CN101293086A - An analgesic and antitumor drug and its preparation method - Google Patents

Abstract

The invention discloses a drug for analgesic and tumor elimination and a preparation method thereof. The active ingredients of the analgesic and antitumor drug are realgar and ginger, and the mass ratio of realgar and ginger is (1-3):(1-3); the particle size of realgar and ginger is 60 -500nm. The medicine also contains a carrier, and the mass ratio of the realgar, the ginger and the carrier is (1-3):(1-3):(4-10). After adding the carrier, the analgesic and antitumor drug of the present invention can be made into an analgesic and antitumor ointment. The analgesic and antitumor drug of the invention has low toxic and side effects and stable blood drug concentration.

Description

An analgesic and antitumor drug and its preparation method

technical field

The invention relates to an analgesic and antitumor drug and a preparation method thereof.

Background technique

At present, the traditional Chinese medicine preparations related to analgesic and antitumor mainly include the following documents: Chinese invention patent CN1133725 discloses cancer tumor reducing swelling and pain relief ointment; Ai Xiaoliu Paste, Chinese invention patent CN1153645 discloses skin cancer powder, skin cancer ointment, Xiaoyao powder flavored and its preparation, and Chinese invention patent CN1615970 discloses an anti-leukemia traditional Chinese medicine compound Huangdai tablet and its preparation method. The main ingredients of the drugs involved in the first four patent documents contain realgar, which is also an ointment, but they are all compound recipes composed of various traditional Chinese medicines, and they are only passed through a 200-mesh sieve. Too large a particle size is not conducive to onset of effect, and The carrier in the ointment is also not safe enough. The compound Huangdai tablet involved in the fifth patent document is an oral tablet, and large and medium doses can cause an increase in ALT, indicating that it has certain liver toxicity, and it is only used in the treatment of blood diseases. Therefore, there is an urgent need for analgesic and antitumor traditional Chinese medicine preparations with low toxic and side effects, safety and good curative effect.

Contents of the invention

The object of the present invention is to provide an analgesic and antitumor drug and a preparation method thereof.

The analgesic and antitumor medicine provided by the present invention, its active ingredient is realgar and ginger, and the mass ratio of described realgar and described ginger is (1-3): (1-3); Said realgar and described ginger The particle size is 60-500nm.

Wherein, the medicine also contains a carrier, and the mass ratio of the realgar, the ginger and the carrier is (1-3): (1-3): (4-10); the realgar and the ginger The particle size is preferably 60-200nm.

After the carrier is added to the analgesic and antitumor drug of the present invention, it can be made into various dosage forms, such as ointment, plaster protective bag or patch.

When the medicine of the present invention is made into an ointment, the carrier is preferably composed of at least one of the following seven substances: glycerol monostearate, hydroxypropylmethylcellulose, isopropyl myristate, Methylparaben, propylparaben, polyoxyl-40-stearate or saline.

The carrier can be composed of the following substances: 8-12% of the glycerol monostearate, 0.5-1.5% of the hydroxypropyl methylcellulose, 8-12% of the isopropyl myristate, the 0.5-1.5% of the methylparaben, 0.5-1.5% of the propylparaben, 9-12% of the polyoxyl-40-stearate and 60-70% of the normal saline; The stated values are all mass percentages.

Wherein, the carrier may specifically be as follows 1) or 2) or 3):

1) 8% of the glycerol monostearate, 1.5% of the hydroxypropyl methylcellulose, 8% of the isopropyl myristate, 1.5% of the methyl paraben, and 1.5% of the paraben Gold propyl ester 1.5%, said polyoxyl-40-stearate 12% and said normal saline 67.5%;

2) 8% of the glycerol monostearate, 1% of the hydroxypropyl methylcellulose, 10% of the isopropyl myristate, 1% of the methyl paraben, and 1% of the paraben Gold propyl ester 1%, said polyoxyl-40-stearate 12% and said normal saline 67%;

3) 9% of the glycerol monostearate, 1% of the hydroxypropyl methylcellulose, 10% of the isopropyl myristate, 0.5% of the methyl paraben, and 0.5% of the paraben Gold propyl ester 0.5%, said polyoxyl-40-stearate 12% and said normal saline 67%;

The above numerical values are all percentages by mass.

When the analgesic and tumor-relieving ointment of the present invention is used, the range of application is determined first, so that the area of application includes tumors and is 10-50 mm larger than the range of tumors.

