CN108992672B - Wogonin pharmaceutical composition for treating tumor - Google Patents
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Abstract
The invention relates to a wogonin pharmaceutical composition for treating neoplastic diseases, which comprises the following components in parts by weight: 20-30 parts of wogonin, 80-120 parts of L-arginine, 30-50 parts of mannitol and 2200-2800 parts of water for injection; the pharmaceutical composition has the advantages of obvious curative effect and small toxic and side effect.
Description
Technical Field
The invention belongs to the field of medicinal preparations, and particularly relates to a wogonin medicinal composition for treating neoplastic diseases.
Background
Malignant tumor is a disease seriously threatening human health, the incidence rate of the disease is increased year by year in recent years, and the death of the malignant tumor is second to heart disease and ranks leading ahead of death due to the direct cause in many developed countries. In China, malignant tumors are also listed as the first and second causes of death. Malignant tumor seriously harms human health, and although comprehensive treatment can be carried out through operation, chemotherapy, radiotherapy and the like, the cure rate is still very low. The selectivity of the anti-cancer western medicines which are commonly used clinically at present is not high, and the anti-cancer western medicines can damage normal cells of an organism, particularly cells with vigorous proliferation while killing tumor cells. The long-term use of the drugs also brings great toxic and side effects to tumor patients, which causes poor compliance of the drugs, so that the search for a high-efficiency low-toxicity drug for treating tumors is very necessary.
Wogonin, as a natural flavonoid compound, is one of the effective components of the traditional Chinese herbal medicine scutellaria baicalensis, and has various effects of resisting bacteria, viruses and tumors, relieving fever and pain, resisting oxidation, scavenging oxygen free radicals, treating cardiovascular diseases and the like. Wogonin has obvious inhibition effect on the growth of a plurality of in vitro cultured tumor cells (human gastric cancer cell strains MGC-803 and BGC-823, human liver cancer cell strain HepG2, human liver cancer 5-FU drug-resistant strain BEL-7402/5-FU, human colon cancer cell strains SW1116 and HCT116, human prostate cancer cell strain DU145, human lung cancer cell strain H460, human breast cancer cell strains MDA-MB-231 and MCF-7, human malignant melanoma cell strain A375, human leukemia cell strains HL60 and K562 and genetic variant cell strain NIH Swiss mouse embryo cell NIH-3T3), but has small influence on normal cells. Because the content of wogonin in the traditional Chinese medicine scutellaria is lower. The wogonin is synthesized by using cheap pyrogallic acid as a raw material (the specific synthesis process can be seen in the patent of CN 1915987A), and the raw material is provided for large-scale production. However, the wogonin is clinically used in the prior tablet, capsule, injection and other dosage forms, and the dosage forms have poor dissolution rate in the gastrointestinal tract and very low bioavailability. Wogonin is unstable in solution and cannot be prepared into injection all the time, so that a preparation with good stability and small toxic and side effects is urgently needed to be developed.
Disclosure of Invention
The invention aims to provide a wogonin medicinal composition for treating neoplastic diseases, which has the advantages of stable and reliable quality, remarkable curative effect and small toxic and side effects.
The wogonin raw material used in the invention is prepared by a chemical synthesis method, and the specific method is that pyrogallic acid is subjected to benzylation, reoxidation, reduction and methylation to prepare an intermediate, and then a series of steps such as removing benzyl by hydrogenolysis and the like are carried out to obtain the wogonin raw material with the purity of more than 99%.
The technical scheme of the patent application of the invention is as follows:
the pharmaceutical composition comprises the following components in parts by weight: 10-40 parts of wogonin, 20-200 parts of cosolvent, 20-60 parts of framework material and 2000-3000 parts of water for injection.
Preferably, the pharmaceutical composition comprises the following components in parts by weight: 20-30 parts of wogonin, 80-120 parts of cosolvent, 30-50 parts of framework material and 2200-2800 parts of injection water.
The cosolvent in the pharmaceutical composition is one or more of sodium bicarbonate, glycerol, sodium phosphate, L-arginine and citric acid.
The skeleton material in the pharmaceutical composition is one or more of lactose, glucose, sorbitol and mannitol.
The pharmaceutical composition comprises the following components in parts by weight: 25 parts of wogonin, 100 parts of L-arginine, 40 parts of mannitol and 2500 parts of water for injection.
The dosage form of the pharmaceutical composition is freeze-dried powder or injection.
