WO2016095649A1 - Preparation containing isochlorogenic acid and application of preparation - Google Patents

Abstract

A preparation containing isochlorogenic acid. The preparation comprises 1 to 100 parts by weight of isochlorogenic acid and 1 to 100 parts by weight of additives. An application of the preparation in preparing drugs for treating a tumor that is any one selected from lung cancer, kidney cancer, liver cancer, uterine cancer, breast cancer, pancreatic cancer, leukemia and brain tumor.

Description

Preparation containing isochlorogenic acid and application thereof Technical field

The present invention relates to a preparation comprising isochlorogenic acid and an application thereof, and belongs to the field of medicine.

Background technique

Cancer is one of the serious diseases that threaten human health and life. In addition to surgery, modern clinical cancer treatment mainly uses radiotherapy and chemotherapy. Because toxic and side effects are usually only intermittent treatment, the compliance is poor, and it is easy to produce drug resistance. Treatment often makes the patient physically unbearable and puts a lot of mental stress on the patient, especially for patients with weak constitution. Therefore, the compliance, continuity and safety of cancer treatment have long been a difficult problem to be solved in clinical practice. At present, for the mechanism of tumor occurrence, multi-channel search for high-efficiency and low-toxic therapeutic drugs is the leading idea and focus of anti-tumor drug research and development in the pharmaceutical industry.

Tumor patients have obvious immune dysfunction and/or decline. The research on anti-tumor biological response regulators for patients with abnormal immune function has become one of the research and development focuses on anti-tumor drugs at home and abroad.

Summary of the invention

It is an object of the present invention to provide a formulation comprising an isochlorogenic acid comprising the following components:

Isochlorogenic acid 1-100 parts by weight

Excipients 1-100 parts by weight.

Isochlorogenic acid is widely present in plants such as Lonicera, Compositae, and Leguminosae. It is formed by the condensation of two caffeic acids with a quinic acid and has obvious pharmacological activity. In the process of screening anti-tumor drugs, the present inventors unexpectedly found that isochlorogenic acid has a significant inhibitory effect on tumors, especially tumors with immunodeficiency, and the inhibitory effect is particularly remarkable. The present invention has been completed based on the above findings.

In the present invention, the isochlorogenic acid specifically means any one or more of isochlorogenic acid a, isochlorogenic acid b or isochlorogenic acid c.

In the present invention, the isochlorogenic acid a is extracted from plants or chemically synthesized, and has the following chemical structural formula:

Molecular formula: C ₂₅ H ₂₄ O ₁₂ , molecular weight: 516.45.

The isochlorogenic acid b is extracted or chemically synthesized from plants and has the following chemical structural formula:

Molecular formula: C ₂₅ H ₂₄ O ₁₂ , molecular weight: 516.45.

Isochlorogenic acid c is extracted from plants or chemically synthesized and has the following chemical structural formula:

Molecular formula: C ₂₅ H ₂₄ O ₁₂ , molecular weight: 516.45.

In the present invention, the preparation may be an oral preparation or an injection; wherein the oral preparation includes, but is not limited to, a tablet, a granule, a granule or a capsule, and the injection includes an injection solution and a powder injection.

In the present invention, the excipients can be appropriately adjusted depending on the dosage form. Specifically, when the preparation is an oral preparation, the auxiliary material includes a filler, a binder, and a lubricant; wherein the filler is selected from the group consisting of mannitol, lactose, starch, dextrin, and microcrystalline cellulose. Or several; the binder is selected from one or more of polyvinylpyrrolidone, cellulose derivatives, starch; the lubricant is selected from magnesium stearate, micronized silica gel, talc, magnesium lauryl sulfate or Several. When the preparation is an injection, the excipient includes a diluent, a scaffold, and an antioxidant; wherein the diluent is selected from one or more of water for injection, physiological saline, aqueous dextrose, glycerin; One or more of mannitol, glucose, and lactose; and the antioxidant is one or more selected from the group consisting of sodium hydrogen sulfite, sodium metabisulfite, and vitamin C.

