TWI810426B - Use and evaluation method of pharmaceutical composition in preparation of medicine for treating cancer - Google Patents

Abstract

The disclosure of the present invention relates to an evaluation method for determining whether a cancer-bearing individual is suitable for using a pharmaceutical composition for cancer treatment. The evaluation is performed by comparing the expression levels of the target genes (CHEK1 and PIK3CA) with the basic multiples, and a population group (high expression of CHEK1 and high expression of PIK3CA) suitable for the use of the pharmaceutical composition is found among cancer-bearing individuals. Furthermore, it is suggested to use the pharmaceutical composition (cell checkpoint kinase (CHK1) inhibitor and phosphatidylinositol 3 kinase (PI3K) inhibitor) to inhibit the survival ability and tumor growth ability of cancer cells in the population to achieve the treatment of cancer The effect.

Description

Use and evaluation method of pharmaceutical composition in preparation of medicine for treating cancer

This disclosure is about the field of personalized precision medicine for treating cancer. The present invention relates to a pharmaceutical composition, and its use and evaluation method for preparing medicines for treating cancer. By means of the detection of target gene expression and the evaluation method of the basic multiple, find out the group of cancer patients who are suitable for using the pharmaceutical composition, so as to achieve the efficacy of cancer treatment.

Cancer is the abnormal growth and rapid division of body cells leading to tumor formation. Proliferating tumor cells invade normal cells and organs of the body, leading to individual death. Cancer is the number one cause of death in the world. In Taiwan, according to statistics from the Ministry of Health and Welfare, cancer has been the number one cause of death for 37 consecutive years since the Republic of China. According to the statistics of the number of cancer deaths in the country in 107, 48,784 people (29,624 males and 19,160 females) died of cancer, accounting for 28.2% of the total deaths, an increase of 1.6% compared to the previous year (an increase of 747 people).

Oral cancer is a globally prevalent cancer, with many cases reported all over the world and it is an increasingly serious cancer disease. In 2018, it is estimated that there will be about 360,000 new oral cancer patients worldwide, and about 177,000 patients will die from oral cancer. Compared with the statistics in 2012, it can be found that the number of individuals suffering from oral cancer is gradually increasing , showing a steady increase in the number of oral cancer patients over the past few years. In Taiwan, according to the statistics of the cause of death in 2017 by the Ministry of Health and Welfare, oral cancer is the fifth leading cause of death from cancer in Taiwan, and it ranks fourth in male oral cancer mortality. The median age of death from all cancers is 69 years old, and the median age of death from oral cancer is only 59 years old, ten years earlier than other cancers. According to statistics, up to 70% of patients suffering from oral cancer have the habit of smoking, drinking or chewing betel nuts. In 2010, the Taiwan government has included the oral cancer screening program in the national cancer The screening subsidy program provides high-risk groups (individuals over 30 years old with chewing betel nuts or smoking habits) with annual free oral examinations. It can be seen that oral cancer is becoming an increasingly serious public health issue.

The oral cavity can be divided into lips, mucous membranes (lining of the lips and cheeks), teeth, the floor of the mouth below the tongue, the front two-thirds of the tongue, the front part of the roof of the mouth (hard palate), the gums, and the small area behind the molars. area. Oral cancer is abnormal hyperplasia in the above-mentioned areas, which invades the surrounding normal tissues, and even metastasizes to other parts of the body, affecting normal functioning and endangering life. The main risk factors for oral squamous cell carcinoma include smoking, alcohol abuse, nonsmoking tobacco use, and betel nut chewing.

Moreover, oral cavity cancer can be divided into squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinoma, sarcoma, and malignant melanoma histologically, among which squamous cell carcinoma (oral cavity squamous cell carcinoma, OSCC) is the majority, It accounts for more than 90% of oral malignant tumors, followed by mucosal, gingiva, jaw, floor of the mouth and lip lesions.

Oral cancer is when the oral mucosa is stimulated by risk factors for a long time, which will cause cell mutation caused by gene regulation imbalance or mutation. The risk factors related to oral cancer include: smoking, drinking, chewing betel nut, ultraviolet radiation, tooth decay, improper dentures, poor oral hygiene, long-term malnutrition, and viral infection (Human Papillomavirus, HPV16), etc.

