CN111481670A

CN111481670A - Application of statin compound in tumor inhibition

Info

Publication number: CN111481670A
Application number: CN202010331596.9A
Authority: CN
Inventors: 宋纯理; 原婉琼; 管志远; 张旺; 王红; 海宝; 曹宝山
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-08-04

Abstract

The invention discloses a new application of statins compound injected into bones locally to treat tumors of mammals and human patients. Compared with other administration routes (oral administration, intravenous injection, subcutaneous injection and intraperitoneal injection), the statin compound can inhibit tumor growth and prolong the life of patients by only locally injecting the statin compound into bones.

Description

Application of statin compound in tumor inhibition

Technical Field

The invention relates to the technical field of medicines, in particular to a new application of HMG-CoA reductase inhibitor (statin compound) in inhibiting tumor growth and prolonging the life of tumor patients.

Background

With the rapid development of social economy, the acceleration of urbanization and industrialization and the aging of population, tumors become one of the most important public health problems affecting the health of residents. In recent years, the incidence rate level of malignant tumors in China shows a trend of rising year by year, and the death rate is the first. Poses serious threat to human life health.

Simvastatin is a first-line medicine for preventing and treating cardiovascular and cerebrovascular events, can effectively reduce cholesterol, is safe and effective, is widely applied clinically at present, and is listed as an OTC non-prescription medicine in many countries. Recent studies have found that simvastatin can also act as an Adjuvant in combination with immunotherapy to exert an anti-tumor effect [ Yun X, et al, the mevalonateppayway Is a drug able Target for Vaccine Adjuvant discovery cell.2018nov 1; 175(4):1059-1073]. The traditional route of administration of simvastatin is oral, however, oral simvastatin needs less than 5% of its first pass effect through the liver, and passes through multiple barriers to reach the target organ, which is much less and less, and severely reduces the bioavailability of the drug [ Vickers S, animal. 18:138-45].

Therefore, the search for more effective routes of administration is of great importance, and there has been no strategy to treat tumors by intervening in local bone to date.

Disclosure of Invention

The inventor of the invention surprisingly finds that the traditional administration routes of statins, such as oral administration and clinically common injection routes, such as intravenous injection, subcutaneous injection and intraperitoneal injection, can not inhibit the growth of tumors, and only the novel administration route of the invention, namely local skeletal injection of statins, can effectively inhibit the growth of the tumors and prolong the survival period of mammals.

The invention aims to provide the application of a statin compound or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting the growth of tumors of mammals or human patients and prolonging the life cycle of the patients.

In a specific embodiment of the present invention, wherein the tumor is: lung cancer, gastric cancer, esophageal cancer, intestinal cancer, liver cancer, cervical cancer, nasopharyngeal carcinoma, breast cancer, etc.

In a specific embodiment of the present invention, wherein the drug is in the form of a local intraosseous injection.

In a specific embodiment of the present invention, wherein the drug is in a sustained release dosage form.

In a specific embodiment of the present invention, wherein, the carrier of the sustained release dosage form is a pharmaceutically acceptable carrier; such as hydrogels, especially temperature sensitive hydrogels.

In one embodiment of the present invention, statin compounds include, but are not limited to, HMG-CoA reductase inhibitors such as simvastatin, atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, pitavastatin, bervastatin, cerivastatin, lovastatin, pravastatin, dalvastatin, mevastatin, or tivastatin. The statin compound may also be in the form of a pharmaceutically acceptable salt, including, for example, but not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate or acetate, sodium, potassium, calcium or magnesium salt of the statin compound.

Preferably, the statin compound is selected from simvastatin, atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and/or pitavastatin; and preferably, their pharmaceutically acceptable salts are selected from the group consisting of hydrochloride, hydrobromide, sulfate, nitrate, phosphate, citrate, mesylate, sodium, potassium, calcium or magnesium salts.

More preferably, the statin compound or the pharmaceutically acceptable salt thereof is selected from simvastatin, atorvastatin calcium or atorvastatin sodium, fluvastatin sodium, pravastatin sodium, rosuvastatin calcium and/or pitavastatin calcium.

