CN113750239B

CN113750239B - Pharmaceutical composition for treating cervical cancer and pharmaceutical preparation and application thereof

Info

Publication number: CN113750239B
Application number: CN202110947820.1A
Authority: CN
Inventors: 戴东方; 朱必清; 武雅琴; 何侠; 王海蓉; 黄健; 杨勇琴
Original assignee: Jiangsu Cancer Hospital
Current assignee: Jiangsu Cancer Hospital
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2022-11-25
Anticipated expiration: 2041-08-18
Also published as: CN113750239A

Abstract

The invention relates to the technical field of medicines, in particular to a pharmaceutical composition for treating cervical cancer, a pharmaceutical preparation and application thereof. The pharmaceutical composition comprises an immune checkpoint inhibitor, an Epidermal Growth Factor Receptor (EGFR) inhibitor and shikonin, and the weight ratio of the pharmaceutical composition to the shikonin is 1. Experiments prove that the combination of the three active ingredients has remarkable synergistic effect on treating the cervical cancer in a specific weight ratio range, so that the dosage of an immune checkpoint inhibitor and an Epidermal Growth Factor Receptor (EGFR) inhibitor can be reduced, the treatment cost is reduced, the treatment effect of the cervical cancer can be remarkably improved, and the pain of a patient can be relieved, thereby having good development potential and application prospect.

Description

Pharmaceutical composition for treating cervical cancer and pharmaceutical preparation and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a pharmaceutical composition for treating cervical cancer, a pharmaceutical preparation and application thereof.

Background

Due to the difference between cervical cancer screening and HPV infection prevention and control levels, the mortality rate of cervical cancer has obvious regional difference. In low income areas, such as east africa, etc., the mortality rate of cervical cancer can be as high as 30.0/10 ten thousand, while in high income areas, such as australia, new zealand, etc., the mortality rate is only 1.7/10 ten thousand, and the difference can be 17 times. In China, the annual incidence rate of cervical cancer is 16.56/10 ten thousand, the mortality rate is as high as 5.04/10 ten thousand, and the physical and psychological health and the life safety of women are seriously threatened.

In 1983, german scientists put forward the hypothesis that the occurrence of cervical cancer may be related to HPV infection for the first time, and clinically verified that the hypothesis has milestone significance for the etiological study of cervical cancer. With the development of research in tumor cell biology and molecular biology, people continuously isolate and identify the HPV genotype. To date, over 150 subtypes of HPV have been found, with 12 types being carcinogenic, with 16 and 18 being the most common. The oncogenicity of HPV is closely related to its viral proteins E6, E7, E6, E7 have no enzymatic activity, and they form complexes by interacting with host intracellular proteins to regulate cellular biological activities. It is reported that the E6 protein is subjected to conformational change after being combined with the cell ubiquitin ligase E6AP, and can be specifically combined with p53 to form an E6/E6AP/p53 complex, so that the p53 is ubiquitinated and degraded. Similarly, E7 proteins can also bind to pRb and inactivate it. Inactivation of the cancer suppressor genes p53 and pRb leads to uncontrolled cell cycle, inhibited apoptosis and excessive cell proliferation to cause canceration.

As described above, human Papillomavirus (HPV) infection is closely related to cervical cancer, and persistent high-risk HPV infection is the most major cause of cervical cancer. Diagnosis of precancerous lesions and HPV infections is not difficult, but rationalization, precision, and individualized treatment of cervical cancer remain problems to be solved urgently. Currently, the commonly used cervical cancer treatment scheme is systemic treatment such as radiotherapy and chemotherapy mainly based on operations, the local focus of early cervical cancer can obtain higher curative effect through the treatment, the 5-year survival rate can reach 91.5%, but the 5-year survival rate is only 16.8% due to the lack of effective treatment means for late-stage, recurrent and metastatic cancer. In addition, effective prevention and treatment as well as improvement of the quality of life of patients and preservation of the fertility function of young patients with fertility requirements are the hot spots of current research. With the use of anti-angiogenesis drugs, immune checkpoint inhibitors and other targeted therapies and immunotherapeutic drugs, the survival time of this part of patients is significantly prolonged, but the final curative effect is not yet achieved, and a need for further molecular level research to reveal new therapeutic targets to guide clinical individualized therapy is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a pharmaceutical composition for treating cervical cancer, a pharmaceutical preparation and an application thereof. The invention combines the immune check point inhibitor, the Epidermal Growth Factor (EGFR) receptor inhibitor and the alkannin to treat the cervical cancer, can obviously improve the treatment effect of single medicine active ingredients, and obviously improves the problems of untoward effect, drug resistance and the like in the treatment process.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a pharmaceutical composition for treating cervical cancer comprising an immune checkpoint inhibitor, an Epidermal Growth Factor (EGFR) receptor inhibitor and shikonin.