Therefore, clinically, it is often seen that the surface of the mucosa is mutated or inappropriately hyperplasia, forming leukoplakia, erythema, erythema, ulcers, verrucous epithelial hyperplasia, or oral mucosal fibrosis, all of which are possible precancerous lesions of the oral cavity. In the case of lesions, it can further evolve into oral cancer due to long-term inflammation and stimulation.

The symptoms of oral cancer are mainly oral ulcers, hard lumps, erythema, white spots or accompanied by cervical lymphadenopathy, inability to open the mouth, difficulty in chewing and swallowing, etc. Most of them have no pain in the early stage. When the tumor grows larger and local necrosis may cause bacterial infection and pain abnormal. In addition, oral cancer often metastasizes to nearby cervical lymph nodes - submandibular region, submental region, upper neck, middle neck, and posterior neck; distant metastasis - lung, liver, bone, subclavian lymph, etc. .

At present, the treatment methods for oral cancer include surgical resection or cervical lymphadenectomy, chemotherapy, and radiation therapy; generally, surgery is the main method, and chemotherapy and radiation therapy are supplemented. The cure rate of early stage I and II oral cancer is about 80-90%. However, the cure rate of advanced stage III and stage IV oral cancer is only about 30-50%. The symptoms of early cancer are not obvious. Statistics show that more than 60% of the patients are stage III or IV patients, because most patients ignore the patients with advanced oral cancer when they are discovered, and the recurrence and metastasis of advanced oral cancer cases also showed a high mortality rate. From the above, it can be understood that the prevention, diagnosis and treatment of oral cancer have unmet medical needs.

At present, there is only one targeted drug for the treatment of oral cancer patients. Erbitux (ERBITUX) can be used. This drug can inhibit the signal transmission of the epidermal growth factor receptor of tumor cells to slow down the proliferation of tumor cells. The US Food and Drug Administration Both the Bureau of Health and Welfare of Taiwan suggest that patients with metastatic and recurrent oral cancer can be treated with Erbitux and chemotherapy drugs. With the chemotherapy drug group, the co-drug use group only extended survival by 3 months. In addition, chemotherapy drugs can cause discomfort and powerful side effects to patients.

Oral cancer patients have unique and different gene regulation imbalances, and different gene regulation imbalances correspond to different drug compositions and chemotherapy for cancer. In view of this, it is a problem that those skilled in the art want to solve by using the genetic detection and basic multiple evaluation methods related to oral cancer to find the corresponding drug treatment to achieve personalized, accurate and effective treatment, avoid ineffective medical treatment and improve survival rate. question.

The main purpose of the present invention is to provide a pharmaceutical composition and its use and evaluation method in the preparation of cancer treatment drugs. Firstly, the group suitable for using the pharmaceutical composition (high expression of CHEK1 and high expression of PIK3CA) among cancer patients is found out by the evaluation method of the group suitable for the pharmaceutical composition.

Furthermore, another object of the present invention is to use the pharmaceutical composition to inhibit the survival ability and tumor growth ability of oral squamous cell carcinoma cells in this group (CHEK1 high expression and PIK3CA high expression).

In order to achieve the above-mentioned purpose, the present invention discloses a pharmaceutical composition used as a medicine for the preparation of oral cancer, wherein the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, the pharmaceutical composition The substance is used to inhibit a cancer cell.

The present invention provides an embodiment, the content of which is the use of the pharmaceutical composition as a medicine for treating cancer, wherein the cancer cell is oral squamous cell carcinoma.

The present invention provides an embodiment, which relates to the use of a pharmaceutical composition as a drug for treating cancer, wherein the pharmaceutical composition inhibits the survival ability of the oral squamous cell carcinoma cells.

The present invention provides an embodiment, which is the use of a pharmaceutical composition as a drug for treating cancer, wherein the pharmaceutical composition inhibits the tumor growth ability of the oral squamous cell carcinoma cells.

Moreover, the present invention also discloses a pharmaceutical composition, which comprises: a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor.