The medicament can be a sustained-release formulation; the carrier of the sustained release dosage form is a medicinal carrier; the statin compound hydrogel is preferably in the form of a statin compound or a pharmaceutically acceptable salt thereof hydrogel for inhibiting the growth of tumors of mammals or humans and promoting survival rate, and comprises the statin compound or the pharmaceutically acceptable salt thereof, water and a hydrogel auxiliary material, wherein the hydrogel auxiliary material is selected from poloxamer, chitosan, polylactic acid, celluloses, hyaluronic acid, fibrin glue and other degradable and absorbable materials. They are commercially available. Of course, the preparation method also comprises the step of directly preparing microcrystalline and nano-particle dosage forms for statins.

The hydrogel adjuvants can also be selected from alginic acid, poly L-lysine, poly L-glutamic acid, etc. synthetic hydrophilic polymer such as polyacrylic acid, polymethacrylic acid, polyacrylamide, poly N-polyacrylamide, etc. can be used.

The preparation method of the hydrogel comprises the steps of dissolving the hydrogel auxiliary material in water at low temperature to form a uniform solution, adding the statin powder with the effective treatment amount into the solution, and continuously stirring the solution to form the uniform solution; preferably, the powder of the hydrogel auxiliary material is dissolved in water to form a uniform solution with the concentration of 25% (W/V) at low temperature (4 ℃), the statin powder is directly added at low temperature (4 ℃), the mixture is continuously stirred to form the uniform solution, and the uniform solution is stored at 4 ℃. The starting materials in the above process are commercially available.

The statin hydrogel composite material provided by the invention comprises the hydrogel of the statin compound or the pharmaceutically acceptable salt thereof. The adjuvants of the hydrogel can be selected from poloxamer, chitosan, polylactic acid, cellulose, hyaluronic acid, fibrin glue, etc. Preferably, the statin hydrogel composite comprises a poloxamer hydrogel of a statin compound or a pharmaceutically acceptable salt thereof.

In one embodiment of the invention, the statin hydrogel composite is effective in inhibiting tumor growth and prolonging the survival of a mammal or a human. The statin hydrogel of the invention can be in the form of injection.

Wherein the tumor, the statin compound or the pharmaceutically acceptable salt thereof and the drugs used in the chemotherapy are as described above.

In the embodiment of the present invention, the statin-based intraosseous injection drug is preferably administered by intraosseous injection or implantation, and the statin-based intraosseous injection drug of the present invention may be in the form of an injection, including but not limited to injection solution, injection suspension, injection emulsion, injection gel, injection solid form, or their sustained-release or controlled-release forms, or in the form of an implant. Herein, the solid form for injection means that it is mixed with a vehicle for injection such as water for injection, physiological saline for injection, or glucose solution for injection at the time of use, for injection application. Or directly by local injection in the form of an implant.

In one embodiment of the invention, the pharmaceutically acceptable excipient is selected from one or more of water-soluble vehicle or oil-based vehicle, dispersing agent, isotonic agent, preservative, solubilizing agent or stabilizing agent, wherein the water-soluble vehicle can be selected from distilled water, physiological saline, Grignard solution or Phosphate Buffered Solution (PBS), the oil-soluble vehicle can be selected from vegetable oil such as olive oil, castor oil, sesame oil, cottonseed oil or corn oil, the dispersing agent can be selected from Tween 20 or Tween 80, polyethylene glycol, carboxymethylcellulose or sodium alginate, the isotonic agent can be selected from sodium chloride, glycerol, sorbitol or glucose, the solubilizing agent can be selected from sodium salicylate, poloxamer or sodium acetate, the preservative can be selected from methylparaben, propylparaben, benzyl alcohol, chlorobutanol, sodium benzoate or phenol, the stabilizing agent can be selected from albumin, bovine serum albumin, the pharmaceutically acceptable excipient can be selected from biodegradable materials such as polylactic acid, polyglycolide, and/or the like, and the pharmaceutically acceptable excipient can be used in the fields of technical field for stabilizing the statin, such as the aqueous suspension, or emulsion, the pharmaceutical composition, wherein the pharmaceutically acceptable excipient can be used in the fields of the invention and/or the fields of the invention (see the fields of the invention).