In the present invention, preferably, the isochlorogenic acid may be 15, 30, 50, 60, 80 or 90 parts by weight; the excipient may be 10, 20, 35, 55, 65, 70, 85 or 95 Parts by weight.

In a specific embodiment of the invention, the formulation comprises:

Isochlorogenic acid a 20 parts by weight

Mannitol 15 parts by weight

Hydroxypropyl cellulose 10 parts by weight

Starch 5 parts by weight

Micronized silica gel 0.5 parts by weight.

In another embodiment of the invention, the formulation comprises:

Isochlorogenic acid a 10 parts by weight

Mannitol 10 parts by weight

Sodium metabisulfite 0.5 parts by weight.

In another embodiment of the invention, the formulation comprises:

Isochlorogenic acid b 50 parts by weight

Vitamin c 5 parts by weight.

In another embodiment of the invention, the formulation comprises:

Isochlorogenic acid b 30 parts by weight

Starch 20 parts by weight

Talc powder 1 part by weight.

In another embodiment of the invention, the formulation comprises:

In another embodiment of the invention, the formulation comprises:

Isochlorogenic acid c 40 parts by weight

Starch 50 parts by weight

Microcrystalline cellulose 20 parts by weight

Talc powder 1 part by weight.

In another embodiment of the invention, the formulation comprises:

In another embodiment of the invention, the formulation comprises:

In another embodiment of the invention, the formulation comprises:

Isochlorogenic acid a 5 parts by weight

Isochlorogenic acid c 25 parts by weight

Lactose 100 parts by weight.

In another embodiment of the invention, the formulation comprises:

Isochlorogenic acid b 20 parts by weight

Isochlorogenic acid c 20 parts by weight

Starch 30 parts by weight

Microcrystalline cellulose 10 parts by weight

PVP 10 parts by weight

Magnesium stearate 0.5 parts by weight.

In another embodiment of the invention, the formulation comprises:

Isochlorogenic acid b 30 parts by weight

Isochlorogenic acid c 50 parts by weight

Sodium metabisulfite 3 parts by weight.

In another embodiment of the invention, the formulation comprises:

In another embodiment of the invention, the formulation comprises:

Another object of the present invention is to provide a use of a formulation for the preparation of a medicament for treating a tumor. Applicants have found that the preparation can activate various immune effector T cells, macrophages, NK cells and B cells by up-regulating the key genes CaN and NFAT in the immune-related signaling pathway, thereby improving Th1. Expression of cytokines (IFN-γ and IL-2), inhibits the expression of Th2 type cytokines (IL-4 and IL-10), changes the state of Th2 hyperthyroidism, enhances the body's immune response and anti-tumor response, and is immune to Defective tumors have a good inhibitory effect. It inhibits the development of tumor by improving the immune response of the body and is suitable for tumors with immunodeficiency. Among them, tumors with immunodeficiency include lung cancer, kidney cancer, liver cancer, uterine cancer, breast cancer, pancreatic cancer, leukemia, and cerebral tumor. .

In the present invention, the oral preparation of the preparation is administered in a clinical dose of 2-20 mg/kg; and the clinical dose of the injection is 1-10 mg/kg.

The beneficial effects of the present invention are that the preparation has a good inhibitory effect on tumors having immunodeficiency. It inhibits the development of tumor by improving the body's immune response and is suitable for tumors with immunodeficiency.

detailed description

Example 1 prescription one

After the isochlorogenic acid a is added to the mannitol or hydroxypropyl cellulose, it is sieved and mixed, added to the starch slurry for granulation, dried, and granulated, and the micro-silica gel is added and compressed.

Example 2 prescription 2

The isochlorogenic acid a, mannitol and sodium metabisulfite are dissolved in an appropriate amount of water, filtered through an ultrafiltration membrane, aseptically filled, freeze-dried, and capped.

Example 3 prescription three

The isochlorogenic acid b and vitamin C are dissolved in water for injection, activated carbon adsorption, ultrafiltration membrane filtration, and aseptic filling.

Example 4 prescription four

The isochlorogenic acid b, starch and talc are sieved and filled to fill the capsules.