Another object of the present invention is to provide a method for preparing the analgesic and antitumor drug.

The method for preparing the analgesic and antitumor drug provided by the present invention is to bake realgar and ginger into powder, process them into 60-500nm granules, and then process the realgar and ginger according to the mass parts Mix to obtain analgesic and antitumor drugs.

After the above method is improved, an analgesic and antitumor cream can be prepared. For example, realgar and ginger are baked into powder, processed into 60-500nm particles, and then the realgar, the ginger and the carrier are prepared according to The parts by mass are mixed and then emulsified to obtain an analgesic and antitumor ointment.

Compared with intravenous administration and oral administration, the analgesic and antitumor ointment of the present invention has the advantage that it can form raw materials into very small nanoparticles, which is convenient for transdermal administration, and at the same time, the carrier it contains can provide a good Adhesion, increase the administration time, play a sustained release effect, easy to achieve and maintain a stable blood concentration, and play a positive role in treatment. On the other hand, transdermal administration avoids the first-pass effect of the drug, reduces the toxicity of the drug to internal organs, and also reduces the toxic and side effects of the drug. Experiments have proved that the analgesic and antitumor ointment of the present invention can significantly reduce toxic and side effects through transdermal administration, can maintain a good blood drug concentration, and can be used to treat various tumors, such as liver cancer, skin cancer and glioma wait.

Description of drawings

Figure 1 is the transmission electron microscope detection picture after nanometerization

Figure 2 shows the product properties

Figure 3 shows the way of administration

Figure 4 shows the effect of Analgesic Xiaoliu Ointment on liver cancer

Figure 5 shows the effect of Analgesic Xiaoliu Ointment on skin cancer

Figure 6 shows the effect of Zhentong Xiaoliu Ointment on skin cancer

Figure 7 shows the effect of Zhentong Xiaoliu ointment on glioma

Figure 8 shows the results of the in vitro transdermal test of Zhentong Xiaoliu ointment

Figure 9 shows the inhibitory effect of the active ingredients of Analgesic Xiaoliu Ointment on cell proliferation

Figure 10 shows the relationship between the active ingredients of Analgesic Xiaoliu Ointment and autophagy

Figure 11 is the result of HE staining

Figure 12 is the result of immunohistochemical staining

Figure 13 shows the distribution of arsenic in organs

Figure 14 is the measurement result of blood arsenic concentration

Detailed ways

Embodiment 1, preparation analgesic and eliminating tumor ointment

Bake realgar (Haozhou Jingde Pharmaceutical Co., Ltd.) and ginger, put them into a low-temperature ball mill, work at 2000-4000rpm for 5 minutes, stop for 15 minutes, work for 5 minutes under the same conditions, repeat 6-7 times, and ensure that the work The temperature is less than 40°C, and the realgar and ginger are processed into nanoparticles. The processed realgar and ginger particles were dissolved in water, and the realgar and ginger particles and particle size were observed under a transmission electron microscope (JEM-2011 JOEL, Tokyo).

The results are shown in Figure 1, showing that the particle size of realgar and ginger is 60-100nm, and the scale bar in Figure 1 is 60nm.

Take processed 50g of realgar granules, 50g of ginger granules and 200g of carrier into the emulsifier, emulsify for 5min at 6000-8000rpm (radius of rotation is 1-2cm), then stop for 15min, then work for 5min under the same conditions, repeat 2- 3 times until a uniform and stable emulsified product is formed, and the properties of the finished product are shown in Figure 2.

The carrier is made by mixing the following ingredients at 50-70°C: 16g glycerol monostearate, 3g hydroxypropyl methylcellulose, 16g isopropyl myristate, 3g methylparaben, 3g paraben propyl Esters, 24g polyoxyl-40-stearate and 135g physiological saline.

Embodiment 2, preparation analgesic and eliminating tumor ointment

Realgar and ginger were made into particles of 60-200nm according to the method in Example 1.

Get processed 50g of realgar granules and 75g of ginger granules and 250g of carrier, mix and emulsify according to the method in Example 1 to form a finished product.

The carrier is made by mixing the following ingredients at 50-70°C: 20g glycerol monostearate, 2.5g hydroxypropyl methylcellulose, 25g isopropyl myristate, 2.5g methylparaben, 2.5 g propylparaben, 30 g polyoxyl-40-stearate and 167.5 g normal saline.

