.4 Conclusion
[0078] A. Lepalvir inhibited the proliferation of 5 human derived tumor cells HepG2, LM3, H460, A549 and HeLa in vitro. It was demonstrated that Lepalvir can inhibit the growth of tumor cells, and its inhibitory effect on human liver cancer cells HepG2 and LM3 was even more pronounced.
[0079] B. Lepalvir can up-regulate the secretion of IL-2, IFN-γ, IL-4, IL-12 and other cytokines in splenic lymphocytes cultured in vitro in mouse, indicating that Lepalvir can activate lymphocytes, promote the proliferation and differentiation of Th1 and Th2 cells, and enhance the body's cellular immune function.
[0080] C. The inhibitory effect of splenic lymphocyte supernatant stimulated by Lepalvir on HepG2 cells was more pronounced than that of Lepalvir alone at the same concentration, further demonstrating that Lepalvir can upregulate cellular immune function by activating lymphocytes and secreting a variety of cytokines, thereby enhancing self-inhibition on tumor cell growth.
Example 2—Pharmacodynamic Study of Lepalvir on Human Liver Cancer-Bearing Nude Mice
[0081] 2.1 Preparation of Human Liver Cancer LM3 Cells
[0082] The LM3 cells in the logarithmic growth phase were removed from the 5% CO.sub.2, 37° C. constant temperature incubator. The original culture medium was aspirated off, and an appropriate amount of PBS was added for washing to remove residual serum, which was then aspirated off. 1 ml of 0.25% trypsin was added for digestion, and then the cells were placed under a microscope. When the cell shape gradually became round, an appropriate amount of DMEM medium containing 10% FBS was added to stop the digestion. The cells were repeatedly pipetted until complete suspension. The cells were then collected in a 15 ml centrifuge tube, centrifuged at 1500 rpm at room temperature for 8 min. The supernatant was discarded, and an appropriate amount of 0.9% sodium chloride injection was added. The cells were gently pipetted until evenly dispersed. The cells were counted, and placed in an ice box for later use. 2.2 Inoculation of subcutaneously transplanted human liver cancer LM3 tumor The prepared LM3 cells were adjusted to a cell concentration of 1×10.sup.7/ml, and inoculated subcutaneously in the axilla of the right forelimb of eight BALB/C nude mice, each injected with 0.2 ml of cell suspension. The injection site was wiped and disinfected with 75% alcohol. The needle was inserted about 1 cm subcutaneously, and the inoculation site can be seen to bulge rapidly. After inoculation, the needle hole was pressed with a cotton swab for a while, and the nude mouse was put back into the breeding box. When the volume of the subcutaneous tumor of the nude mouse grew to 800-1000 mm.sup.3, the mouse was sacrificed by cervical dislocation. After wiping with 75% alcohol, the mouse was moved into an ultra-clean table to remove the tumor tissue aseptically, which was cleaned with 0.9% sodium chloride injection, and then cut with scissors into a small piece of about 1 mm.sup.3. The piece was subcutaneously inoculated into 50 BALB/c nude mice under the axilla of the right forelimb using a trocar. The injection site was wiped and disinfected with 75% alcohol. The trocar was subcutaneously inserted into about 1 cm deep, and the inoculation site can be seen to bulge rapidly. After inoculation, press the needle hole was pressed with a cotton swab for a while, and the nude mouse was put back into the breeding box. The mental state, diet, activity, and defecation of the nude mouse were observed every day and the tumor growth was recorded.
[0083] 2.3 Dosing Groups for Animals
[0084] The subcutaneously transplanted human liver cancer LM3 tumor model for fifty BALB/c nude mice was established, and the tumor growth was observed and recorded every day. When the tumor volume grew to 100-300 mm.sup.3, the mice were randomly divided and dosed according to the tumor volume. Fifty nude mice were randomly divided into 5 groups, namely blank control group (10 mice), positive control group (10 mice), and low, medium and high-dose Lepalvir group (10 mice in each group). The blank control group was given 0.9% sodium chloride injection, i.p. 0.2 ml/d; the positive control group was given cyclophosphamide, i.p. 20 mg/kg, once every other day; the low-dose Lepalvir group, i.p. 0.01 u/g/d; the medium dose Lepalvir group, i.p. 0.02 u/g/d; the high-dose Lepalvir group, i.p. 0.04 u/g/d. The administration was continued for 15 days. From the first day of administration, the mental state, diet, activity, and defecation of the nude mice were observed every day. The body weight and tumor growth of the nude mice were recorded every three days. The experiment was ended on the 16.sup.th day, and the nude mice of each group were weighed. Then, the mouse was sacrificed by cervical dislocation. The tumor tissue and spleen tissue were completely stripped, weighed and measured, and the tumor inhibition rate and spleen index were calculated.