Furthermore, the present invention discloses an evaluation method using a pharmaceutical composition, the steps of which include: using a real-time quantitative reverse transcription polymerase chain reaction to detect a CHEK1 gene and a PIK3CA gene in a tumor tissue sample to obtain a first CHEK1 gene expression level, a first PIK3CA gene expression level and detecting the CHEK1 gene and the PIK3CA gene in a normal tissue sample to obtain a second CHEK1 gene expression level and a second PIK3CA gene expression level; comparing the first CHEK1 gene expression amount and the second CHEK1 gene expression level and the first PIK3CA gene expression level and the second PIK3CA gene expression level to obtain a CHEK1 difference multiple and a PIK3CA difference multiple; when the CHEK1 difference multiple is higher than a CHEK1 basic multiple and the When the PIK3CA difference multiple is higher than a PIK3CA basic multiple, a comparison result is formed; and a pharmaceutical composition is used according to the comparison result, and the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a phosphatidylinositol 3 Kinase inhibitor; wherein the basic ploidy of CHEK1 is 1.7, and the basic ploidy of PIK3CA is 2.2.

The present invention provides an embodiment, which is an evaluation method, wherein in the step of detecting a CHEK1 gene and a PIK3CA gene in a tumor tissue sample, the tumor tissue sample is a cancer tumor tissue.

In addition, the present invention discloses a method for evaluating a group of suitable pharmaceutical compositions, the steps of which include: (a) obtaining a tumor tissue sample and a normal tissue sample from an individual; (b) detecting the tumor tissue sample and the normal tissue sample respectively; The expression level of a CHEK1 gene and the expression level of a PIK3CA gene in the tissue sample; (c) calculate a CHEK1 gene difference multiple and a PIK3CA gene difference based on step (b) multiple; and (d) determine whether the individual is a group suitable for the pharmaceutical composition based on the multiple of the CHEK1 gene difference and the multiple of the PIK3CA gene in step (c); wherein, when the multiple of the CHEK1 gene difference is higher than 1.7 and the PIK3CA gene When the multiple of difference is higher than 2.2, the individual belongs to the group for which the pharmaceutical composition is applicable.

S1~S4: Step process

Figure 1: It is a flow chart of the steps of an embodiment of the present invention; Figure 2: It is a basic multiple figure of CHEK1 and PIK3CA in an embodiment of the present invention; Figure 3A: It is an embodiment of the present invention The concentration result graph of the cell checkpoint kinase inhibitor; Fig. 3B: it is the concentration result graph of the phosphatidylinositol 3-kinase inhibitor of one embodiment of the present invention; Fig. 4: it is one embodiment of the present invention Figure 5A: It is an oral cancer according to an embodiment of the present invention. Figure 5B: It is a graph of the control experiment results of the tumor growth ability of the oral cancer mouse model of an embodiment of the present invention; and Figure 5C: It is the result of the experiment of the present invention An experimental result diagram of the body weight of the oral cancer mouse model of an embodiment.

In order to enable your review committee members to have a further understanding and understanding of the characteristics of the present invention and the achieved effects, the examples and accompanying descriptions are provided as follows: In view of the fact that different oral cancer patients have different oral cancer gene characteristics Manifestations, corresponding drug treatment is needed, in order to achieve more precise treatment, improve survival rate and reduce the Side effects of chemotherapy drugs. Accordingly, the present invention proposes a pharmaceutical composition and its application and evaluation method in the preparation of drugs for treating oral cancer, so as to solve the problems caused by the prior art.

The following will further illustrate the features, structures and methods of the present invention: First, please refer to FIG. 1 , which is a flow chart of the steps of an embodiment of the present invention. As shown in the figure, an evaluation method using a pharmaceutical composition of the present invention, the steps include: S1: Use real-time quantitative reverse transcription polymerase chain reaction to detect the CHEK1 gene and PIK3CA gene in tumor tissue samples, and obtain the first CHEK1 gene expression level , the first PIK3CA gene expression level and the detection of CHEK1 gene and PIK3CA gene in normal tissue samples to obtain the second CHEK1 gene expression level and the second PIK3CA gene expression level; S2: compare the first CHEK1 gene expression level with the second CHEK1 gene expression level and the first PIK3CA gene expression level and the second PIK3CA gene expression level to obtain the CHEK1 difference multiple and PIK3CA difference multiple; S3: When the CHEK1 difference multiple is higher than the CHEK1 basic multiple and the PIK3CA differential multiple is higher than the PIK3CA basic multiple, a comparison is made Result; and S4: judging to use the pharmaceutical composition according to the comparison result, the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor.

Among them, the target gene used in the present invention is the CHEK1 gene. The CHEK1 gene transcribes the CHK1 protein, which regulates the cell cycle checkpoint signal. When errors occur in the replication of cellular nucleic acid substances, the CHK1 protein will be activated to cause cell cycle arrest, and Repair function. If the activity of CHK1 protein is inhibited in tumor cells, it can hinder the repair of tumor cells, and then achieve the effect of killing tumor cells.