Embodiment 3, preparation analgesic and eliminating tumor ointment

Realgar and ginger were made into particles of 60-200nm according to the method in Example 1.

Get processed 75g of realgar granules and 50g of ginger granules and 250g of carrier and mix and emulsify according to the method in Example 1 to form a finished product.

The carrier is made by mixing the following ingredients at 50-70°C: 22.5g glycerol monostearate, 2.5g hydroxypropyl methylcellulose, 25g isopropyl myristate, 1.25g methylparaben, 1.25g propylparaben, 30g polyoxyl-40-stearate, 167.5g normal saline.

Embodiment 4, the effect of the analgesic and eliminating tumor ointment of embodiment 1 on liver cancer

1×10 ⁶ HepG2 liver cancer cells were implanted subcutaneously in the dorsal side of nude mice (6-8 weeks) to form small tumor masses.

The nude mice implanted with HepG2 liver cancer cells were randomly divided into 3 groups, directly applied to the tumor skin group, not directly applied to the tumor skin group and a control group, with 6 mice in each group. Directly apply the analgesic and eliminating tumor ointment of embodiment 1 on the tumor skin of nude mice 16 days after tumor planting in tumor skin group; The analgesic and antitumor ointment of embodiment 1 is coated on the non-tumor skin; the nude mice that the control group is treated with cream matrix after tumor planting; · 2 square centimeters) were transdermally administered for 3 consecutive days, and the tumor growth and body weight changes were recorded. The experiment was repeated three times.

The experimental results are shown in Figure 4. From the 22nd day after the tumor was implanted (6 days after the application of the analgesic and antitumor ointment in Example 1), there was a difference in the size of the tumor. The subcutaneous tumors of mice had no obvious growth, and the tumor sizes were 497.77±127.25 (mm3) and 434.94±178.16 (mm3) at 25 days and 28 days after tumor implantation; The subcutaneous tumors of the mice all had obvious growth, and the tumor sizes were 2054.89±467.11 (mm3) and 3406.99±629.83 (mm3) at 25 days after tumor implantation, and 2420.38 at 28 days after tumor implantation. ±691.15 (mm3) and 5680.24±643.22 (mm3).

The analgesic eliminating tumor ointment of embodiment 5, embodiment 2 are to the effect of skin cancer

a) Effect of the analgesic and antitumor ointment of embodiment 2 on skin cancer

1×10 ⁷ B16 melanoma cells were implanted subcutaneously in the dorsal side of male C57BL/6 mice (6-8 weeks, body weight 20-24 grams), and a small tumor mass formed after about a week.

Male C57BL/6 mice implanted with B16 melanoma cells were randomly divided into 3 groups, a group directly applied to the tumor skin, a group not directly applied to the tumor skin, and a control group, with 6 mice in each group. Apply the analgesic and eliminating tumor ointment of embodiment 2 directly on the tumor skin of male C57BL/6 mice 10 days after tumor planting in the tumor skin group; The non-tumor skin of C57BL/6 mice was coated with the analgesic and tumor-reducing ointment of Example 2; in the control group, male C57BL/6 mice were treated with cream matrix after tumor implantation. The Analgesic Xiaoliu Ointment of Example 2 was transdermally administered at a dose of 100 microliters/(20 g body weight·2 square centimeters) per day for 3 consecutive days, and the tumor growth and body weight changes were recorded. The experiment was repeated three times.

The experimental results are shown in Figure 5. On the 16th day after the tumor was planted (the analgesic and antitumor ointment of Example 2 was applied for 6 days), there was a difference in the size of the tumor, and the male C57BL/6 male C57BL/6 directly applied to the tumor skin group The subcutaneous tumors of the mice did not grow significantly, and the tumor sizes were 577.77±147.05 (mm3) and 534.94±99.36 (mm3) at 19 days and 22 days after tumor implantation; the tumor skin group and the control group were not directly applied The subcutaneous tumors of male C57BL/6 mice all had obvious growth, and the tumor sizes were 1954.89±427.61((mm3) and 6406.99±589.86((mm3) 19 days after tumor implantation. The tumor sizes were 2320.38±594.36 (mm3) and 7680.24±884.88 (mm3) at day 22. The progress of tumor growth is shown in Figure 6.

b) the analgesic and antitumor ointment of embodiment 2 is to the effect of angiogenesis in the tumor

Take the male C57BL/6 mice in a) that were directly applied to the tumor skin group, not directly applied to the tumor skin group, and the control group 20 days after the analgesic Xiaoliu ointment was applied for HE staining and immunohistochemical staining analysis. The antibody used in the immunohistochemical analysis was goat anti-mouse VEGF antibody (Beijing Boaosen Biotechnology Co., Ltd.).