[0085] 2.4 Determination of Tumor Inhibition Rate and Spleen Index
[0086] At the end of the experiment on the 16.sup.th day, the nude mice of each group were sacrificed by cervical dislocation, and the subcutaneous tumor tissue was completely stripped out for weighing and measurement. The long diameter (a) and short diameter (b) of the tumor were measured using a vernier caliper. The tumor volume was calculated as follows: tumor volume=a×b×b/2. Tumor inhibition rate (%)=[(average tumor weight of the control group-average tumor weight of the administration group)/average tumor weight of the control group]×100%.
[0087] At the end of the experiment on the 16.sup.th day, the nude mice were weighed. After the nude mice of each group were sacrificed by cervical dislocation, the spleen tissue was completely stripped and weighed. The formula for calculating the spleen index was as follows: spleen index=spleen weight (mg)/mouse weight (g)×10.
[0088] 2.5 The Effect of Lepalvir on the Body Weight of Nude Mice with Human Liver Cancer
[0089] For BALB/C nude mice inoculated with LM3 transplanted tumor, the weight of the blank control group was 19.81±0.49 g before the start of the experiment, and the weight at the end of the experiment was 21.48±0.76 g. The body weight profile of the nude mice for the low, medium or high-dose Lepalvir group was substantially the same as the blank control group, and there was no statistical difference between the groups (P>0.05). However, the weight of the nude mice in the cyclophosphamide (CTX) group decreased significantly, and the weight was 18.91±0.46 g at the end of the experiment. Compared with the control group, the difference was statistically significant (P<0.05) (
[0090] 2.6 the Inhibitory Effect of Lepalvir on Subcutaneously Transplanted Tumor of the Nude Mice with Human Liver Cancer
[0091] Human liver cancer cells LM3 were subcutaneously inoculated into the BALB/C nude mice. When the tumor volume grew to 100-300 mm.sup.3 after inoculation, the mice were randomly divided into five groups and dosed according to the tumor volume, namely blank control group, positive control group, and low, medium and high-dose Lepalvir group (10 mice in each group). The long diameter (a) and short diameter (b) of the tumor were measured using a vernier caliper. The tumor volume was calculated and the tumor growth curve was prepared. Judged from the tumor volume growth of each group, the positive control group and the low, medium or high-dose Lepalvir group already had statistical differences compared with the blank control group on the six.sup.th day of administration (P<0.05) (
TABLE-US-00002 TABLE 2 Effect of Lepalvir on tumor weight and tumor inhibition rate of nude mice transplanted with LM3 tumor Tumor Tumor weight inhibition rate Group Number Dose (g) (%) Blank control group 6 — 1.01 ± 0.24 — Positive control group 6 20 mg/kg 0.21 ± 0.10** 79.33 ± 0.10 Low-dose group 6 0.01 U/g 0.75 ± 0.13* 25.24 ± 0.13 Medium dose group 6 0.02 U/g 0.66 ± 0.08** 34.71 ± 0.08 High dose group 6 0.04 U/g 0.50 ± 0.07** 49.86 ± 0.07 Note: *P < 0.05, **P < 0.01, compared with the blank control group;
[0092] 2.7 Effects of Lepalvir on the Immune Organs of Nude Mice with Human Liver Cancer
[0093] At the end of the experiment on the 16.sup.th day, after the nude mice were sacrificed by cervical dislocation, the spleen tissue was completely stripped to remove the excess adhesive tissues, first rinsed with 0.9% sodium chloride injection, and then dried with absorbent paper and weighed. The spleen index was calculated. The results showed (Table 3), the spleen index of the blank control group was 0.84±0.12, the spleen index of the positive control group was 0.70±0.10, the spleen index of the low-dose (0.01 U/g), medium-dose (0.02 U/g) and high-dose (0.04 U/g) group were 1.15±0.05, 1.23±0.07 and 1.17±0.02, respectively. Compared with the blank control group, there were statistical differences in each treatment group (P<0.01).
