Patent Description:
Deoxycytidine kinase (DCK) is as an enzyme with broad substrate specificity, which can phosphorylate pyrimidine and purine deoxynucleosides, and is a key enzyme in the remedial pathway of deoxynucleotide biosynthesis. It is capable of maintaining normal DNA metabolism and phosphorylating a variety of antiviral and anticancer nucleoside analog drugs, which can only be activated after phosphorylation, thereby inhibiting tumor growth. In the past decades, apoptosis has been widely studied, and radiotherapy strategies targeting apoptosis have become one of the important means of tumor treatment.

Azvudine is a broad-spectrum RNA virus inhibitor. As a synthetic nucleoside analog of viral RNA-dependent RNA polymerase (RdRp), it is metabolized in cells into <NUM>'-triphosphate metabolite (azvudine triphosphate) with antiviral activity, which can specifically act on the novel coronavirus polymerase (RdRp). It targets virus RdRp, and can block the synthesis and replication of RNA chain by inhibiting the activity of RdRp in the host cell. In July <NUM>, azvudine tablet was approved for marketing in China for the treatment of HIV-<NUM> infected adult patients with high viral load. In July <NUM>, azvudine was approved for the treatment of novel coronavirus infection.

Patent document <CIT> discloses use of azvudine in the treatment of tumors, such as colon cancer, liver cancer, gastric cancer, esophageal cancer, lung cancer, breast cancer, cervical cancer, leukemia, and lymphoma. It has been found that azvudine has significant inhibitory effect on various human cancer cells and transplanted tumors in animals.

Among the traditional tumor treatment methods, chemotherapy has become the most widely used clinical tumor treatment method due to its strong therapeutic effect and high drug efficacy. However, most of the chemotherapeutic drugs currently used clinically have poor selectivity, which may cause great damage to normal tissues of the human body while treating tumor tissues, thereby leading to serious toxic and side effects.

Single drug therapy has defects such as poor physical and chemical properties, low bioavailability, and poor therapeutic effect. If chemotherapeutic drugs can be combined with azvudine, the anti-tumor effect can be improved. Based on the complementary synergy between the anti-tumor mechanisms of the two drugs, synergistic enhancement of anti-tumor efficacy with chemotherapy and immunity can be achieved by double-targeting tumor cells.

The present disclosure provides a pharmaceutical composition comprising azvudine (FNC) and avastin® (bevacizumab); or a chemotherapeutic agent selected from the group consisting of cyclophosphamide, paclitaxel and a combination thereof. The present disclosure also provides said pharmaceutical composition for use in preventing or treating a tumor disease.

Compared with each single drug, the pharmaceutical composition of the present invention has the following advantages:.

In order to solve the technical problem of the present invention, the present invention provides a pharmaceutical composition, comprising:.

In the present invention, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, paclitaxel and a combination thereof.

In addition, the present invention further provides another pharmaceutical composition, comprising:.

In a preferred embodiment of the present invention, the (i) and (ii) are administered simultaneously, separately, or sequentially, or the (i) and (ii) exist in the same dosage form.

In a preferred embodiment of the present invention, the pharmaceutical composition is used for treating a tumor-related disease selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, melanoma, brain tumor, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, lung cancer, kidney cancer, skin cancer, glioblastoma, neuroblastoma, sarcoma, liposarcoma, osteochondroma, bone tumor, osteosarcoma, seminoma, testicular tumor, uterine cancer, head and neck tumor, multiple myeloma, malignant lymphoma, polycythemia vera, leukemia, thyroid tumor, ureter tumor, bladder tumor, gallbladder cancer, non-small cell lung cancer, cholangiocarcinoma and choriocarcinoma.

In some embodiments, the azvudine is administered at an amount selected from <NUM>-<NUM>, and the chemotherapeutic agent is administered at an amount selected from <NUM>-<NUM>.

In some embodiments, the azvudine is administered at an amount selected from <NUM>-<NUM>, and the avastin is administered at an amount selected from <NUM>-<NUM>.

In the present disclosure, the azvudine is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

In the present disclosure, the chemotherapeutic agent is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> , <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

In some embodiments, the azvudine is administered at an amount selected from <NUM>-<NUM>, and at a frequency of once a day, twice a day or three times a day, and the chemotherapeutic agent is administered at an amount selected from <NUM>-<NUM>, and at a frequency of once a day, twice a day or three times a day.

In some embodiments, the azvudine is administered at an amount selected from <NUM>-<NUM>, and at a frequency of once a day or twice a day, and the chemotherapeutic agent is administered at an amount selected from <NUM>-<NUM>, and at a frequency of once a day.