Example 5 prescription five

After the isochlorogenic acid c and lactose, mannitol and dextrin are sieved and mixed, pvp aqueous solution is added, wet granulation, drying, granulation and bagging.

Example 6 prescription six

The isochlorogenic acid c is added to the starch, and the microcrystalline cellulose is sieved and mixed, granulated, granulated, and talc powder is added and compressed.

Example 7 prescription seven

The isochlorogenic acid a, the isochlorogenic acid b, the lactose, the mannitol, and the methyl cellulose are sieved, granulated, and granulated, and the magnesium lauryl sulfate is added and tableted.

Example 8 prescription eight

The isochlorogenic acid a, the isochlorogenic acid b, the lactose, and the dextrin are sieved and mixed, and then added to the starch slurry for wet granulation, dried, granulated, and bagged.

Example 9 prescription nine

After adding isochlorogenic acid a and isochlorogenic acid c to lactose, it is dissolved in an appropriate amount of water, filtered through an ultrafiltration membrane, aseptically filled, freeze-dried, and rolled.

Example 10 prescription ten

The isochlorogenic acid a and the isochlorogenic acid c are sieved and mixed, and the capsules are filled.

Example 11 prescription eleven

The isochlorogenic acid b and the isochlorogenic acid c are added to starch, microcrystalline cellulose, pvp and magnesium stearate, and then sieved and mixed, and tableted.

Example 12 prescription twelve

The isochlorogenic acid b and the isochlorogenic acid c are added to sodium metabisulfite, dissolved in physiological saline, filtered through an ultrafiltration membrane, and aseptically filled.

Example 13 prescription thirteen

After adding isochlorogenic acid a, isochlorogenic acid b and isochlorogenic acid c to sodium hydrogen sulfite, it is dissolved in an appropriate amount of water, filtered through an ultrafiltration membrane, aseptically filled, freeze-dried, and rolled.

Example 14 prescription fourteen

Adding isochlorogenic acid a, isochlorogenic acid b and isochlorogenic acid c to lactose, low-substituted hydroxypropyl cellulose, pvp, sieving and mixing, wet granulation, drying, granulating, adding micro-silica gel, tableting .

Example 15 prescription fifteen

After adding isochlorogenic acid a, isochlorogenic acid b and isochlorogenic acid c to lactose, mannitol and pvp, sieving, granulating, granulating and bagging.

Example 16

Animal C57BL/6 mice, half male and half female, weighing 13-20 g.

The cell line Lewis cells are mouse lung cancer cell lines, which are adherently grown in RPMI-1640 medium containing 10% calf serum, 100 U·ml ^-1 penicillin, 100 μg·ml ^-1 streptomycin at 37 ° C, 5 Incubate in %C0 ₂ incubator and change it every 2 to 3 days. After the Lewis cells in the logarithmic growth phase were digested with 0.25% trypsin, the digestion was terminated, RPMI-1640 medium was added, the cells were blown to make a suspension, centrifuged at 1000 rpm for 5 minutes, washed twice, and trypan blue staining was performed. Count the number of viable cells. The cell suspension was inoculated subcutaneously into the left forelimb of the mouse, 0.2 ml·only ^-1 (about 1×10 ⁷ cells), and the mice were used as experimental mice when the tumor grew 1 cm×1 cm×1 cm. The mice were aseptically stripped and washed with Hank's solution for 3 times to remove blood stains, fat and necrotic tissue. The tumor was cut into pieces of 1 mm × 1 mm × 1 mm, and Hank's solution was washed twice, and physiological saline (1 g) was added in proportion. : 3 ml), and then ground in a glass homogenizer, and filtered through an 80-100 mesh sieve to prepare a single cell suspension, and the number of viable cells was counted by trypan blue staining.