TABLE-US-00003 TABLE 3 Effects of Lepalvir on the immune organs of nude mice transplanted with LM3 tumor Spleen quality Spleen index Group Number Dose (g) (mg/g) Blank control group 6 — 0.15 ± 0.03 0.94 ± 0.12 Positive control group 6 20 mg/kg 0.12 ± 0.02** 0.70 ± 0.10** Low-dose group 6 0.01 U/g 0.21 ± 0.02** 1.15 ± 0.05** Medium dose group 6 0.02 U/g 0.23 ± 0.02** 1.22 ± 0.07** High dose group 6 0.04 U/g 0.22 ± 0.01** 1.17 ± 0.02** Note: **P < 0.01, compared with the negative control group;
[0094] 2.8 Conclusion
[0095] A. The BALB/C nude mouse model subcutaneously transplanted with LM3 tumor was successfully established, which has laid the foundation for the in vivo pharmacodynamic study of Lepalvir.
[0096] B. Lepalvir had no significant effect on the weight gain of BALB/C nude mouse subcutaneously transplanted with LM3 tumor, showing its high safety characteristics.
[0097] C. Lepalvir had a significant inhibitory effect on the growth of BALB/C nude mouse subcutaneously transplanted with LM3 tumor, and had some correlation with the dose, indicating its practical value in cancer treatment.
[0098] D. Lepalvir can increase the weight of the immune organ (spleen) of BALB/C nude mouse subcutaneously transplanted with LM3 tumor, and can increase the spleen index of nude mouse, suggesting that Lepalvir can improve the immune function of the body.
Example 3—Pharmacodynamic Study of Lepalvir Combined with Cyclophosphamide on Human Liver Cancer-Bearing Nude Mice
[0099] See Example 2 for the preparation of human liver cancer LM3 cells and the establishment of BALB/C nude mouse model subcutaneously transplanted with LM3 tumor.
[0100] 3.1 Dosing Groups for Animals
[0101] The subcutaneously transplanted human liver cancer LM3 tumor model for fifty BALB/c nude mice was established, and the tumor growth was observed and recorded every day. When the tumor volume grew to 100-300 mm.sup.3, the mice were randomly divided and dosed according to the tumor volume. Fifty nude mice were randomly divided into 5 groups, namely blank control group (10 mice), Lepalvir group (10 mice), low-dose cyclophosphamide group (10 mice), and high-dose cyclophosphamide group (10 mice), Lepalvir combined with cyclophosphamide administration group (10 mice). The control group was given 0.9% sodium chloride injection, i.p. 0.2 ml/d; Lepalvir group, i.p. 0.02 U/g/d; cyclophosphamide low-dose group, i.p. 10 mg/kg, given once every two days; cyclophosphamide high-dose group, i.p. 20 mg/kg, given once every two days; Lepalvir and cyclophosphamide combined administration group, i.p. 0.02 U/g+10 mg/kg, Lepalvir was given once a day, cyclophosphamide was given once every two days. The administration was continued for 15 days. From the first day of administration, the mental state, diet, activity, and defecation of the nude mice were observed every day. The body weight and tumor growth of the nude mice were recorded every three days. The experiment was ended on the 16.sup.th day, and the nude mice of each group were weighed. Then, the mouse was sacrificed by cervical dislocation. The tumor tissue and spleen tissue were completely stripped, weighed and measured, and the tumor inhibition rate and spleen index were calculated.
[0102] 3.2 Determination of Tumor Inhibition Rate and Spleen Index
[0103] At the end of the experiment on the 16.sup.th day, the nude mice of each group were sacrificed by cervical dislocation, and the subcutaneous tumor tissue was completely stripped out for weighing and measurement. The long diameter (a) and short diameter (b) of the tumor were measured using a vernier caliper. The tumor volume was calculated as follows: tumor volume=a×b×b/2. Tumor inhibition rate (%)=[(average tumor weight of the control group-average tumor weight of the administration group)/average tumor weight of the control group]×100%.
[0104] At the end of the experiment on the 16.sup.th day, the nude mice were weighed. After the nude mice of each group were sacrificed by cervical dislocation, the spleen tissue was completely stripped and weighed. The formula for calculating the spleen index was as follows: spleen index=spleen weight (mg)/mouse weight (g)×10.