In some embodiments, the chemotherapeutic agent is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day or twice a day. The chemotherapeutic agent is administered at an amount selected from <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day.

In some embodiments, the azvudine is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day or twice a day. The azvudine is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day.

In some embodiments, the chemotherapeutic agent is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day or twice a day. The chemotherapeutic agent is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day.

In some embodiments, the chemotherapeutic agent is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day or twice a day. The chemotherapeutic agent is administered at an amount selected from the group consisting of <NUM>, <NUM>, <NUM> and <NUM>, and at a frequency of once a day.

Route of the combined administration in the present invention is selected from the group consisting of oral administration, parenteral administration and transdermal administration, wherein the parenteral administration includes but is not limited to intravenous injection, subcutaneous injection and intramuscular injection, preferably oral administration.

The present invention further provides a pharmaceutical composition comprising the above azvudine and chemotherapeutic agent and one or more pharmaceutically acceptable carriers, excipients and diluents. The pharmaceutical composition can be made into any pharmaceutically acceptable dosage form. For example, it can be formulated into a tablet, a capsule, a pill, a granule, a solution, a suspension, a syrup, an injection (including an injection solution, a sterile powder for injection and a concentrated solution for injection), a suppository, an inhalant or a spray. The pharmaceutical composition can also be made into the same dosage form, for example, the azvudine and chemotherapeutic agent can be formulated into a composite tablet, a composite capsule, a composite pill, a composite granule, a composite solution, a composite suspension, a composite syrup, a composite injection (including an injection solution, a sterile powder for injection and a concentrated solution for injection), a composite suppository, a composite inhalant or a composite spray.

The present invention further provides a pharmaceutical kit for use in a medicament for treating a tumor disease, which comprises the pharmaceutical composition of azvudine and chemotherapeutic agent described in the present disclosure.

In the present invention, azvudine is administered in combination with a chemotherapeutic agent, thereby enhancing the effect of drugs for treating a tumor disease.

The "combination" described in the present invention is a mode of administration that refers to the administration of at least one dose of azvudine and at least one dose of chemotherapeutic agent within a certain period of time, wherein both substances show pharmacological effects. The period of time can within one administration cycle, preferably within <NUM> weeks, within <NUM> weeks, within <NUM> weeks, within <NUM> week, or within <NUM> hours, more preferably within <NUM> hours. The azvudine and chemotherapeutic agent can be administered simultaneously or sequentially. Such a treatment that azvudine and a chemotherapeutic agent are administered by the same route of administration or by different routes of administration is included in this period of time.

The following will describe the present disclosure in more detail in conjunction with examples, and the examples of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the scope of the present disclosure.

Experimental animals were <NUM> (<NUM> plus <NUM> spare mice) BALB/c female nude mice, <NUM>-<NUM> weeks old (the age of mice at the time of tumor cell inoculation) and weighing <NUM>-<NUM>, which were purchased from Beijing VitalRiver Experimental Animal Technology Co. Breeding environment was SPF grade. The experimental animals were all kept in an independent ventilated box with constant temperature and humidity. The temperature of the breeding room was <NUM>-<NUM>, the humidity was <NUM>-<NUM>%. The breeding room was ventilated <NUM>-<NUM> times per hour. The alternating time of day and night was <NUM>/<NUM>. The mice were continuously supplied with cobalt <NUM> radiation sterilized mouse complete pellet feed, and had free access to the feed. The mice drank tap water (available after sterilization with high-pressure steam), which was continuously supplied using a water-drinking bottle, and had free access to water. The mouse box was a polysulfone mouse box, which was used after sterilization under high pressure, with a size of <NUM> × <NUM> × <NUM>. Example <NUM> Test results and discussion of the anti-tumor effect of azvudine in combination with capecitabine on the Colo <NUM> human colon cancer model (reference example).

Colo <NUM> cells were cultured in RPMI1640 medium containing <NUM>% fetal bovine serum. Colo <NUM> cells in the exponential growth phase were collected, resuspended in PBS to an appropriate concentration and mixed with Matrigel at a ratio of <NUM>:<NUM>. The resulting mixture was used for subcutaneous inoculation of tumor in mice. <NUM>×<NUM><NUM> Colo <NUM> cells were subcutaneously inoculated on the right side of female mice. The day of inoculation was defined as Day <NUM>. When the average tumor volume reached <NUM><NUM>, the mice were randomly divided into groups according to tumor size.

Relative tumor proliferation rate, T/C %, refers to the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point, which was calculated as follows:.