The preparation method of the pharmaceutical composition comprises the following process steps:
the preparation method comprises the steps of mixing wogonin and a cosolvent, adding a small amount of water for injection to wet, adding the water for injection, and stirring to completely dissolve a medicine to obtain a mixed solution;
adding a framework material into the mixed solution obtained in the step, stirring for dissolving, adding activated carbon, stirring for removing a pyrogen, removing carbon, performing suction filtration, and adding a proper amount of injection water for constant volume configuration; filtering with filter membrane for sterilization to obtain filtrate, and lyophilizing to obtain lyophilized powder.
The preparation method comprises the following process steps of freeze drying: freezing for 3-6 h at a freezing temperature of-60 to-40 ℃; and maintaining the freezing temperature of-30 to-0 ℃, the vacuum degree of 10 to 20pa and the freezing time of 15 to 30 hours; continuously keeping the vacuum degree at 10-20 pa, freezing at 0-20 ℃, and freezing for 4-8 h; and then gradually heating to 25-35 ℃, drying for 5-8 h, and carrying out vacuum tamponade to obtain the freeze-dried powder.
The preparation method is carried out in a nitrogen environment.
After a large number of experiments, the inventor of the present application finds out a wogonin pharmaceutical composition prescription with stable quality and good curative effect, and the following is a part of research data preferred by the experiments of the present invention.
1.1 Co-solvent screening
Taking the prescription dose of wogonin and cosolvent, putting into a container, adding a proper amount of water for injection, heating to dissolve, and adding the water for injection to a sufficient amount.
TABLE 1 screening of Co-solvents
Results and analysis: during the test, various cosolvents were used and the amounts were adjusted so that the sodium bicarbonate and arginine were able to dissolve all of the wogonin. However, when sodium bicarbonate is used, the drug solution is placed at room temperature for a period of time, and the drug is quickly crystallized, which indicates that the dissolution assisting effect is not good; the arginine has good solubilizing effect, so that a clear and transparent drug solution which is stable and does not precipitate at room temperature can be obtained, but the pH value of the solution is too high when the amount of the arginine is too large, so that the arginine is possibly not suitable for clinical use; when the weight ratio of wogonin to arginine is 1:4, the medicine can be completely dissolved, and the pH value of the liquid medicine is proper, so that the cosolvent in the prescription is arginine, and the dosage of the cosolvent is 100mg per bottle.
1.2 framework materials screening
Taking 25mg of wogonin and 100mg of L-arginine according to the prescription amount, putting into a container, adding boiled water for injection, stirring, and keeping the temperature at 100 ℃ until the wogonin and the L-arginine are dissolved; cooling, adding appropriate amount of mannitol, stirring for dissolving, and adding injectable water until the volume of injectable water is 2.5 mL. Measuring the osmotic pressure of each liquid medicine; and (3) subpackaging the liquid medicines into penicillin bottles, conveying the penicillin bottles into a freeze dryer for freeze drying, and inspecting the formability and the redissolution property of the wogonin.
TABLE 2 relationship between mannitol content in wogonin solution and osmotic pressure, moldability and re-solubility
Description of the drawings: the checking method of re-solubility is to add 2.5mL of distilled water into the freeze-dried powder, shake and observe the dissolution condition of the freeze-dried powder. As a result, all of the lyophilized powders prepared by the above formulations were dissolved, but the dissolution rates were different. We denote by ×) slow dissolution; indicates slower dissolution; indicates faster dissolution; indicates rapid dissolution.
The results of the above experiments, Table 2, show that the osmotic pressure gradually increases with increasing mannitol dosage. When the dosage of the mannitol is 40-60mg, the osmotic pressure of the solution is between 280-320mOsm/kg, the osmotic pressure requirement is met, and the dosage of the mannitol in the formula is finally determined to be 40mg added in each 2.5mL of the solution in consideration of indexes such as formability, re-solubility and the like.
1.3 microporous Membrane adsorption test investigation
Taking 25mg of wogonin and 100mg of L-arginine according to the prescription amount, putting into a suitable container, and adding boiled water for injection to 2.5ml for dissolving. Adding 40mg of mannitol, stirring uniformly, dividing the sample solution into two parts, one part is not treated, the other part is filtered by a 0.22 mu m microporous membrane, after the two parts of solution are diluted properly, measuring ultraviolet absorption at 275nm, and inspecting whether the medicine is lost before and after the microporous membrane filtration. The results are shown in Table 3.
TABLE 3 examination results of microporous membrane adsorption test
The test results show that the medicament is not lost before and after the filtration by the microporous filter membrane, and the microporous filter membrane with the diameter of 0.22 mu m does not adsorb the main medicament, so the microporous filter membrane can be used for the aseptic filtration of the liquid medicament in the process.