Another target gene used in the present invention is PIK3CA. The PIK3CA gene transcribes the PI3K alpha protein, which regulates the phosphatidylinositol 3-kinase (PI3Ks) signaling pathway and participates in the regulation of various cell functions such as cell proliferation and apoptosis. The overactivation of this protein in tumor cells leads to the rapid proliferation of tumor cells all the time. Therefore, if the activity of PI3K alpha protein is inhibited in tumor cells, the growth of tumor cells can be hindered, and the effect of killing tumor cells can be achieved.

First, as shown in step S1, a real-time quantitative reverse transcription polymerase chain reaction is used to detect a CHEK1 gene and a PIK3CA gene in a tumor tissue sample to obtain a first CHEK1 gene expression level and a first PIK3CA gene expression level. Grind the tumor tissue sample (a cancer tumor tissue) that has been inspected and obtained according to medical routine and placed in the experimental device, use Trizol reagent to lyse the cells in the tumor tissue sample, and then add chloroform to make the substances in the cells The layers were separated, and the aqueous layer containing RNA was precipitated by isopropanol, and then redissolved in secondary water treated with diethylpyrocarbonate (DEPC), and the RNA in the cells was extracted. Finally, the CHEK1 gene and the PIK3CA gene in the tumor tissue sample are detected by means of the real-time quantitative reverse transcription polymerase chain reaction, and the first CHEK1 gene expression level and the first PIK3CA gene expression level are obtained.

And in the same manner as above, detect the CHEK1 gene in a normal tissue sample with the real-time quantitative reverse transcription polymerase chain reaction (usually the activation of Chk1 will lead to cell cycle arrest, and start cell cycle checkpoints and DNA repair, to prevent the death of damaged cells (including cancer cells) and the PIK3CA gene (usually the activation of PI3K is often related to tumorigenesis), and obtain a second CHEK1 gene expression level and a second PIK3CA gene expression level.

Next, as shown in step S2, compare the first CHEK1 gene expression level (obtained from the tumor tissue sample) with the second CHEK1 gene expression level (obtained from the normal tissue sample), and obtain a CHEK1 difference multiple ( Divide the expression level of the first CHEK1 gene by the expression level of the second CHEK1 gene). In addition, comparing the expression level of the first PIK3CA gene (obtained from the tumor tissue sample) with the expression level of the second PIK3CA gene (obtained from the normal tissue sample), a PIK3CA multiple difference (obtained from the first PIK3CA gene expression level divided by the second PIK3CA gene expression level).

Furthermore, it is further matched with Fig. 2, which is a basic multiple diagram of CHEK1 and PIK3CA according to an embodiment of the present invention. As shown in step S3, when the CHEK1 difference multiple is higher than a CHEK1 basic multiple and the PIK3CA difference multiple is higher than a PIK3CA basic multiple, a comparison result is formed. As shown in Figure 2, the basic multiple of CHEK1 (CHEK1) is 1.7, and the basic multiple of PIK3CA (PIK3CA) is 2.2. The basic multiple of CHEK1 is 1.7, which is determined based on the average of the differential multiples of CHEK1 gene expression in the oral cancer patient group (37 persons). The basic multiple of PIK3CA is 2.2, which is based on the population of oral cancer patients (31 persons) were determined by the average value of the CHEK1 gene expression fold difference. Therefore, when the CHEK1 difference multiple is higher than 1.7 and the PIK3CA difference multiple is higher than 2.2, the comparison result is formed.

Finally, as shown in step S4, it is determined to use a pharmaceutical composition according to the comparison result, and the pharmaceutical composition contains a cell checkpoint kinase inhibitor (the preferred embodiment of the present invention in subsequent oral cancer experiments is PF477736) And a phosphatidylinositol 3-kinase inhibitor (the preferred embodiment of the present invention in subsequent oral cancer experiments is BYL719). The pharmaceutical composition is used for inhibiting a cancer cell, wherein the cancer cell is an oral squamous cell carcinoma (a preferred embodiment of the present invention).