(3) Administration experiment

The prepared cell suspension was inoculated into the left anterior axilla of C57BL/6 mice by 0.2 ml·only ^-1 (about 1×10 ⁷ cells), and randomly divided into groups according to body weight, each group was 10, which were different. Chlorogenic acid group a, isochlorogenic acid b group, isochlorogenic acid group c, isochlorogenic acid ab combination group, isochlorogenic acid ac combination group, isochlorogenic acid bc combination group, isochlorogenic acid abc combination Group, negative group (NS saline). On the second day after inoculation, each group was intraperitoneally injected (ip) with a volume of 0.2 ml·10 g ^-1 once daily for 12 consecutive administrations. The tumor volume was measured on the 6th day after the inoculation (V=0.52×the long diameter of the transplanted tumor L×the shortest diameter W ^{2 of the} transplanted tumor), and the average tumor of the negative group was greater than 1.0 g (the tumor volume was about 0.5 cm ³ ). The experiment was stopped, the mice were sacrificed by cervical dislocation and weighed, the tumor was removed, and the tumor inhibition rate was calculated.

Tumor inhibition rate%=[1-(mean tumor weight of the administration group/average tumor weight of the negative group)]×100%

(4) Test results

Table 1 Effect of isochlorogenic acid on the weight and tumor inhibition rate of Lewis lung cancer C57BL/6 mice

Compared with the negative group *p<0.05, **p<0.01

(5 Conclusion

The results showed that isochlorogenic acid a, isochlorogenic acid b, isochlorogenic acid c and their compositions had significant inhibitory effects on Lewis lung cancer C57BL/6 mice xenografts.

Example 17 Liver Cancer

(1) Animal Kunming mice, 1 male and female, weighing 16 ~ 27g.

The cell line H22 cell line is a mouse hepatoma cell, which is suspended in RPMI-1640 medium and contains 10% calf serum, 100 U·ml ^-1 penicillin and 100 μg·ml ^-1 streptomycin at 37 ° C, 5%. C0 ₂ incubator culture, every 2 to 3 days change the liquid and pass it once. H22 cells in the logarithmic growth phase were harvested, centrifuged at 1000 rpm for 5 minutes, washed twice, and the number of viable cells was counted by trypan blue staining. Inoculated in the peritoneal cavity of KM mice as soon as possible, 7 to 10 days later, ascites was taken in the left forelimb of the mouse, and the ascites cell suspension was inoculated 0.2 ml·only ^-1 (about 2×10 ⁷ cells), and the tumor grew 1 cm. When used at 1 cm × 1 cm, it was used as a breeding mouse for experiments. The mice were aseptically stripped and washed with Hank's solution for 3 times to remove blood stains, fat and necrotic tissue. The tumor was cut into 1 mm × 1 mm × 1 mm pieces, Hank's solution was washed twice, and physiological saline was added in proportion (1 g: 3 ml). Then, it was ground in a glass homogenizer, filtered through an 80-100 mesh screen to prepare a single cell suspension, and the number of viable cells was counted by trypan blue staining.

Drug delivery experiment

The prepared cell suspension was inoculated into the left anterior axilla of 69 KM mice according to 0.2 ml·only ^-1 (about 1×10 ⁶ cells), and randomly divided into groups according to body weight, each group was 10, which were different. Chlorogenic acid group a, isochlorogenic acid b group, isochlorogenic acid group c, isochlorogenic acid ab combination group, isochlorogenic acid ac combination group, isochlorogenic acid bc combination group, isochlorogenic acid abc combination Group and negative group (NS). The intragastric administration was started on the second day after the inoculation, once a day for 9 days. The tumor volume was measured on the 5th day after the inoculation (V=0.52×the long diameter of the transplanted tumor L×the shortest diameter W ^{2 of the} transplanted tumor), and the average tumor of the negative group was greater than 1.0 g (the tumor volume was about 0.5 cm ³ ). The experiment was stopped, the mice were sacrificed by cervical dislocation and weighed, the tumor was removed, and the tumor inhibition rate was calculated.

Tumor inhibition rate%=[1-(mean tumor weight of the administration group/average tumor weight of the negative group)]×100%

(4) Test results

Table 2 Effect of isochlorogenic acid on the weight of transplanted tumor of H22 liver cancer KM mice

Compared with the negative group *p<0.05, **p<0.01

(5 Conclusion

The results showed that isochlorogenic acid a, isochlorogenic acid b, isochlorogenic acid c and their compositions had significant inhibitory effects on H22 liver cancer KM mouse xenografts.