1.4 activated carbon adsorption test investigation
Dividing the medicinal solution prepared according to the prescription into four parts, respectively adding activated carbon for injection according to 0.0%, 0.1%, 0.3% and 0.5%, stirring for 20 min, removing carbon, properly diluting the solution, measuring ultraviolet absorption at 275nm, and inspecting the adsorption condition of the activated carbon to the medicine. The results are shown in Table 4.
TABLE 4 results of activated carbon adsorption test
The above test results show that the adsorption amount of the drug is gradually increased as the concentration of the activated carbon is increased, so that the activated carbon with a low concentration should be selected as much as possible while removing pyrogens. From the practical point of view, 0.1 percent of activated carbon is finally selected. Meanwhile, in order to ensure the drug content in the final product, the dosage of the wogonin is increased by 10 percent.
1.5 Freeze-drying Curve plotting
Taking 25mg of wogonin and 100mg of L-arginine according to the prescription amount, putting into a suitable container, and adding boiled water for injection to 2.5ml for dissolution. Adding mannitol 40mg, stirring, filtering with microporous membrane to obtain wogonin solution for injection, placing the sample in freeze dryer, inserting eutectic point and temperature probe, closing the box door, opening the freeze dryer, cooling with heat-conducting oil, and measuring the eutectic point at about-11 deg.C. Reducing the temperature of the sample to be lower than-38 ℃, preserving the heat for 2 hours, then starting a condenser, starting a vacuum system when the condensation temperature is lower than-40 ℃, and controlling the vacuum to be 15Pa +/-4 Pa; and (4) heating the heat-conducting oil to-11 ℃, preserving the heat for 14 hours, and after the temperature of the product rises to the eutectic point temperature for 1 hour, finishing sublimation. After sublimation, the temperature of the heat-conducting oil is raised to 40 ℃, the temperature of the product is raised to 30 ℃, heat is preserved for 6 hours in a timing mode, and the product is subjected to plugging, air release and box discharge.
1.6 Pilot sample preparation
On the basis of prescription process screening, three batches of pilot samples are prepared, and the prescription process is as follows: placing wogonin 25mg and L-arginine 100mg in a suitable container, adding boiling water for injection 2200ml, stirring to dissolve; adding mannitol 40mg, and stirring to dissolve; adding 0.1% of active carbon for a needle, stirring for 20 minutes, decarburizing by using a titanium sand core, performing suction filtration on the decarburized active carbon into a clean container, adding 2500ml of water for injection, stirring the solution for 5 minutes till the solution is uniform, filtering the solution by using a 0.22 mu m microporous membrane, filling filtrate into 10ml penicillin bottles, wherein each bottle is 2.5ml, partially plugging butyl rubber plugs, conveying the bottles onto a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Lyophilization was performed according to the lyophilization profile and the results of three sample runs are shown in Table 5 below.
TABLE 5 three sample yield and appearance inspection
1.7 photostability experiment
Taking appropriate amount of wogonin and L-arginine according to the formula proportion, placing in a container, adding boiling water for injection, stirring, keeping at 100 deg.C until it is dissolved, taking down, cooling to room temperature, adding mannitol, stirring for dissolving, and adding water for injection to a sufficient amount. Recording as zero; the sample was placed under the illumination of 4500Lx for 24h, and samples were taken at intervals to determine the content, substances of interest and pH of the sample. And (5) investigating the stability of the wogonin solution to light. The results are shown in Table 6.
TABLE 6 photostability of wogonin solution
The above results in Table 6 show that after 24h of strong light irradiation, the content of the product has no obvious change, the related substances have slight increase, and the pH of the solution has no obvious change, which indicates that the wogonin solution is relatively stable to light, and the production process of the wogonin preparation does not need to be protected from light.
1.8 production Process stability Studies
Taking 25mg of wogonin, 100mg of L-arginine, 40mg of mannitol and 2.5mL of water for injection according to the formula proportion, putting a proper amount of wogonin and L-arginine into a container, adding boiled water for injection, stirring, keeping at 100 ℃ until the materials are dissolved, taking down, cooling to room temperature, adding mannitol, stirring to dissolve, and adding water for injection to a sufficient amount. The wogonin solution prepared above is placed at 25 ℃, samples are taken at intervals, and the content, related substances and pH value of the samples are measured. The results are shown in Table 7.
TABLE 7 stability of wogonin solution during production
And (4) analyzing results: the prescription and the production process of the wogonin pharmaceutical composition for injection are preliminarily determined through the prior process exploration. Considering that a period of time may be required from preparation of the drug solution to filling and boxing for freezing in an actual production process, stability of the drug solution in the process needs to be examined. As a result, the solution of the product is placed at 25 ℃ for 24 hours, the content of the solution is not obviously changed, related substances meet the limit requirements, and the pH value of the solution is not obviously changed, which indicates that the product has better production stability and feasible process.