Next, please refer to Figures 3A-3B, which are the concentration results of the cell checkpoint kinase inhibitor in one embodiment of the present invention and the concentration results of the phosphatidylinositol 3-kinase inhibitor in one embodiment of the present invention Graph (Y-axis is the cell survival rate (%), X-axis is the Log ₁₀ concentration (respectively has nM and μ M). Carry out the cell viability analysis method (MTT assay) by this oral squamous epithelial carcinoma cell (SAS cell), test The cell checkpoint kinase inhibitor (the present invention is selected from one or a combination of PF477736 (column A), AZD7762 (column B) and LY2606368 (column C), but does not use the above-mentioned cell checkpoint kinase inhibitors) and the phosphatidylinositol 3-kinase inhibitor (a BYL719 (column A), a GDC0941 (column B) and a GSK1059615 (column C) or a combination thereof, but not Phosphatidylinositol 3-kinase inhibitors are limited), and the effects of inhibiting the growth of the oral squamous cell carcinoma cells (SAS cells and OEC-M1 cells) were observed respectively.

Seed 1×10 ⁴ oral squamous cell carcinoma cells (SAS cells and OEC-M1 cells) into 96-well plates, culture for 6-8 hours and start adding different concentrations of different checkpoint kinase inhibitors (PF477736, LY2606368, AZD7762) or different phosphatidylinositol 3-kinase inhibitors (BYL719, GDC0941, GSK1059615), each group was repeated three times. After 48 hours, the cell culture medium containing the cell checkpoint kinase inhibitor (PF477736, LY2606368, AZD7762) or the phosphatidylinositol 3-kinase inhibitor (BYL719, GDC0941, GSK1059615) was removed and washed with phosphate buffered saline ( After washing twice with PBS), 200 μL of 10% MTT (20 μL MTT and 180 μL cell culture medium) was added. After incubating in a 5% CO ₂ , 37°C incubator for one hour, remove the MTT and add 200 μL of DMSO for color development. After mixing evenly, take 100 μL from each well to a 96-well plate for detection, and use an ELISA reader ( Thermo Fisher Scientific, USA) detects the reading at a wavelength of 540nm. The groups of each concentration of the cell checkpoint kinase inhibitor (PF477736, LY2606368, AZD7762) or the phosphatidylinositol 3-kinase inhibitor (BYL719, GDC0941, GSK1059615) were treated without the cell checkpoint kinase inhibitor (PF477736 , LY2606368, AZD7762) or the phosphatidylinositol 3-kinase inhibitor (BYL719, GDC0941, GSK1059615) the absorbance value of the control group was divided, and then converted into cell viability.

Finally, as can be seen from Figures 3A and 3B, the addition of the cell checkpoint kinase inhibitors (PF477736, LY2606368, AZD7762) will obviously cause the death of the oral squamous cell carcinoma cells (SAS cells and OEC-M1 cells), and the PF477736 The drug concentration when causing SAS cell death to 50% is 120nM (Fig. 3A). OEC-M1 cells) died, and the BYL719 caused the SAS cell death to 50% at a drug concentration of 9 μM (Fig. 3B).

Next, as shown in Figure 4, it is the experimental result of the viability of oral cancer cells using a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor (pharmaceutical composition) at the same time according to an embodiment of the present invention picture. Carry out the cell viability analysis method (MTT assay) again, this time except the control group (the symbols are all -), the cell checkpoint kinase inhibitor (the present invention uses the PF477736, 120nM, the symbols are +, -), the In addition to the phosphatidylinositol 3-kinase inhibitor (this invention chooses the BYL719 at this time, 9 μ M, the symbols are -, +), there is also the pharmaceutical composition (in order to add the cell checkpoint kinase inhibitor (PF477736, 120 nM) and The phosphatidylinositol 3-kinase inhibitor (BYL719, 9 μM), all symbols are +) in the oral squamous epithelial carcinoma cells (SAS cells). Each group was repeated three times, and this experimental procedure was repeated twice, and the two data were combined. The results are shown in Figure 4, using the pharmaceutical composition (i.e. adding the cell checkpoint kinase inhibitor (PF477736, 120nM) and the phosphatidylinositol 3-kinase inhibitor (BYL719, 9μM) half inhibitory dose ( 50%) after treating the oral squamous cell carcinoma cells (SAS cells), the average cell survival rate (Y-axis) is 38.2%, which is far lower than 50%, so it means that the pharmaceutical composition (the cell checkpoint kinase inhibits The drug plus the phosphatidylinositol 3-kinase inhibitor) can effectively inhibit the growth of the cancer cell (the oral squamous cell carcinoma (SAS cell)), and the effect is more significant than that of using each inhibitor alone.