Efficacy evaluation criteria <MAT> (TRTV: the average RTV of the treatment group; CRTV: the average RTV of the vehicle control group; RTV=Vt/V<NUM>, where V<NUM> is the tumor volume of the animal when grouped, and Vt is the tumor volume of the animal after treatment);
or <MAT> (TTW: the average tumor weight of the treatment group at the end of the experiment; CTW: the average tumor weight of the vehicle control group at the end of the experiment).

Relative tumor inhibition rate, TGI (%), was calculated according to the formula as follows: TGI% = (<NUM>-T/C) × <NUM>%. (T and C are the relative tumor volumes (RTV) or tumor weights (TW) of the treatment group and the control group at a specific time point).

On the <NUM>th day after tumor inoculation (on the <NUM>th day after grouping), the mice in the vehicle group had an average tumor volume of <NUM><NUM>, and the single drug azvudine <NUM>/kg treatment group had an average tumor volume of <NUM><NUM>, which had no statistically significant difference compared with the control group (p=<NUM>), with a relative tumor inhibition rate TGI (%) of <NUM>%. The capecitabine <NUM>/kg treatment group had an average tumor volume of <NUM><NUM>, which had statistically significant difference compared with the control group (p<<NUM>), with a relative tumor inhibition rate TGI (%) of <NUM>%. The combination group of azvudine <NUM>/kg and capecitabine <NUM>/kg had an average tumor volume of <NUM><NUM>, which had statistically significant difference compared with the control group (p<<NUM>), with a relative tumor inhibition rate TGI (%) of <NUM>%.

Human colon cancer cells LoVo (Cat. No.: ECACC-<NUM>) were cultured in vitro as a monolayer in F12K medium containing <NUM>% fetal bovine serum, <NUM> U/mL penicillin and <NUM>µg/mL streptomycin, in a <NUM> and <NUM>% CO<NUM> incubator. The cells were routinely digested with trypsin-EDTA and passaged twice a week. When the cell saturation reached <NUM>%-<NUM>% and the cell number achieved the requirement, the cells were collected, counted and inoculated.

<NUM> (<NUM>×<NUM><NUM>) of LoVo cells were subcutaneously inoculated on the right back of each mouse. The grouping administration was started when the average tumor volume reached about <NUM><NUM>.

The tumor volume changes and the tumor weight changes in each group (on the <NUM>st day of administration) after administration of test drugs for treating BALB/c nude mice with Lovo cell subcutaneous xenograft tumor are shown in Table <NUM> and Table <NUM> respectively.

In this experiment, the in vivo efficacy of the test drugs in the LoVo xenograft tumor model was evaluated. The tumor volume and tumor weight of each group on the <NUM>st day of administration are shown in Table <NUM>, Table <NUM> and Table <NUM>, respectively. On the <NUM>th day after the start of administration, the tumor volume of the tumor-bearing mice in the blank control group reached <NUM><NUM>. Compared with the blank control group, the test drug azvudine (<NUM>/kg) group showed a small tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T /C of <NUM>%, TGI of <NUM>%, and p-value of <NUM>. Compared with the blank control group, the test drug capecitabine (<NUM>/kg) group showed a small tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/C of <NUM>%, TGI of <NUM>%, and p value of <NUM>.

Compared with the blank control group, the test drugs combination group of azvudine + capecitabine (<NUM>+<NUM>/kg) showed a significant tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/C of <NUM>%, TGI of <NUM>% and p-value of <NUM>. The combined administration of azvudine + capecitabine can improve the antitumor effect of single drug capecitabine in the LoVo colorectal tumor, and can increase TGI from <NUM>% to <NUM>%.

Human Burkitt's lymphoma cells Daudi (Cat. No.: DSMZ-ACC129) were in vitro suspended and cultured in RPMI <NUM> medium containing <NUM>% fetal bovine serum, <NUM> glutamine, <NUM> U/mL penicillin and <NUM>µg/mL streptomycin, in a <NUM> and <NUM>% CO<NUM> incubator. When the cell saturation achieved <NUM>%-<NUM>% and the cell number reached the requirement, the cells were collected, counted and inoculated.

<NUM> (<NUM>×<NUM><NUM>) of Daudi cells (added with Matrigel at a volume ratio of <NUM>:<NUM>) were subcutaneously inoculated on the right back of each mouse. The grouping administration was started when the average tumor volume reached about <NUM><NUM>.

The changes of tumor volume and tumor weight in each group after administration of test drugs for treating CB <NUM> SCID mice with Daudi cell subcutaneous xenograft tumor are shown in Table <NUM> and Table <NUM> respectively.