The beneficial effect of this application does:
based on the prior art, the inventor of the application carries out further research and screening on a wogonin injection prescription, and finally screens out a wogonin pharmaceutical composition with stable quality and reliability. The pharmaceutical composition of the invention has reasonable prescription proportion, and pharmacological experiments prove that: the wogonin medicine composition has obvious growth inhibiting effect on various tumor cells such as human gastric cancer MGC-803 tumor cells, human lung cancer H460 nude mouse xenograft tumor, liver cancer SMMC-7721 tumor cells, human gastric cancer BGC-823 nude mouse xenograft tumor, human liver cancer SMMC-7721 nude mouse xenograft tumor and the like, and the medicine composition has obvious curative effect. Toxicological experiment results show that the pharmaceutical composition has the advantage of small toxic and side effects.
The wogonin injection preparation prepared by the optimal preparation method has reliable quality and good stability.
Detailed Description
The following are specific examples of the present disclosure, which are used to illustrate technical solutions to be solved in the present disclosure and help those skilled in the art understand the present disclosure, but the present disclosure is not limited to these examples. The preparation process in the embodiments 1 to 9 should be carried out under nitrogen atmosphere.
Example 1
Placing 25g wogonin and 100g L-arginine in a container, adding boiling water for injection, and stirring to dissolve; adding 40g of mannitol, stirring for dissolving, adding 0.1% of activated carbon for a needle, stirring for 20 minutes, decarburizing by using a titanium sand core, pumping into a clean container, adding water for injection to 2500ml, stirring the solution for 5 minutes to be uniform, filtering by using a 0.22 mu m microporous filter membrane, filling filtrate into penicillin bottles with 2.5ml of each bottle, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing at-50 deg.C for 4 h; maintaining the freezing temperature at-20 deg.C, vacuum degree at 15pa, and freezing time at 15 h; continuously keeping the vacuum degree at 15pa, freezing at-10 ℃ for 6 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 2
Placing 30g wogonin and 120g L-arginine in a suitable container, adding boiling water for injection, and stirring to dissolve; adding 50g of mannitol, stirring for dissolving, adding 0.1% of activated carbon for a needle, stirring for 20 minutes, decarburizing by using a titanium sand core, pumping into a clean container, adding water for injection to 2200ml, stirring the solution for 5 minutes to be uniform, filtering by using a 0.22-micron microporous membrane, filling filtrate into penicillin bottles with 2.5ml per bottle, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing at-60 deg.C for 3 h; maintaining the freezing temperature at 0 deg.C, vacuum degree at 20pa, and freezing time at 30 h; continuously keeping the vacuum degree at 10pa, freezing at 0 deg.C for 4 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 3
Placing 20g wogonin and 80g L-arginine in a container, adding boiling water for injection, and stirring to dissolve; adding 30g of mannitol, stirring for dissolving, adding 0.1% of activated carbon for a needle, stirring for 20 minutes, decarburizing by using a titanium sand core, pumping into a clean container, adding water for injection to 2800ml, stirring the solution for 5 minutes to be uniform, filtering by using a 0.22-micron microporous membrane, filling filtrate into penicillin bottles with 2.5ml per bottle, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing at-40 deg.C for 6 h; maintaining the freezing temperature at-30 deg.C, vacuum degree at 10pa, and freezing time at 15 h; continuously keeping the vacuum degree at 20pa, the freezing temperature at-20 ℃, and the freezing time at 8 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 4
Placing 40g wogonin and 200g L-arginine in a suitable container, adding boiling water for injection, and stirring to dissolve; adding 60g of mannitol, stirring for dissolving, adding 0.1% of activated carbon for a needle, stirring for 20 minutes, decarburizing by using a titanium sand core, pumping into a clean container, adding water for injection to 3000ml, stirring the solution for 5 minutes to be uniform, filtering by using a 0.22-micron microporous filter membrane, filling filtrate into penicillin bottles with 2.5ml per bottle, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing at-50 deg.C for 5 h; maintaining the freezing temperature at-20 deg.C, vacuum degree at 15pa, and freezing time at 20 h; continuously keeping the vacuum degree at 15pa, freezing at-10 ℃ for 5 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 5