Example 18 breast cancer

(1) Animal BABLc mice, half male and half female, weighing 17-21 g.

The cell line EMT-6 cell line was mouse breast cancer cells, which were adherently grown in RPMI-1640 with 10% calf serum, 1 mmol/L glutamine, 100 U·ml ^-1 penicillin and 100 μg·ml ^-1 . In the medium, culture in 37 ° C, 5% CO ₂ incubator, change the liquid every 2 to 3 days. The EMT-6 cells in the logarithmic growth phase were digested with 0.25% trypsin, the digestion was terminated, the RPMI-1640 medium was added, the cells were blown to make a suspension, centrifuged at 1000 rpm for 5 minutes, and washed twice. The number of viable cells was counted by pan blue staining. As soon as possible, the cell suspension of the left forelimb of the mouse was inoculated subcutaneously with 0.2 ml·only ^-1 (about 1×10 ⁷ cells), and the mice were used as experimental mice when the tumor grew 1 cm×1 cm×1 cm. The mice were aseptically stripped and washed with Hank's solution for 3 times to remove blood stains, fat and necrotic tissue. The tumor was cut into 1 mm × 1 mm × 1 mm pieces, Hank's solution was washed twice, and physiological saline was added in proportion (1 g: 3 ml). Then, it was ground in a glass homogenizer, filtered through an 80-100 mesh screen to prepare a single cell suspension, and the number of viable cells was counted by trypan blue staining.

Drug delivery experiment

The prepared cell suspension was inoculated into the left anterior axilla of 70 BABLc mice by 0.2 ml·only ^-1 (containing about 1×10 ⁶ cells), and randomly divided into groups according to body weight, each group was 10, which were different. Chlorogenic acid group a, isochlorogenic acid b group, isochlorogenic acid group c, isochlorogenic acid ab combination group, isochlorogenic acid ac combination group, isochlorogenic acid bc combination group, isochlorogenic acid abc combination Group and negative group (NS). The intravenous (iv) administration was started on the second day after the inoculation, and the volume was 0.2 ml·10 g ^-1 once a day for 9 days. The tumor volume was measured on the 6th day after inoculation (V=0.52×transplantation tumor long diameter L×transplantation tumor shortest diameter W ² ), and the experiment was stopped when the average tumor volume of the negative group was greater than 1.0 g (tumor volume was about 0.5 cm ³ ). The mice were sacrificed by cervical dislocation and weighed, the tumor was removed, and the tumor inhibition rate was calculated.

Tumor inhibition rate%=[1-(mean tumor weight of the administration group/average tumor weight of the negative group)]×100%

Table 3 Effect of isochlorogenic acid on the weight and tumor inhibition rate of transplanted tumor of EMT-6 breast cancer BABLc mice

Compared with the negative group *p<0.05, **p<0.01

(5 Conclusion

The results showed that isochlorogenic acid a, isochlorogenic acid b, isochlorogenic acid c and their compositions had significant inhibitory effects on transplanted tumors of EMT-6 breast cancer BABLc mice.

Claims (10)