In this experiment, the in vivo efficacy of the test drugs in the Daudi xenograft tumor model was evaluated. The tumor volumes of each group at different time points are shown in Table <NUM> and Table <NUM>. On the <NUM>th day after the start of administration, the tumor volume of the tumor-bearing mice in the blank control group reached <NUM>,<NUM><NUM>. Compared with the blank control group, the test drug azvudine (<NUM>/kg) group showed a significant tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/C of <NUM>%, TGI of <NUM>% and p-value of < <NUM>. Compared with the blank control group, the test drug cyclophosphamide (<NUM>/kg) showed a significant tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/C of <NUM>%, TGI of <NUM>% and p value of <<NUM>. Compared with the blank control group, the test drugs combination group of azvudine+cyclophosphamide (<NUM>+<NUM>/kg) showed a significant tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/C of <NUM>%, TGI of <NUM>%, and p-value of < <NUM>.

In this experiment, the test drugs azvudine (<NUM>/kg) in combination with cyclophosphamide can improve the tumor inhibitory effect of single drug CTX in the human Burkitt's lymphoma cell Daudi, and increase TGI from <NUM>% to <NUM>%.

Human acute lymphoblastic leukemia cells MOLT4 (Cat. No.: ECACC-<NUM>) were in vitro suspended and cultured in RPMI <NUM> medium containing <NUM>% fetal bovine serum, <NUM> U/mL penicillin and <NUM>µg/mL streptomycin, in a <NUM> and <NUM>% CO<NUM> incubator. When the cell saturation achieved <NUM>%-<NUM>% and the cell number reached the requirement, the cells were collected, counted and inoculated.

<NUM> (<NUM>×<NUM><NUM>) of MOLT4 cells (added with Matrigel at a volume ratio of <NUM>:<NUM>) were subcutaneously inoculated on the right back of each mouse. The grouping administration was started when the average tumor volume reached about <NUM><NUM>.

The changes of tumor volume and tumor weight in each group after administration of test drugs for treating SCID Beige mice with MOLT4 cell subcutaneous xenograft tumor are shown in Table <NUM> and Table <NUM>.

In this experiment, the in vivo efficacy of the test drugs in the MOLT4 xenograft tumor model was evaluated. The tumor volumes of each group at different time points are shown in Table <NUM> and Table <NUM>. On the <NUM>th day after the start of administration, the tumor volume of the tumor-bearing mice in the blank control group reached <NUM>,<NUM><NUM>. Compared with the blank control group, the test drug azvudine (<NUM>/kg) group showed a tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/ C of <NUM>, TGI of <NUM>%, and p-value of < <NUM>. Compared with the blank control group, cyclophosphamide CTX (<NUM>/kg) group showed a significant tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/C of <NUM>%, TGI of <NUM>%, and p value of <<NUM>. Compared with the blank control group, the test drugs combination group of azvudine+cyclophosphamide (<NUM>+<NUM>/kg) showed a significant tumor inhibitory effect, with a tumor volume of <NUM><NUM>, T/C of <NUM>%, TGI of <NUM>%, and p-value of <<NUM>. The analysis and statistical results of the tumor weight in the test drugs combination group were basically consistent with the tumor volume data.

In this experiment, the test drugs azvudine (<NUM>/kg), cyclophosphamide (<NUM>/kg) and azvudine+cyclophosphamide (<NUM>+<NUM>/kg) under test doses had significant inhibitory effect on the growth of MOLT4 xenograft tumor. In this experiment, the test substances azvudine (<NUM>/kg) in combination with cyclophosphamide can improve the tumor inhibitory effect of single drug cyclophosphamide in MOLT4 human acute lymphoblastic leukemia tumor, and increase TGI from <NUM>% to <NUM>%.

OVCAR-<NUM> cells were cultured in RPMI1640 medium containing <NUM>% fetal bovine serum. OVCAR-<NUM> cells in the exponential growth phase were collected, resuspended in PBS to an appropriate concentration and mixed with Matrigel at a ratio of <NUM>:<NUM>. The resulting mixture was used for subcutaneous inoculation of tumor in mice.

<NUM>×<NUM><NUM> OVCAR-<NUM> cells were inoculated subcutaneously on the right side of female mice. When the average tumor volume achieved <NUM><NUM>, the mice were randomly divided into groups according to tumor size.

The experimental protocols for animal experiments in this experiment were reviewed and approved by the CrownBio IACUC committee. During the experiment, the animal experiments were performed in accordance with the requirements of AAALAC. After tumor inoculation was completed, the impact of tumor growth and treatment on normal behavior of animals was routinely monitored, specifically the activity of experimental animals, food intake and water intake, body weight gain or loss (body weight was measured twice a week), eye, hair and other abnormalities. The clinical symptoms observed during the experiment were recorded in the original data. The tumor volume was calculated according to the following formula: tumor volume (mm<NUM>) = <NUM>/<NUM> × (a × b<NUM>) (where a represents long diameter and b represents short diameter).