Placing 10g wogonin and 20g sodium phosphate in a suitable container, adding boiling water for injection, and stirring to dissolve; adding 20g of mannitol, stirring for dissolving, adding 0.1% of activated carbon for a needle, stirring for 20 minutes, decarburizing by using a titanium sand core, pumping into a clean container, adding water for injection to 2000ml, stirring the solution for 5 minutes to be uniform, filtering by using a 0.22-micron microporous filter membrane, filling filtrate into penicillin bottles with 2.5ml per bottle, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing for 3-6 h at the freezing temperature of-55 ℃; maintaining the freezing temperature at-10 deg.C, vacuum degree at 15pa, and freezing time at 15 h; continuously keeping the vacuum degree at 20pa, the freezing temperature at-5 ℃ and the freezing time at 6 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 6
Placing 30g wogonin and 120g citric acid in a suitable container, adding boiling water for injection, and stirring to dissolve; adding 50g of glucose, stirring for dissolving, adding 0.1% of needle activated carbon, stirring for 20 minutes, decarburizing by using a titanium sand core, pumping into a clean container, adding water for injection to 2500ml, stirring the solution for 5 minutes to be uniform, filtering by using a 0.22 mu m microporous filter membrane, filling filtrate into penicillin bottles, wherein 2.5ml of each penicillin bottle is filled, then partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing at-45 deg.C for 4 h; maintaining the freezing temperature at-25 deg.C, vacuum degree at 20pa, and freezing time at 15 h; continuously keeping the vacuum degree at 10pa, the freezing temperature at-10 ℃ and the freezing time at 7 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 7
Placing 20g wogonin and 80g citric acid in a suitable container, adding boiling water for injection, and stirring to dissolve; adding 30g of glucose, stirring to dissolve, adding 0.1% of needle activated carbon, stirring for 20 minutes, decarburizing by using a titanium sand core, pumping into a clean container, adding water for injection to 2000ml, stirring the solution for 5 minutes to be uniform, filtering by using a 0.22-micron microporous filter membrane, filling 2.5ml of filtrate into penicillin bottles, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing for 3-6 h at a freezing temperature of-60 to-40 ℃; and maintaining the freezing temperature of-30 to-0 ℃, the vacuum degree of 10 to 20pa and the freezing time of 15 to 30 hours; continuously keeping the vacuum degree at 10-20 pa, the freezing temperature at 0-minus 20 ℃, and the freezing time at 4-8 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 8
Placing 40g wogonin and 200g sodium bicarbonate in a suitable container, adding boiling water for injection, and stirring to dissolve; adding 60g of lactose, stirring for dissolving, adding 0.1% of active carbon for a needle, stirring for 20 minutes, decarburizing titanium sand cores, pumping into a clean container, adding water for injection to 2200ml, stirring the solution for 5 minutes to be uniform, filtering by a 0.22-micron microporous membrane, filling filtrate into penicillin bottles with 2.5ml per bottle, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing at-50 deg.C for 5 h; maintaining the freezing temperature at-20 deg.C, vacuum degree at 15pa, and freezing time at 20 h; continuously keeping the vacuum degree at 15pa, freezing at-10 ℃ for 7 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
Example 9
Placing 10g wogonin and 20g sodium bicarbonate in a suitable container, adding boiling water for injection, and stirring to dissolve; adding 20g of sorbitol, stirring for dissolving, adding 0.1% of activated carbon for a needle, stirring for 20 minutes, decarburizing titanium sand cores, performing suction filtration into a clean container, adding water for injection to 2500ml, stirring the solution for 5 minutes to be uniform, filtering by a 0.22-micron microporous membrane, filling filtrate into penicillin bottles with 2.5ml per bottle, partially plugging butyl rubber plugs, conveying to a plate layer in a freeze-drying box, inserting a temperature probe, and closing a box door. Freezing at-60 deg.C for 3 h; maintaining the freezing temperature at-30 deg.C, vacuum degree at 20pa, and freezing time at 15 h; continuously keeping the vacuum degree at 15pa, freezing at-20 ℃ for 5 h; finally the drying temperature was 30 ℃ and held for 6 hours. Sealing, deflating, taking out of the box, and capping.
To further verify the pharmacological and toxicological effects of the preferred group of 1 wogonin pharmaceutical compositions of the present invention, the following relevant pharmacological and toxicological test data of the wogonin pharmaceutical compositions of the present application are provided.
2. Pharmacological experiments
2.1 the wogonin pharmaceutical composition (group 1 of the present invention) of the present invention is used in combination with chemotherapeutic drugs to inhibit the growth of MGC-803 tumor cells of human gastric carcinoma cultured in vitro
The growth inhibition effect of the wogonin pharmaceutical composition and chemotherapeutic drugs of fluorouracil (5-FU), cisplatin (DDP), adriamycin (EPB) and Paclitaxel (PTX) on MGC-803 in vitro cultured human gastric cancer is investigated.