Furthermore, as shown in FIG. 5A , it is an experimental result graph of the tumor growth ability of oral cancer cells according to an embodiment of the present invention (the X axis is the number of days). As shown in the figure, the humanized xenograft mouse model established with the cancer tumor tissue (oral cancer tumor tissue) was used for drug efficacy testing, and the tumor growth and size were detected. The calculation method of the tumor size is: 1/2×length×width×width. When the tumor of the xenograft animal model reaches 300-400mm ³ , the mice are randomly divided into groups and start their respective treatments. The course of treatment was two weeks. In the first week, the cell checkpoint kinase inhibitor was injected intraperitoneally (0.5 mg/time, 4 times/week), and at the same time, the phosphatidylinositol 3-kinase inhibitor was orally fed (1.25 mg , 5 times/week), in the second week of treatment, only the phosphatidylinositol 3-kinase inhibitor was fed orally (1.25 mg, 5 times/week), during the treatment period, the tumor size was measured twice a week, and after treatment The tumor size was observed for one week, and the tumor volume was expressed as mean ± standard error of the mean. The results show that if the transplanted oral cancer tumor tissue belongs to the CHEK1 high-expression group and the PIK3CA high-expression group (by this evaluation method, it is known that the CHEK1 difference is greater than 1.7, and the PIK3CA difference is greater than 2.2), the cell checkpoint kinase In the group where the inhibitor and the phosphatidylinositol 3-kinase inhibitor (the pharmaceutical composition of the present invention is group D, the number of mice is 4) are used in combination, the growth of the tumor is obviously inhibited, compared with the control group (group A, Without drug administration, the number of mice 4) has a significant difference (*, p<0.05). And it can be more efficient than the cell checkpoint kinase inhibitor (group B, number of mice 3) and the phosphatidylinositol 3-kinase inhibitor (group C, number of mice 4) used alone inhibition of tumor growth.

Please also refer to FIG. 5B , which is a control experiment result graph of the tumor growth ability of the oral cancer mouse model of an embodiment of the present invention (the X axis is the number of days). Figure 5B is the same as Figure 5A, with the same experimental procedure. The difference is that in the experiment in Figure 5B, the transplanted cancer tumor tissue (oral cancer tumor tissue) is not a CHEK1 high-expression group and a PIK3CA high-expression group (indicating that the CHEK1 difference multiple is less than 1.7, and the PIK3CA The multiple of difference is less than 2.2).

As can be seen from the results in Fig. 5B, whether it is the control group (group A, no drug administration, number of mice is 6), the phosphatidylinositol 3-kinase inhibitor alone (group B, number of mice is 5) or the cell check Neither the dot kinase inhibitor nor the phosphatidylinositol 3-kinase inhibitor (the pharmaceutical composition of the present invention, which is group C, the number of mice is 4) has no effect on inhibiting tumor growth. It shows that the pharmaceutical composition of the present invention has a good therapeutic effect only on the CHEK1 high-expression group and the PIK3CA high-expression group.

Furthermore, it is an experimental result diagram of the body weight of the oral cancer mouse model in one embodiment of the present invention (the Y axis is the multiple of body weight change, and the X axis is the number of days). As shown in Figure 5C, the body weight of the mice was measured once a week, and the weight difference of the mice after spraying was calculated based on the body weight measured before spraying. The results show that relative to the control group (without adding drugs, it is group A), no matter whether the cell checkpoint kinase is used alone Inhibitor (group B) and the phosphatidylinositol 3-kinase inhibitor (group C), or use the pharmaceutical composition (the cell checkpoint kinase inhibitor and the phosphatidylinositol 3-kinase inhibitor, for D group) treatment, the body weight of the mice did not significantly decrease, showing that the pharmaceutical composition (the cell checkpoint kinase inhibitor and the phosphatidylinositol 3-kinase inhibitor combined) treatment can not only effectively inhibit tumor growth, but also does not affect the mice There will be side effects of dehydration and weight loss (because traditional chemotherapy drugs will cause cell death through DNA cross-linking, which will cause patients to have severe side effects such as dehydration and weight loss).