In an embodiment of the invention, for local injection of statins in the bone of a mammal or a human, the statin dose used may be between 0.1mg and 50mg, preferably between 0.1mg and 10mg, of statin compound per time. It is within the teachings of the present invention for the clinician to adjust or modify the number and dosage of administrations as needed for the effect of the clinical treatment.

In an embodiment of the invention, the statin is injected locally into the bone of the mammal or human, and the drug is administered at an interval of 7 to 600 days, preferably 10 to 500 days, more preferably 20 to 400 days, and particularly preferably 30 to 300 days.

Experiments prove that the statin local injection in bones at a small dose once can inhibit the growth of tumors of mammals or human patients and prolong the life cycle of the mammals or the human tumor patients.

Drawings

FIG. 1 shows that the tumor volume of the breast cancer tumor-bearing mice reaches 80-100mm²Later, intervention was performed by different routes of administration of simvastatin: oral administration (A), intravenous injection (B), subcutaneous injection (C), intraperitoneal injection (D) and bone injection (E) simvastatin (the concrete operation is shown in test example 1) for the change of the body weight of the breast cancer tumor-bearing mice. The difference is statistically significant (P)<0.05), it was shown by intragroup and intragroup comparison that only bone injection of simvastatin could better maintain the life status of mice (n-8).

FIG. 2 shows the change of tumor volume of mice bearing tumor of breast cancer after two weeks of simvastatin intervention by oral administration (A), intravenous injection (B), subcutaneous injection (C), intraperitoneal injection (D) and bone injection (E) (see test example 1 for details). Compared with a corresponding control group, only the bone injection of simvastatin can inhibit the tumor volume increase, the difference has statistical significance (P <0.05), and the administration routes of other simvastatin cannot change the tumor volume of a mouse, so that the bone injection of simvastatin can inhibit the tumor, and the administration route has a good effect (n is 8).

FIG. 3 shows fluorescence photographs (A) and (B) of tumors imaged in vivo in mice bearing tumor of breast cancer in the simvastatin group by oral administration, intravenous injection, subcutaneous injection, intraperitoneal injection and skeletal injection two weeks after simvastatin intervention (see test example 1 for details). The difference in skeletal injection of simvastatin alone was statistically significant (P <0.05, n ═ 4) compared to the corresponding control group, further indicating that only skeletal injection of simvastatin was able to inhibit tumor growth.

FIG. 4 shows the change of tumor weight of mice bearing tumor of breast cancer caused by oral administration, intravenous injection, subcutaneous injection, intraperitoneal injection and skeletal injection of simvastatin after two weeks of simvastatin intervention (see test examples 1 and 4 for details). Compared with a corresponding control group, the tumor weight is reduced by only injecting the simvastatin into the skeleton, which further indicates that the tumor can be reduced and the tumor growth can be inhibited by only injecting the simvastatin into the skeleton, and other administration routes have no inhibition effect on the tumor, thus reflecting the importance of injecting the simvastatin into the skeleton.

FIG. 5 shows the prognosis of simvastatin dryness, survival of mice bearing breast cancer with oral administration (A) and bone injection (B) of simvastatin (see test example 5 for details). Compared with a control group, the oral simvastatin group can not improve the survival of mice, and the difference between the bone injection simvastatin group and the control group has statistical significance (P is less than 0.05, and n is 8-9), which shows that the bone injection simvastatin group can prolong the survival time of the mice, and is an effective administration way for reducing the death caused by tumors.