1. A formulation comprising an isochlorogenic acid comprising the following components:

   Isochlorogenic acid 1-100 parts by weight

   Excipients 1-100 parts by weight.
2. The preparation according to claim 1, wherein the isochlorogenic acid is selected from any one or more of isochlorogenic acid a, isochlorogenic acid b or isochlorogenic acid c.
3. The preparation according to claim 1 or 2, wherein the isochlorogenic acid may be 15, 30, 50, 60, 80 or 90 parts by weight; the excipient may be 10, 20, 35, 55, 65, 70 , 85 or 95 parts by weight.
4. The preparation according to any one of claims 1 to 3, wherein the preparation is an oral preparation or an injection; preferably, the oral preparation is a tablet, granule, granule or capsule, and preferably the injection is an injection or a powder injection.
5. The preparation according to any one of claims 1 to 4, wherein when the preparation is an oral preparation, the adjuvant is a filler, a binder, and/or a lubricant; when the preparation is an injection, the adjuvant is Diluent, scaffolding and/or antioxidants.
6. The preparation according to claim 4, wherein said filler is selected from one or more of mannitol, lactose, starch, dextrin, and microcrystalline cellulose; and the binder is selected from the group consisting of polyvinylpyrrolidone and cellulose derivatives. One or more of the starch; the lubricant is selected from the group consisting of magnesium stearate, micronized silica gel, talc, magnesium lauryl sulfate, and the diluent is selected from the group consisting of water for injection, physiological saline, One or more of glucose water liquid and glycerin; the scaffolding agent is one or more selected from the group consisting of mannitol, glucose, and lactose; and the antioxidant is selected from one of sodium hydrogen sulfite, sodium metabisulfite, and vitamin C. Several.
7. The formulation of any of claims 1-6, wherein the formulation comprises:

   10 parts by weight of isochlorogenic acid, 15 parts by weight of mannitol, 10 parts by weight of hydroxypropyl cellulose, 5 parts by weight of starch, 0.5 parts by weight of micronized silica gel;

   Or include: 10 parts by weight of isochlorogenic acid a, 10 parts by weight of mannitol, 0.5 parts by weight of sodium metabisulfite;

   Or include: 50 parts by weight of isochlorogenic acid b, and 5 parts by weight of vitamin C;

   Or include: 30 parts by weight of isochlorogenic acid b, 20 parts by weight of starch, and 1 part by weight of talc;

   Or include: 5 parts by weight of isochlorogenic acid c, 20 parts by weight of lactose, 10 parts by weight of mannitol, 10 parts by weight of dextrin, and 1 part by weight of PVP;

   Or comprising: 40 parts by weight of isochlorogenic acid c, 50 parts by weight of starch, 20 parts by weight of microcrystalline cellulose, and 1 part by weight of talc;

   Or include: 5 parts by weight of isochlorogenic acid a, 15 parts by weight of isochlorogenic acid b, 30 parts by weight of lactose, 20 parts by weight of mannitol, 15 parts by weight of methyl cellulose, 0.5 parts by weight of magnesium lauryl sulfate;

   Or it may include: 3.5 parts by weight of isochlorogenic acid a, 1.5 parts by weight of isochlorogenic acid b, 25 parts by weight of lactose, 20 parts by weight of dextrin, and 2 parts by weight of starch.
8. The preparation according to any one of claims 1 to 6, wherein the preparation comprises: 5 parts by weight of isochlorogenic acid, 25 parts by weight of isochlorogenic acid c, and 100 parts by weight of lactose;

   Or include: 20 parts by weight of isochlorogenic acid b, 20 parts by weight of isochlorogenic acid c, 30 parts by weight of starch, 10 parts by weight of microcrystalline cellulose, 10 parts by weight of PVP, 0.5 parts by weight of magnesium stearate;

   Or include: 30 parts by weight of isochlorogenic acid b, 50 parts by weight of isochlorogenic acid c, and 3 parts by weight of sodium metabisulfite;

   Or include: 5 parts by weight of isochlorogenic acid a, 10 parts by weight of isochlorogenic acid b, 35 parts by weight of isochlorogenic acid c, and 1 part by weight of sodium hydrogen sulfite;

   Or include: isochlorogenic acid a 20 parts by weight, isochlorogenic acid b 10 parts by weight, isochlorogenic acid c 10 parts by weight, lactose 30 parts by weight, mannitol 20 parts by weight, low substituted hydroxypropyl cellulose 10 weight Parts, PVP 3 parts by weight, 0.5 parts by weight of micronized silica gel.
9. Use of a formulation according to any one of claims 1-8 for the manufacture of a medicament for the treatment of a tumor.
10. The use according to claim 9, wherein the tumor is selected from the group consisting of lung cancer, kidney cancer, liver cancer, uterine cancer, breast cancer, pancreatic cancer, leukemia, and cerebral tumor.