Therefore, the pharmaceutical composition of the present invention (the combination of the cell checkpoint kinase inhibitor (PF477736) and the phosphatidylinositol 3-kinase inhibitor (BYL719) can replace traditional chemotherapy drugs (Cisplatin, Platinum, Cisplatin, commonly known as Platinum, usually treated by intravenous injection, its main side effects are severe vomiting, nephrotoxicity and neurotoxicity) new treatment method, and can reduce the discomfort caused by chemotherapy drugs (severe vomiting, nephrotoxicity and neurotoxicity) Toxicity), can provide more effective and precise treatment and improve the survival rate of oral cancer patients in the CHEK1 high-expression group and PIK3CA high-expression group (for the CHEK1 multiplicity higher than 1.7 and the PIK3CA multiplier higher than 2.2) .

Finally, the present invention also discloses a method for evaluating the population of suitable pharmaceutical compositions, the steps include: (a) obtaining a tumor tissue sample and a normal tissue sample from an individual; (b) detecting the tumor tissue sample and the normal tissue respectively The expression level of a CHEK1 gene and the expression level of a PIK3CA gene in the sample; (c) calculating a CHEK1 gene difference multiple and a PIK3CA gene difference multiple based on step (b); and (d) the CHEK1 gene difference based on step (c) The gene difference multiple and the PIK3CA gene difference multiple are used to determine whether the individual is suitable for a group of pharmaceutical compositions; wherein, when the CHEK1 gene difference multiple is higher than 1.7 and the PIK3CA gene difference multiple is higher than 2.2, then the individual is suitable for the group A family of pharmaceutical compositions.

The subject in step (a) is a mammal, preferably a human. In addition, the tumor tissue is not limited to oral cancer tumor tissue, and the tumor tissue applicable to cancer mediated by the activity of CHEK1 gene and PIK3CA gene includes (but not limited to), the cancer includes: adult and children tumors, solid tumors Growth/malignancy, myxoid and round cell carcinomas, locally growing neoplasms, metastatic cancers, human soft tissue sarcomas, including Ewing sarcoma, cancer metastases, including lymphoid metastases, squamous cell carcinomas, especially Fingers and neck, squamous cell carcinoma of the esophagus, oral carcinoma, hematologic malignancies (including multiple myeloma), leukemia (including acute lymphoblastic leukemia, acute lymphoblastic leukemia, Chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia), leaky lymphoma (body cavity-based lymphoma), thymic lymphoma lung cancer (including small cell carcinoma, cutaneous T-cell lymphoma, Hawking's ( Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the renal cortex, ACTH-producing tumors, non-small cell carcinoma, breast cancer, including small cell carcinoma and ductal carcinoma), gastrointestinal cancer (including gastric cancer, colon cancer, colon cancer Rectal cancer and polyps associated with colorectal neoplasia), pancreatic cancer, liver cancer, urinary tract cancer (including bladder cancer, such as: primary superficial bladder tumor, invasive transformed cell carcinoma of the bladder and muscle-invasive bladder cancer), prostate cancer, malignant diseases of the female reproductive tract (including ovarian cancer, primary peritoneal epithelial tumor, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine cancer and ovarian follicles solid tumors), malignant diseases of the male reproductive tract (including testicular cancer and penile cancer), kidney cancer (including renal cell carcinoma), brain cancer (including endogenous brain tumors, neuroblastoma, and astrocytic brain tumors) , glioma and metastatic tumor cells invading the central nervous system), bone cancer (including osteoma and osteosarcoma), skin cancer (including malignant melanoma, tumor development of human skin keratinocytes and squamous cell carcinoma), thyroid Carcinoma, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms' tumor, gallbladder carcinoma, trophoblastic tumor, hemangiopericytoma, and Kaposi's )sarcoma.

The subsequent detection method used in step (b) is real-time quantitative reverse transcription polymerase chain reaction. The calculation method in step (c) is to divide the expression level of the CHEK1 gene of the tumor tissue sample by the expression level of the CHEK1 gene of the normal tissue sample, and similarly is the expression of the PIK3CA gene of the tumor tissue sample The expression level of the PIK3CA gene in the normal tissue sample is divided by the expression level of the PIK3CA gene to calculate the multiple of the CHEK1 gene difference and the multiple of the PIK3CA gene difference.