In FIG. 6, a CD8 is shown⁺Proportion of T cells in bone marrow (A), peripheral blood (B), lymph nodes (C), spleen (D) and tumor infiltrating lymphocytes (E) of breast cancer tumor-bearing mice. Statistical analysis results showed that bone injection of simvastatin increased CD8⁺The proportion of T cells among the above tissue-derived lymphocytes increases (p)<0.05); FIG. 6F shows the detection of CD8 by immunohistochemical staining⁺Expression of T cells in tumor-bearing mouse tumors.The above fully shows that the injection of simvastatin into bone increases CD8 from' source⁺T cell ratio, causing the systemic immune organ CD8⁺Increased proportion of T cells, eventually clearing and moistening CD8 in the tumor⁺The proportion of T cells increases.

FIG. 7 shows tumor-infiltrated CD8⁺Perforin (Perforin), granzyme B (granzyme B) and the levels of tumor suppressor cytokines TNF α, IFN-gamma secreted by T cells in response to bone injection of simvastatin to increase the level of CD8⁺Tumor killing ability of T cells.

Detailed Description

The following examples further illustrate the applicability of the invention but are not to be construed as limiting the invention in any way. It will be apparent to those skilled in the art from the teachings of the prior art that equivalent modifications or substitutions of features can be made in the embodiments of the present invention without departing from the scope of the invention as claimed.

Example 1 preparation of temperature sensitive Poloxamer hydrogels

Dissolving poloxamer 407 powder in double distilled water to form a uniform solution with the concentration of 25% at a low temperature, and continuously stirring at the low temperature (4 ℃) to form the uniform solution.

Example 2 simvastatin hydrogel composite preparation

Under the ice-bath state, 2.5mg of simvastatin was added to 100ul of poloxamer 407 hydrogel solution, respectively, to form a poloxamer 407 hydrogel solution with simvastatin. When the simvastatin containing poloxamer 407 hydrogel solution is used, the simvastatin containing poloxamer 407 hydrogel solution is fully and uniformly mixed, and the temperature is raised to form the simvastatin hydrogel composite material.

Test example 1

Establishing a breast cancer mouse model, carrying out drug intervention, and recording the weight change of a tumor-bearing mouse

Luciferase-transfected 4T1 mouse breast cancer cells at log phase growth were seeded into mouse mammary fat pads. The volume of breast cancer in mice was recorded every other day using a vernier caliper. Tumor volume fraction was calculated as tumor longest diameter (a) and perpendicular shortest diameter (b):formula V (mm)³)＝a²b/2(V is the approximate volume of the tumor). mm means millimeter. When the tumor reaches 80-100mm³Drug intervention was started, tumor-bearing mice were randomly divided into ten groups of 8 mice each:

(1) oral control group: 1% methylcellulose (made up with sterile distilled water, daily injection for 2 weeks)

(2) Oral simvastatin group: SIM 10mg/kg/day (SIM dissolved in 1% methylcellulose, injected daily for 2 weeks)

(3) Intravenous injection control group: 2% DMSO (saline formulation, injection every other day for 2 weeks, total 7 times)

(4) Group of simvastatin injected intravenously: SIM 5mg/kg (SIM dissolved in 2% DMSO, injected every other day for 2 weeks, 7 times in total)

(5) Subcutaneous injection control 20 mu L Poloxamer 407 thermo-sensitive intelligent hydrogel (every other day injection, continuous injection for 2 weeks, total 7 times)

(6) Subcutaneous injection simvastatin group: SIM 5mg/kg (SIM is dissolved in poloxamer 407 temperature sensitive intelligent hydrogel, injected every other day, continuously injected for 2 weeks, totally 7 times)

(7) Control group for intraperitoneal injection: 1% methylcellulose (made up of sterile distilled water, injected every other day for 2 weeks, 7 times total)

(8) Intraperitoneal injection of simvastatin group: SIM 11mg/kg/day (SIM solvent is 1% methylcellulose, injected every other day for 2 weeks, 7 times in total)

(9) Bone injection empty vector control group 20 mu L poloxamer 407 thermo-sensitive intelligent hydrogel (single local injection)

(10) Bone injection simvastatin group 50 ug SIM/20 ug L poloxamer 407 thermo-sensitive intelligent hydrogel (single local injection)

The bone intervention operation comprises the following steps of weighing the weight of a mouse, carrying out intraperitoneal injection on the mouse (0.3m L/kg) subjected to anesthesia with a compound anesthetic, fixing the mouse in a supine position after anesthesia is effective, carrying out conventional skin preparation on the proximal end of a single-side tibia, carrying out alcohol disinfection, enabling the tibia to be perpendicular to a table top, touching the tuberosity of the tibia with fingers, penetrating a 1m L injector into a medullary cavity along the medullary cavity direction at the inner side edge of a patellar ligament, and slowly injecting a corresponding reagent into the medullary cavity according to the experimental design.