Relative tumor proliferation rate, T/C %, refers to the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point, which was calculated as follows:
<MAT> (TRTV: the average RTV of the treatment group; CRTV: the average RTV of the vehicle control group; RTV=Vt/V<NUM>, where V<NUM> is the tumor volume of the animal when grouped, and Vt is the tumor volume of the animal after treatment);
or <MAT> (TTW: the average tumor weight of the treatment group at the end of the experiment; CTW: the average tumor weight of the vehicle control group at the end of the experiment).

To compare tumor volumes of different treatment groups on a given day, first the assumption of homogeneity of variance among all groups was tested using the Bartlett test. When the p-value of Bartlett's test was not less than <NUM>, whether all group means were equal was tested using one-way ANOVA. If the p-value of the one-way ANOVA was less than <NUM>, pairwise comparisons among all groups were conducted using Tukey's HSD test, or pairwise comparisons between each treatment group and control group were conducted using Dunnett's t-test. When the p-value of Bartlett's test was less than <NUM>, whether the medians of all groups were equal was tested using Kruskal Wallis test. If the p-value of the Kruskal Wallis test was less than <NUM>, pairwise comparisons among all groups or between each treatment group and control group were conducted using Conover test, and the corresponding p-value correction was performed according to the number of groups in the multiple test.

In addition, for the purpose of exploratory data analysis, pairwise comparisons among all groups at any time point were performed. Since this comparison was only directed to tumor volume data of two test groups at specific time points, no multiple test correction was required. First, the assumption of homogeneity of variance between two groups was verified using Bartlett test. When the p value of the Bartlett test was not less than <NUM>, whether the means of two groups were equal was tested using Welch's t test. When the p value of the Bartlett test was less than <NUM>, whether the medians of two groups were equal was tested using Mann Whitney U test.

All statistical analysis and graphing were performed in the R language environment (version <NUM>. Unless otherwise specified, all tests were two-tailed tests, and p value less than <NUM> was considered statistically significant.

On the <NUM>th day of administration, the tumor volume of the vehicle control group was <NUM><NUM>. The average tumor volumes of the test drug azvudine <NUM>/kg treatment group, the positive drug paclitaxel <NUM>/kg treatment group, and the positive drug avastin <NUM>/kg treatment group were <NUM><NUM>, <NUM><NUM>, and <NUM><NUM> respectively, which showed no statistically significant difference compared with the control group (p=<NUM>, <NUM>, and <NUM>), with relative tumor inhibition rates TGI (%) of <NUM>%, <NUM>%, and <NUM> %, respectively. The average tumor volume of the combination treatment group of test drugs azvudine <NUM>/kg and paclitaxel <NUM>/kg was <NUM><NUM>, which was statistically significantly different from the control group (p=<NUM>), with a relative tumor inhibition rate TGI (%) of <NUM>%. The combined administration of the test drugs azvudine and paclitaxel significantly improved the anti-tumor effect of single drug paclitaxel (with a TGI of <NUM>%). The average tumor volume of the combination treatment group of test drugs azvudine <NUM>/kg and avastin <NUM>/kg was <NUM><NUM>, which was statistically significantly different from that of the control group (p=<NUM>), with a relative tumor inhibition rate TGI (%) of <NUM>%. The combined administration of test drugs azvudine and avastin significantly improved the antitumor effect of single drug avastin (with a TGI of <NUM>%). The tumor growth of each treatment group and control group is shown in Table <NUM>.

Human melanoma cells A2058 (Cat. No.: CRL-<NUM>) were cultured in vitro as a monolayer in DMEM medium containing <NUM>% fetal bovine serum, <NUM> U/mL penicillin and <NUM>µg/mL streptomycin, in a <NUM> and <NUM>% CO<NUM> incubator. The cells were routinely digested with trypsin-EDTA and passaged twice a week. When the cell saturation reached <NUM>%-<NUM>% and the cell number achieved the requirement, the cells were collected, counted and inoculated.

<NUM> (<NUM>×<NUM><NUM>) of A2058 cells were added with Matrigel, and the mixture was subcutaneously inoculated on the right back of each mouse. The grouping administration was started when the average tumor volume reached about <NUM><NUM>.

Claim 1:
A pharmaceutical composition comprising:
(i) azvudine or a pharmaceutically acceptable salt, a stereoisomer or an isotopic derivative thereof;
(ii) a chemotherapeutic agent or avastin® (bevacizumab);
wherein the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, paclitaxel and a combination thereof.