Preferably, the immune checkpoint inhibitor is selected from the group consisting of: ipilimumab, atuzumab, nivolumab, pembrolizumab, and/or parbolzumab.

Preferably, the immune checkpoint inhibitor is selected from ipilimumab.

Preferably, the Epidermal Growth Factor (EGFR) receptor inhibitor is selected from the group consisting of: cetuximab, matuzumab, erlotinib, gefitinib, and/or lapatinib.

Preferably, the Epidermal Growth Factor (EGFR) receptor inhibitor is selected from cetuximab.

Preferably, the weight ratio of the immune checkpoint inhibitor, the Epidermal Growth Factor (EGFR) receptor inhibitor and the shikonin is 1.

Preferably the weight ratio of the immune checkpoint inhibitor, the Epidermal Growth Factor (EGFR) receptor inhibitor and the shikonin is 1.

Furthermore, the invention also provides the application of the alkannin in preparing a sensitizer used as an immune checkpoint inhibitor and/or an Epidermal Growth Factor (EGFR) receptor inhibitor.

Further, the invention also provides a pharmaceutical preparation for treating cervical cancer, which comprises the pharmaceutical composition and pharmaceutically acceptable auxiliary components.

Preferably, the pharmaceutical formulation is an injection.

Furthermore, the invention also provides an application of the combination of an immune checkpoint inhibitor, an Epidermal Growth Factor Receptor (EGFR) inhibitor and shikonin in preparing a medicament for treating cervical cancer, wherein the weight ratio of the immune checkpoint inhibitor, the Epidermal Growth Factor Receptor (EGFR) inhibitor and the shikonin is (1).

Immune checkpoints are molecules that play an inhibitory role in the immune system, and common checkpoints include PD-1, CTLA-4, etc., which normally maintain autoimmune tolerance and prevent the development of autoimmune responses by modulating T cell function. However, during tumorigenesis, they become a helper for tumor immune escape. Immune checkpoint inhibitors restore effector T cell function by targeting PD-1 or CTLA-4 and have demonstrated anti-tumor activity in a variety of tumors. CTLA-4 is considered to inhibit the body anti-tumor immune factor, block CTLA-4 can enhance the body to weak immunogenic tumor immune response effectively, CTLA-4 is the single clone medicine of the target point is the hot spot and direction of the present tumor targeting immunotherapy. The ipilimumab is an anti-CTLA-4 fully human immunoglobulin G1 monoclonal antibody, and is the first targeted immune checkpoint therapeutic drug approved by the FDA in the United states.

The Epidermal Growth Factor (EGFR) receptor pathway is a basic signaling pathway of epithelial cells and plays a major role in maintaining cell growth, proliferation, survival, and the like. The EGFR specific monoclonal antibody can prevent the combination of EGFR and ligand, inhibit the phosphorylation of receptor tyrosine kinase, internalize EGFR and inhibit the growth of tumor cells. Researches show that the expression rate of EGFR in cervical cancer is as high as 87.5-100%, and the high expression of EGFR is closely related to poor prognosis of tumor and low treatment response rate, so that the targeted inhibition of EGFR is expected to become a new strategy for treating recurrent metastatic cervical cancer. Cetuximab (Cetuximab) is a human/mouse chimeric monoclonal antibody IgG2 that inhibits adenovirus binding and receptor activation by binding to extracellular fragments of EGFR-expressing cancer cells, thereby inhibiting downstream signaling by EGFR and thus blocking the growth and replication functions of cancer cells. Preclinical studies have demonstrated that cervical cancer cells are highly sensitive to cetuximab-mediated tumor cytotoxicity and growth inhibition.