The body weight of the mice was monitored and recorded every two days, and after two weeks, the body condition and body weight maintenance of the breast cancer tumor-bearing mice by simvastatin by different administration routes were analyzed (fig. 1).

Test example 2

Measurement of tumor size in tumor-bearing mice

Constructing a breast cancer mouse model by the same early stage steps of test example 1 until the tumor reaches 80-100mm³Drug intervention was initiated (see test example 1 for details of steps and groups). Drug dry prognosis mouse breast cancer tumor volume was measured every two days using a vernier caliper. Using the formula V (mm)³)＝a²b/2 tumor volumes were counted and statistically analyzed (FIG. 2). The effect of simvastatin on tumor size by different routes of administration was compared.

Test example 3

Tumor collection of tumor-bearing mice

Constructing a mouse breast cancer model until the tumor reaches 80-100mm³The drug intervention is started (the steps and the groups are detailed in test example 1), after simvastatin treatment is carried out for two weeks, a substrate L uciferin (200 mu l/mouse) of luciferase is injected into the abdominal cavity of a mouse by using a micro-syringe, and after 10 minutes, the luciferase substrate reaches a tumor part through blood circulation to fully react with luciferase of tumor cells.

Test example 4

Tumor weight detection of tumor-bearing mice

Constructing a mouse breast cancer model until the tumor reaches 80-100mm³The method comprises the following steps of firstly carrying out drug intervention on mice by different drug administration ways (see test example 1 in detail in steps and groups), killing the mice by a cervical dislocation method after simvastatin treatment for two weeks, taking out tumors of the mice, respectively measuring the weights of ten groups of tumors of the mice by using a balance, carrying out statistical analysis, and determining the influence of the administration of simvastatin by different ways on the tumor weight of the tumor-bearing mice (figure 4). Specific numerical valueAs in the table below. In-group analysis: the tumor weight of mice in the group of simvastatin-injected skeleton groups was lower than that of mice in the group of control-administered skeleton groups, compared to the corresponding control group; analysis between groups: the simvastatin can not inhibit the growth of the breast cancer tumor after oral administration, tail vein injection, subcutaneous injection and intraperitoneal injection, and the simvastatin can obviously inhibit the growth of the breast cancer tumor after skeletal injection. Embodies the advantages of treating tumors by injecting simvastatin into bones.

Tumor weight in grams (g)

Test example 5

Detection of survival time of tumor-bearing mice

Constructing a mouse breast cancer model until the tumor reaches 80-100mm³Drug intervention was initiated (see test example 1 for details), and tumor-bearing mice were randomly divided into four groups of 8-9 mice each:

(3) Bone injection empty vector control group 20 mu L poloxamer 407 thermo-sensitive intelligent hydrogel (single local injection)

(4) Bone injection simvastatin group 50 ug SIM/20 ug L poloxamer 407 thermo-sensitive intelligent hydrogel (single local injection)

The status of the mice was observed and the survival of the mice was recorded, counted and analyzed (fig. 5). The effect of traditional oral statistics and skeletal administration of simvastatin on the survival of breast cancer tumor-bearing mice was compared.