Finally, as shown in step (d), it is judged whether the individual belongs to the group suitable for the pharmaceutical composition according to the CHEK1 gene difference multiple and the PIK3CA gene difference multiple. It can be known from the above oral cancer examples that when the CHEK1 gene differential multiple is higher than 1.7 and the PIK3CA differential multiple is higher than 2.2, the individual is suitable for the pharmaceutical composition (the cell checkpoint kinase inhibitor and the phosphatidylinositol 3 combinations of kinase inhibitors) group.

Therefore, the present invention is novel, progressive and can be used in the industry. It should meet the patent application requirements of my country's patent law. I file an invention patent application in accordance with the law. I pray that the bureau will grant the patent as soon as possible. I am sincerely praying.

However, the above-mentioned ones are only preferred embodiments of the present invention, and are not used to limit the scope of the present invention. For example, all equal changes and modifications are made according to the shape, structure, characteristics and spirit described in the scope of the patent application of the present invention. , should be included in the patent application scope of the present invention.

S1~S4: Step process

Claims (5)

A pharmaceutical composition used as a medicine for treating cancer, wherein the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, the pharmaceutical composition is used to inhibit a cancer cell, wherein the The cell checkpoint kinase inhibitor is PF477736, and the phosphatidylinositol 3-kinase inhibitor is BYL719. The cancer cell line comes from tumor tissue samples of oral cancer patients, and the expression of CHEK1 gene in tumor tissue samples is compared with normal tissue samples A CHEK1 differential multiple and a PIK3CA differential multiple were obtained after the PIK3CA gene expression level and the CHEK1 differential multiple were greater than 1.7 and the PIK3CA differential multiples of the cancer cell were greater than 2.2, and the cancer cell line was an oral squamous cell carcinoma cell line. The pharmaceutical composition described in item 1 of the patent application is used as the preparation of a drug for treating cancer, wherein the pharmaceutical composition inhibits the survival ability of the oral squamous cell carcinoma cells. The pharmaceutical composition described in item 1 of the scope of the patent application is used as the preparation of a drug for treating cancer, wherein the pharmaceutical composition inhibits the tumor growth ability of the oral squamous cell carcinoma cells. An evaluation method using a pharmaceutical composition, the steps comprising: using a real-time quantitative reverse transcription polymerase chain reaction to detect a CHEK1 gene and a PIK3CA gene in a tumor tissue sample, and obtaining a first CHEK1 gene expression level, a first PIK3CA gene Gene expression and detecting the CHEK1 gene and the PIK3CA gene in a normal tissue sample to obtain a second CHEK1 gene expression and a second PIK3CA gene expression; Comparing the expression level of the first CHEK1 gene with the expression level of the second CHEK1 gene and the expression level of the first PIK3CA gene with the expression level of the second PIK3CA gene to obtain a CHEK1 difference multiple and a PIK3CA difference multiple; when the CHEK1 difference multiple When it is higher than a basic multiple of CHEK1 and the differential multiple of PIK3CA is higher than a basic multiple of PIK3CA, a comparison result is formed; and according to the comparison result, it is judged to use a pharmaceutical composition comprising a cell checkpoint kinase inhibitor agent and a phosphatidylinositol 3-kinase inhibitor, the cell checkpoint kinase inhibitor is PF477736, and the phosphatidylinositol 3-kinase inhibitor is BYL719; wherein the tumor tissue sample is a cancer tumor tissue, the cancer tumor The tissue is an oral squamous epithelial carcinoma cell, the basic ploidy of CHEK1 is 1.7, and the basic ploidy of PIK3CA is 2.2. A method for evaluating a group of suitable pharmaceutical compositions, the steps comprising: (a) obtaining a tumor tissue sample and a normal tissue sample, the tumor tissue sample is a cancer tumor tissue, and the cancer tumor tissue is an oral squamous epithelium Cancer cells; (b) respectively detecting the expression level of a CHEK1 gene and the expression level of a PIK3CA gene in the tumor tissue sample and the normal tissue sample; (c) calculating a CHEK1 gene difference multiple and a PIK3CA gene based on step (b) and (d) based on the CHEK1 gene difference and the PIK3CA gene difference in step (c) to determine whether the individual is a group suitable for the pharmaceutical composition; Wherein, when the multiple of difference of the CHEK1 gene is higher than 1.7 and the multiple of the difference of PIK3CA is higher than 2.2, then the individual belongs to the group suitable for the pharmaceutical composition, and the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, the cell checkpoint kinase inhibitor is PF477736, and the phosphatidylinositol 3-kinase inhibitor is BYL719.

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