The effective component of radix Arnebiae is L-alkannin belonging to naphthoquinone, and has various pharmacological effects, such as antiinflammatory, anti-tumor, hemostasis, wound healing promotion, and epidermal cell hyperproliferation inhibition. The research in recent years shows that the traditional Chinese medicine can play an anti-tumor role through various mechanisms of inhibiting the proliferation of tumor cells, inducing the apoptosis of the tumor cells, inducing the differentiation of the tumor cells, inhibiting the generation of tumor vessels, reversing the multidrug resistance of the tumor cells, enhancing the immunity of organisms and the like. Shikonin is a main medicinal component extracted from lithospermum, and many researches show that the shikonin can inhibit the growth of various tumors, such as liver cancer, colorectal cancer, colon cancer, leukemia and the like.

Compared with the prior art, the invention has the beneficial effects that: the combination of the immune checkpoint inhibitor, the Epidermal Growth Factor Receptor (EGFR) inhibitor and the alkannin for treating the cervical cancer can obviously improve the treatment effect of single active ingredients, and obviously improve the problems of adverse reaction, drug resistance and the like in the treatment process. Through research on inhibition experiments on the growth of cervical cancer cells when the immune checkpoint inhibitor, the Epidermal Growth Factor Receptor (EGFR) inhibitor, the alkannin and the three active ingredients are singly used in combination, the results show that the combination of the three active ingredients has obvious synergistic effect on treating the cervical cancer in a specific weight ratio range. The application doses of an immune checkpoint inhibitor and an Epidermal Growth Factor Receptor (EGFR) inhibitor can be reduced, the treatment cost is reduced, the treatment effect of cervical cancer can be obviously improved, and the pain of a patient can be relieved, so that the cervical cancer treatment composition has good development potential and application prospect.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. The materials, reagents and the like used are all commercially available reagents and materials unless otherwise specified.

Effect embodiment: inhibition effect of pharmaceutical composition on mouse cervical carcinoma U14 tumor strain

Experimental animals: the female mice of Kunming species had a total of 70 mice, weighing approximately (20. + -.2) g.

Pharmaceutical agent: (1) Immune checkpoint inhibitor ipilimumab, available from behmeet-schrobo, usa; (2) Cetuximab, an Epidermal Growth Factor Receptor (EGFR) inhibitor, available from merccrion pharmaceuticals, germany; (3) Shikonin, available from Mirrida technologies, inc., beijing.

Establishment of cervical cancer U14 tumor-bearing mouse model: inoculating the tumor strain to Kunming mice, generating ascites after 7 days of growth, extracting the ascites by using an empty needle with the specification of 1ml under the aseptic condition, and adding physiological saline according to the volume ratio of 1. 0.2ml of the diluted U14 cell suspension was injected into the right anterior axillary region of the mice. 70 mice are completely inoculated, and the inoculation success rate is 100 percent through inspection.

Experimental groups and dosing regimens: 70 mice successfully inoculated with the tumor strains were randomly divided into 1 control group +6 administration groups of 10 mice each. The control group was gavaged with sodium carboxymethylcellulose solution (0.5 ml/20 g), and each administration group was subjected to femoral vein instillation (400 mg/m) according to the administration schedule in Table 1 ² ) The administration was continued for 10 days.

TABLE 1 dosing regimens for each dosing group

Index observation and experimental results:

(1) Tumor growth conditions of tumor-bearing mice: before administration, the body mass of the tumor-bearing mice is weighed, the body mass of the tumor-bearing mice is measured every other day after administration, and the tumor volume is measured and recorded every other day starting on the 6 th day after inoculation. Tumor volume (cm) ³ ) Calculating according to the formula: tumor volume = ab ² Pi/6 (tumor diameter measured with a vernier caliper, a is the longest diameter cm, b is the shortest diameter cm). The results of the experiment are shown in table 2.