[0105] 3.3 The Effect of Lepalvir on the Body Weight of Nude Mice with Human Liver Cancer
[0106] For BALB/C nude mice inoculated with LM3 transplanted tumor, the weight of the control group was 18.13±0.54 g before the start of the experiment, and the weight at the end of the experiment was 19.83±0.51 g. The body weight profile of the nude mice for the Lepalvir group and Lepalvir combined with cyclophosphamide group was substantially the same as the control group, and there was no statistical difference between the groups (P>0.05). At the end of the experiment, the body weight of nude mice in the cyclophosphamide low and high dose groups were 18.58±0.41 g and 17.54±0.28 g, respectively. Compared with the control group, the difference was statistically significant (P<0.05) (
[0107] 3.4 The Inhibitory Effect of Lepalvir on Subcutaneously Transplanted Tumor of the Nude Mice with Human Liver Cancer
[0108] Human liver cancer cells LM3 were subcutaneously inoculated into the BALB/C nude mice. When the tumor volume grew to 100-300 mm.sup.3 after inoculation, the mice were randomly divided into five groups and dosed according to the tumor volume, namely control group, Lepalvir group, cyclophosphamide low-dose group, cyclophosphamide high-dose group, and Lepalvir combined with cyclophosphamide group. The long diameter (a) and short diameter (b) of the tumor were measured using a vernier caliper. The tumor volume was calculated and the tumor growth curve was prepared. Judged from the tumor volume growth of each group, Lepalvir group, cyclophosphamide low-dose, high-dose group, and Lepalvir combined with cyclophosphamide group already had statistical differences on the ninth day of administration, compared with the control group (P<0.05) (
TABLE-US-00004 TABLE 4 Effect of Lepalvir combined with cyclophosphamide on tumor weight and tumor inhibition rate of nude mice transplanted with LM3 tumor Tumor Tumor weight inhibition rate Group Number Dose (g) (%) Control group 6 — 0.75 ± 0.04 — Lepalvir group 6 0.02 U/g 0.58 ± 0.06** 22.25 ± 4.48 CTX low-dose group 6 10 mg/kg 0.50 ± 0.02** 33.21 ± 1.41 CTX high-dose group 6 20 mg/kg 0.28 ± 0.06** 62.67 ± 5.81 Lepalvir + CTX 6 0.02 U/g + .sup. 0.29 ± 0.05**.sup.##ΔΔ 60.95 ± 4.36 10 mg/kg Note: **P < 0.01, compared with the control group; .sup.##P < 0.01, compared with the Lepalvirue group; .sup.ΔΔP < 0.01, compared with the cyclophosphamide low-dose group (10 mg/kg);
[0109] 3.5 Effects of Lepalvir on the Immune Organs of Nude Mice with Human Liver Cancer
[0110] At the end of the experiment on the 16.sup.th day, after the nude mice were sacrificed by cervical dislocation, the spleen tissue was completely stripped to remove the excess adhesive tissues, first rinsed with 0.9% sodium chloride injection, and then dried with absorbent paper and weighed. The spleen index was calculated. The results showed (Table 5), compared with the spleen index of the control group, there were statistical differences in the Lepalvir group (0.02 U/g), the cyclophosphamide low (10 mg/kg) and high dose group (20 mg/kg). There was no significant difference in the Lepalvir combined with cyclophosphamide group (0.02 U/g+10 mg/kg) (P>0.05). Compared with the spleen index of the combined administration group, there were statistical differences in the Lepalvir group, the cyclophosphamide low (10 mg/kg) and high dose group (P<0.05).
TABLE-US-00005 TABLE 5 Effects of Lepalvir combined with cyclophosphamide on the immune organs of nude mice subcutaneously transplanted with LM3 tumor Spleen quality Spleen index Group Number Dose (g) (mg/g) Control group 6 — 0.21 ± 0.02 1.16 ± 0.16 Lepalvir group 6 0.02 U/g 0.24 ± 0.01**.sup.## 1.22 ± 0.13**.sup.## CTX low-dose group 6 10 mg/kg 0.19 ± 0.01*.sup.# 1.08 ± 0.12*.sup.# CTX high-dose group 6 20 mg/kg 0.17 ± 0.01**.sup.## 1.01 ± 0.08**.sup.## Lepalvir + CTX 6 0.02 U/g + 0.21 ± 0.01 1.14 ± 0.09 10 mg/kg Note: *P < 0.05, **P < 0.01, compared with the blank control group; .sup.#P < 0.05, .sup.##P < 0.01, compared with the Lepalvir combined with cyclophosphamide group;
[0111] 3.6 Conclusion
[0112] A. Lepalvir can reduce the toxic and side effects of cyclophosphamide on the body and improve the health of the body.
[0113] B. Lepalvir had an anti-tumor synergistic effect on cyclophosphamide, and Lepalvir can be used in combination with cyclophosphamide to reduce the amount of cyclophosphamide to achieve the same anti-tumor effect. This indicated the practical value of Lepalvir as an anti-cancer adjuvant.
[0114] C. Compared with the group administered with the same dose of cyclophosphamide alone, the combined administration of Lepalvir and cyclophosphamide can significantly increase the spleen index and reduce the inhibitory effect of cyclophosphamide on the spleen. Lepalvir can improve the body's immune function and play a role in reducing toxicity and increasing efficacy of cyclophosphamide.