Test example 6

Detection of CD8 from different sources⁺Proportion of T cells

After the modeling is finished, collecting peripheral blood of a mouse in an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, separating lymphocytes by using a lymphocyte separation solution for later use, stripping muscles around the tibia of the mouse, exposing the tibia, injecting PBS (containing 2% FBS) into a 1m L syringe, blowing out bone marrow cells, centrifuging, resuspending cell sediment by using a erythrocyte lysate, lysing erythrocytes, and screening for later use, collecting draining lymph nodes of the mouse, grinding, screening for later use, collecting spleen of the mouse, grinding, separating lymphocytes by using a mouse spleen lymphocyte separation solution kit, lysing erythrocytes, screening for later use, collecting tumor tissues of the mouse, digesting by DNaesI and Collagen D, centrifuging by Percoll, separating tumor-infiltrated lymphocytes, lysing erythrocytes, and screening for later use.

Blocking the cells at 4 deg.C for 20min, adding flow antibody at 4 deg.C for 30min, washing with PBS twice, and analyzing CD8 in the cells from the above five sources with flow cytometer⁺T(CD45⁺CD3⁺CD8⁺) Proportion of cells (FIGS. 6A-E). Definitive skeletal administration of simvastatin to CD8⁺The effect of the proportion of T cells; in addition, mouse tumor tissues were embedded and sectioned, and CD8 was detected by immunohistochemical assay⁺T cell expression, comparison of CD8 in tumors⁺Proportion of T cells.

Test example 7

Tumor infiltration CD8⁺Secretion of tumor killing factor by T cell

Collecting mouse tumor tissue, digesting with DNaesI and Collagen D, centrifuging with Percoll, separating tumor-infiltrated lymphocytes, lysing erythrocytes, stimulating cells with 50ng/m L PMA and 500 ng/m L Ionomycin, adding GolgiStop, culturing at 37 deg.C in a cell culture box for 6h, collecting cells, sealing at 4 deg.C for 20min, adding flow antibody at 4 deg.C for 30min, washing with PBS twice, analyzing tumor-infiltrated CD8 with flow cytometer⁺Perforin in T cells (CD 45)⁺CD3⁺CD8⁺Perforin⁺)、Granzyme B(CD45⁺CD3⁺CD8⁺Granzyme B⁺)、TNFα(CD45⁺CD3⁺CD8⁺TNFα⁺) And (CD 45)⁺CD3⁺CD8⁺IFN-γ⁺) The level of secretion of (a).

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Claims

1. Use of a statin compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting tumor growth and/or prolonging the life of a mammalian or human patient by intraosseous administration.

2. The use according to claim 1, wherein the statin compound includes, but is not limited to, simvastatin, atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, pitavastatin, bervastatin, cerivastatin, lovastatin, pravastatin, dalvastatin, mevastatin and/or tivastatin; preferably simvastatin, atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and/or pitavastatin; more preferably, simvastatin; the pharmaceutically acceptable salt is selected from hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, citrate, mesylate, trifluoroacetate or acetate, sodium, potassium, calcium or magnesium salt; most preferably, the statin compound or the pharmaceutically acceptable salt thereof is selected from simvastatin, atorvastatin calcium or atorvastatin sodium, fluvastatin sodium, pravastatin sodium, rosuvastatin calcium and/or pitavastatin calcium.

3. The use according to claim 1, wherein the tumor is a solid tumor; preferably lung cancer, gastric cancer, esophageal cancer, intestinal cancer, liver cancer, cervical cancer, nasopharyngeal carcinoma or breast cancer; more preferably breast cancer.

4. The use according to claim 1, wherein the medicament is in a topical injectable pharmaceutical dosage form; preferably, the pharmaceutical dosage form is injected intraosseously.

5. The use according to claim 1, wherein the medicament is in a sustained release dosage form; the carrier of the sustained release preparation is a medicinal carrier.

6. Use according to claim 1, wherein the medicament is a hydrogel, preferably a temperature-sensitive hydrogel.

7. Use according to claim 1, wherein the medicament is administered once in an interval of 7 to 600 days, preferably once in 10 to 500 days, more preferably once in 20 to 400 days, particularly preferably once in 30 to 300 days.

8. Use according to claim 1, wherein the dose of the medicament is 0.1 to 50mg, preferably 0.1 to 10mg, of statin compound per dose.

9. The use according to claim 1, wherein the medicament is to be administered only once.