TABLE 2 comparison of tumor volumes (cm) of U14 tumor-bearing mice at different times ³ )

As can be seen from the experimental results in Table 2, the volume of the implanted tumor in each group of mice gradually increased with time, and the tumor volume of the control group of mice increased most obviously and at the highest speed. Although each single medicine group can inhibit the increase of the tumor volume to a certain degree, the combined administration group has more obvious inhibition effect. Specifically, the tumor volume of the combination group peaked at day 8 and then showed a downward trend. Of these, the combined administration of 3 groups (ipilimumab: cetuximab: alkannin =1: 0.2) had the best tumor suppression effect, with the tumor volume at the end of the treatment being only 0.34 ± 0.04cm ³ Significantly lower than 1.97 +/-0.30 cm of a control group ³ 。

(2) The tumor inhibition rate is as follows: weighing the mass of the mouse body on the 11 th day after inoculation, taking off the vertebra to kill the mouse, weighing the tumor mass, and calculating the tumor inhibition rate of each administration group respectively. The tumor inhibition rate (%) is calculated according to the formula: tumor inhibition rate = (control group average tumor mass-administration group average tumor mass)/control group average tumor mass × 100%. The results of the experiment are shown in table 3.

TABLE 3 comparison of tumor mass (g) and tumor inhibition (%) of U14 tumor-bearing mice in each group

As can be seen from the experimental results in table 3, the tumor mass of mice in each administration group was significantly lower than that in the control group, which indicates that each administration group has tumor suppression effect. The tumor inhibition rate is calculated according to the tumor mass, and the tumor inhibition rate (both higher than 76%) of each combined medicine group is obviously higher than that of each single medicine group (both are not more than 40%), which shows that the combined medicine of the ipilimumab, the cetuximab and the alkannin has obvious synergistic effect on the aspect of inhibiting the proliferation of cervical cancer cells. Wherein, the combined administration of 3 groups (ipilimumab: cetuximab: shikonin = 1.

In conclusion, the pharmaceutical composition containing the immune checkpoint inhibitor, the Epidermal Growth Factor Receptor (EGFR) inhibitor and the alkannin in a specific weight ratio has a remarkable tumor inhibition effect on U14 tumor-bearing mice, can effectively reduce the dosage of each pharmaceutical component when used alone, has the effects of enhancing the curative effect, reducing the drug resistance and relieving the side effect, has an important significance for clinically treating cervical cancer, and has good development potential and application prospect.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications or equivalents may be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalents should also be regarded as the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating cervical cancer, comprising an immune checkpoint inhibitor, an Epidermal Growth Factor (EGFR) receptor inhibitor and shikonin, wherein the weight ratio of the immune checkpoint inhibitor, the Epidermal Growth Factor (EGFR) receptor inhibitor and the shikonin is 1.1-0.5;

the immune checkpoint inhibitor is selected from ipilimumab;

the Epidermal Growth Factor (EGFR) receptor inhibitor is selected from cetuximab.

2. The pharmaceutical composition of claim 1, wherein the immune checkpoint inhibitor, epidermal Growth Factor (EGFR) receptor inhibitor and shikonin are present in a weight ratio of 1.

3. A pharmaceutical preparation for treating cervical cancer, comprising the pharmaceutical composition of claim 1 or 2 and a pharmaceutically acceptable adjuvant component.

4. The pharmaceutical formulation of claim 3, wherein the pharmaceutical formulation is an injection solution.

5. The application of the combination of an immune checkpoint inhibitor, an Epidermal Growth Factor (EGFR) receptor inhibitor and shikonin in preparing a medicament for treating cervical cancer, wherein the weight ratio of the immune checkpoint inhibitor, the Epidermal Growth Factor (EGFR) receptor inhibitor and the shikonin is (1);

the immune checkpoint inhibitor is selected from ipilimumab;