The results show that the wogonin pharmaceutical composition and the combination administration of the wogonin pharmaceutical composition and 5-FU according to the IC50 ratio (3: 1) have synergistic effect when the wogonin pharmaceutical composition and the fluorouracil are combined in the dosage range of 22.25 mu M +8.9 mu M to 200 mu M +80 mu M. The wogonin pharmaceutical composition and its co-administration with cisplatin at an IC50 ratio (1: 4) have synergistic effects in the dosage range of the wogonin pharmaceutical composition with cisplatin 60 μ M +45 μ M to a final concentration of 2.42 μ M +1.82 μ M. The wogonin pharmaceutical composition is co-administered with epirubicin at an IC50 ratio (1: 2) to provide a synergistic effect in a dosage range of the wogonin pharmaceutical composition and epirubicin of 60 μ M +0.8 μ M to a final concentration of 1.69 μ M +0.02 μ M. The wogonin drug combination is administered in combination with paclitaxel at an IC50 ratio (1: 4) to provide a synergistic effect in the dosage range of 9.53 μ M +3.91nM to a final concentration of 4.95 μ M +2.03 nM.
2.2 Wogonin pharmaceutical composition (group 1 of the present invention) used in combination with chemotherapeutic agents for inhibiting the growth of human hepatoma SMMC-7721 tumor cells cultured in vitro
The growth inhibitory effect of the combination of wogonin and Paclitaxel (PTX), a chemotherapeutic drug, Epirubicin (EPB), on SMMC-7721 cultured in vitro human liver cancer shows that the combination of wogonin and paclitaxel at IC50 ratio (1: 3) has synergistic effect in the dose range of 6.74 μ M +0.34 μ M to 50 μ M +2.5 μ M. The wogonin drug combination is administered in combination with epirubicin at an IC50 ratio (1: 4) to provide a synergistic effect in the dosage range of 42.0 μ M +1.02 μ M to a final concentration of 13.74 μ M +0.33 μ M of the wogonin drug combination and paclitaxel.
2.3 Wogonin pharmaceutical composition (group 1 of the present invention) having inhibitory effect on human gastric cancer BGC-823 nude mouse xenograft tumor
The wogonin pharmaceutical composition for injection has obvious growth inhibition effect on human gastric cancer BGC-823 nude mouse transplantation tumor. The wogonin pharmaceutical composition for injection is administrated by intravenous injection at 60mg/kg, 30mg/kg and 15mg/kg once every other day, and the T/C (%) of the nude mouse transplantation tumor of human gastric cancer BGC-823 after 21 days of administration is 37.22%, 51.53%, 88.26% (first result) and 34.22%, 47.90% and 78.07% (second result), respectively, the tumor inhibition rate reaches 62.84%, 48.57%, 16.49% (first result) and 67.62%, 52.00% and 22.86% (second result). T/C (%) of human gastric cancer BGC-823 nude mouse transplanted tumors by fluorouracil injected at 20mg/kg tail vein under the same conditions was 31.43% (first results) and 27.92% (second results), and tumor inhibition rates were 70.38% and 72.10% (second results). The results of the two experiments are similar. The result shows that the wogonin medicine composition has similar tumor inhibiting effect compared with the positive medicine 5-FU, but has smaller influence on the body weight of the experimental animal.
2.4 Wogonin pharmaceutical composition (group 1 of the present invention) having inhibitory effect on human hepatoma SMMC-7721 nude mouse xenograft tumor
The wogonin medicinal composition for injection has obvious growth inhibition effect on human liver cancer SMMC-7721 nude mouse transplantation tumor. The wogonin pharmaceutical composition for injection is administered by intravenous injection at 60mg/kg, 30mg/kg and 15mg/kg once every other day, and the T/C (%) of the transplanted tumor of human liver cancer SMMC-7721 nude mice is 34.91%, 55.70%, 85.77% (first result) and 34.75%, 49.12% and 79.12% (second result) after 21 days of administration, and the tumor inhibition rate reaches 68.50%, 49.29%, 23.88% (first result) and 63.48%, 45.93% and 22.61% (second result). Under the same conditions, the T/C (%) of fluorouracil injected at 20mg/kg tail vein of human liver cancer SMMC-7721 nude mouse transplantable tumor is 28.46% (first batch result) and 24.31% (second batch result), and the tumor inhibition rate is 75.41% (first batch result) and 71.30% (second batch result). The results of the two experiments are similar. The results show that the wogonin pharmaceutical composition has similar tumor inhibition effect compared with the positive drug 5-FU, but has smaller influence on the body weight of the experimental animal.
2.5 Wogonin pharmaceutical composition (inventive example 1 group) inhibiting effect on human Lung cancer H460 nude mouse xenograft tumor
The experiment establishes a human non-small cell lung cancer H460 nude mouse xenograft tumor model, and the model is utilized to evaluate the antitumor activity of the wogonin pharmaceutical composition for injection. Experimental results show that the wogonin pharmaceutical composition for injection has a remarkable growth inhibition effect on human non-small cell lung cancer H460 nude mouse transplanted tumors. The wogonin pharmaceutical composition for injection is administered by intravenous injection at 60mg/kg, 30mg/kg and 15mg/kg once every other day, and the T/C (%) of transplanted tumor of human non-small cell lung cancer H460 nude mouse is 34.78%, 48.33%, 76.53% (first batch) and 35.09%, 54.71%, Han dynasty after 21 days of administration
3 toxicology study
3.1 Effect of wogonin drug combinations on the hematopoietic System of Normal and tumor-bearing mice, and the Effect of wogonin drug combinations on the immune System of Normal mice
The results show that the intravenous injection (60, 30mg/kg) of the wogonin pharmaceutical composition has no obvious influence on the hematopoietic system of normal mice, including leukocytes in peripheral blood and the number of bone marrow nucleated cells, and has no obvious influence on the immune system of normal mice, including thymus index and spleen index. The wogonin pharmaceutical composition (60, 30mg/kg) injected intravenously has no obvious effect on the total number of leucocytes in peripheral blood of tumor-bearing mice.
3.2 study of acute toxicity test of injectable Wogonin pharmaceutical composition
The healthy clean SD rat is continuously injected 3 times intravenously (iv) in 8 hours with 150mg/(kg. times) doses of the wogonin pharmaceutical composition for injection, the doses of 450mg/kg are accumulated, and the animal death is not caused except toxic and side effects such as tachypnea, eye closure, lying on the stomach and motionlessness, diuresis, activity reduction and the like in 60 minutes after each administration. In the (iv) single intravenous injection of the wogonin pharmaceutical composition for injection to the healthy and clean ICR mice, 250mg/kg of the wogonin pharmaceutical composition for injection is given, so that toxic and side effects such as tachypnea, eye closure, lying on the stomach and motionlessness and reduced movement appear within 3 hours after the administration, the symptoms are similar to those of rats, and the animals are not killed. Under the condition of the experimental dosage, the maximum tolerated dose of the wogonin pharmaceutical composition for injection to the rat intravenous injection (iv) is as follows: 450mg/kg (administered in 3 divided doses of 150mg/kg each time); the maximum tolerated dose administered intravenously (iv) to mice was: 250 mg/kg.
3.3 Wogonin pharmaceutical composition for injection rat three month toxicity test
SD rats were administered with 120, 60 and 30mg/(kg.d) doses of the injectable wogonin pharmaceutical composition by tail vein injection for three consecutive months, respectively, and had significant effects on the respiratory system and motor functions of the rats during the administration period. The main manifestations are decreased activity, groveling and immobility, weakness of limbs (flaccid paralysis), shortness of breath, eye closure, polyuria reaction and 27% of animal deaths occurring immediately to 30 minutes after each dose, high and medium dose of side reactions, high dose of death dose, and no abnormal reaction of low dose and control group during the whole test period.
The three dosage groups of the pharmaceutical composition have no obvious influence on weight gain, food intake and water drinking of SD rats, and the check of one half month (middle administration period), three months (next day after drug withdrawal) and one month after drug withdrawal (end of recovery period) of administration has no obvious influence on hematology, organ weight coefficient and general appearance of organ pathology, and is basically the same as that of a control group. There was an increasing effect on individual blood biochemical indicators (CK) in the three dose groups. The biopsy at each stage and the pathological histology examination of the half-way dead rat find that the heart of the high-dose group rat has certain damage effect, mainly manifested by myocardial fibrosis, interstitial fibroblasts, histiocytes and a small amount of inflammatory cell infiltration which are accompanied with fibrosis with different degrees, and the pathological changes still exist but are relieved after the drug is stopped for one month. The medium and low doses do not show similar pathological changes in the autopsy at each stage, and the three dose groups have no obvious pathological changes in other organ tissues and are similar to the control group. Under the condition of the experimental dosage, the main toxic and side effects of the injection wogonin medicine composition given by continuously injecting three intravenous injection routes to SD rats are reduced activity, lying on the stomach, myasthenia of limbs, shortness of breath, eye closure, polyuria reaction and increase of creatine phosphokinase (CK). The target organ to be poisoned is the heart, and the disease still exists after stopping the medicine for one month, but the degree is reduced. The toxic death dose is 120mg/(kg.d), the toxic reaction appearing dose is 60mg/(kg.d), and the safe (non-toxic) dose is 30 mg/(kg.d).
In conclusion, the wogonin pharmaceutical composition developed by the invention has obvious growth inhibition effect on human gastric cancer MGC-803 tumor cells, human lung cancer H460 nude mouse xenograft tumor, liver cancer SMMC-7721 tumor cells, human gastric cancer BGC-823 nude mouse xenograft tumor, human liver cancer SMMC-7721 nude mouse xenograft tumor and the like, which also shows that the pharmaceutical composition has obvious curative effect. Toxicology experiments show that the wogonin pharmaceutical composition has no obvious influence on the total number of white blood cells in peripheral blood of a tumor-bearing mouse, and the pharmaceutical composition has no abnormal reaction on the mouse. The result also shows that the pharmaceutical composition has the advantage of small toxic and side effects.
Claims (5)
1. The wogonin medicine composition for treating tumor diseases is characterized by being prepared from the following components in parts by weight: 25 parts of wogonin, 100 parts of L-arginine, 40 parts of mannitol and 2500 parts of water for injection.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in the form of a lyophilized powder.
3. A process for the preparation of a pharmaceutical composition according to claim 1 or 2, characterized in that it comprises the process steps of:
(1) mixing wogonin and L-arginine, adding a small amount of water for injection to moisten, adding water for injection, and stirring to completely dissolve the medicine to obtain a mixed solution;
(2) adding mannitol into the mixed solution obtained in the step (1), stirring for dissolving, adding activated carbon, stirring for removing pyrogen, removing carbon, performing suction filtration, adding a proper amount of water for injection, and performing constant volume preparation; filtering with filter membrane for sterilization to obtain filtrate, and lyophilizing to obtain lyophilized powder.
4. A process for preparing a pharmaceutical composition according to claim 3,
the freeze drying process conditions in the preparation method process step (2) are as follows: freezing for 3-6 h at a freezing temperature of-60 to-40 ℃; and maintaining the freezing temperature of-30 to-0 ℃, the vacuum degree of 10 to 20pa and the freezing time of 15 to 30 hours; continuously keeping the vacuum degree at 10-20 pa, the freezing temperature at 0-20 ℃, and the freezing time at 4-8 h; and then gradually heating to 25-35 ℃, drying for 5-8 h, and carrying out vacuum tamponade to obtain the freeze-dried powder.
5. The method of claim 4, wherein the method is performed under a nitrogen atmosphere.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556101A (en) * | 2003-12-31 | 2004-12-22 | 中国药科大学 | Extraction technology of Hanbaicalein, medicinal composition and preparation technology of medicine |
| CN1562147A (en) * | 2004-03-24 | 2005-01-12 | 杭州华东医药集团生物工程研究所有限公司 | Radix scutellariae extract freeze-dried powder injection and its preparing method |
| TW201311263A (en) * | 2011-09-05 | 2013-03-16 | Univ Nat Taiwan Normal | Wogonin-containing pharmaceutical composition for inhibiting cancer stem cells growth and application thereof |
| AU2015255189A1 (en) * | 2010-03-12 | 2015-11-26 | Allergan Industrie Sas | A fluid composition comprising a hyaluronan polymer and mannitol for improving skin condition |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556101A (en) * | 2003-12-31 | 2004-12-22 | 中国药科大学 | Extraction technology of Hanbaicalein, medicinal composition and preparation technology of medicine |
| CN1562147A (en) * | 2004-03-24 | 2005-01-12 | 杭州华东医药集团生物工程研究所有限公司 | Radix scutellariae extract freeze-dried powder injection and its preparing method |
| AU2015255189A1 (en) * | 2010-03-12 | 2015-11-26 | Allergan Industrie Sas | A fluid composition comprising a hyaluronan polymer and mannitol for improving skin condition |
| TW201311263A (en) * | 2011-09-05 | 2013-03-16 | Univ Nat Taiwan Normal | Wogonin-containing pharmaceutical composition for inhibiting cancer stem cells growth and application thereof |
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| "汉黄芩素单药及联合用药对人胃癌的生长抑制作用研究";王婷婷 等;《第二届中国女医师大会论文集 肿瘤》;20121031;第280-283页 * |
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