The results of HE staining experiments showed that after the analgesic and antitumor ointment was directly applied to the tumor skin, the number of blood vessels in the tumor tissue was significantly reduced (Figure 11A). Compared with the control group (Figure 11B), the number of blood vessels in each field of view (n =6) was significantly reduced with a significant difference (Fig. 11C).

The experimental results of immunohistochemical analysis showed that compared with the control group (Figure 12A) and the group not directly applied to the tumor skin (Figure 12C), the expression of vascular endothelial growth factor in the tumor skin group (Figure 12B) was significantly increased. reduce.

The above experimental results show that the analgesic and antitumor ointment of the present invention can effectively inhibit tumor angiogenesis.

Effect of the analgesic and antitumor ointment of embodiment 6 and embodiment 3 on glioma

1×10 ⁹ C6 glioma cells were implanted subcutaneously in the dorsal side of male Wistar rats (body weight 150-200 grams), and a small tumor mass would form after a few weeks.

Male Wistar rats implanted with C6 glioma cells were randomly divided into 3 groups, directly applied to tumor skin group, not directly applied to tumor skin group and control group, with 6 rats in each group. Directly applied to the tumor skin of the tumor skin group on the tumor skin of male Wistar rats 22 days after tumor planting, the analgesic and tumor-removing ointment of embodiment 3 was coated; The analgesic and antitumor ointment of Example 3 was coated on the non-tumor skin of the rat; the male Wistar rats in the control group were treated with the ointment base after the tumor was planted. The analgesic and antitumor ointment of Example 3 was transdermally administered at a dose of 200 μl/(100 g body weight·3 cm2) per day for 3 consecutive days, and the tumor growth and body weight changes were recorded. The experiment was repeated three times.

The experimental results are shown in Figure 7. On the 24th day after the tumor was implanted (the analgesic and antitumor ointment of Example 3 was applied for 2 days), there were differences in the size of the tumors, which were directly applied to the male Wistar rats of the tumor skin group. There was no obvious growth of the subcutaneous tumor, and the tumor size was 653.77±100.82 (mm3) and 661.15±780.92 (mm3) at 26 days and 28 days after the tumor was implanted; the male Wistar of the tumor skin group and the control group were not directly applied The subcutaneous tumors of the rats all had obvious growth. The tumor sizes were 785.12±714.12 (mm3) and 1005.16±313.4 (mm3) 26 days after the tumor implantation, and 802.79±313.4 (mm3) after the tumor implantation. 737.79 (mm3) and 1318.2±256.22 (mm3).

Embodiment 7, in vitro transdermal experiment

The analgesic and antitumor ointment prepared in Example 1 was used for in vitro transdermal experiment.

Male Wistar rats with a body weight of 200±20 g were taken, anesthetized, the fluff was removed, the skin was carefully peeled, the subcutaneous fat tissue and capillaries were separated, washed several times with normal saline, and stored in the refrigerator for later use.

The skin of male Wistar rats is 3.14 square centimeters, and the in vitro transdermal experiment is carried out.

The in vitro transdermal test method is as follows: put the skin into a transdermal absorption instrument (Tianjin Zhengtong), 37°C, 200rpm, take samples at 0, 2, 4, 6, 8, 10, 16, and 24 hours to determine the in vitro properties of the ointment. Transdermal Kinetic Analysis.

The analysis method of in vitro transdermal kinetics is as follows: adopt graphite furnace atomic absorption spectrometry (Perkin-Elmer Corp., USA) to measure.

The experimental results shown in Figure 8 show that the permeation of arsenic has a time-dependent relationship.

Example 8, the inhibitory effect of analgesic and antitumor drugs on cell proliferation

According to the method of Example 1, realgar and ginger are made into nanoparticles (particle diameter is 60-200nm). Realgar and ginger nanoparticles are mixed according to the mass ratio of 1:1 to obtain tumor-removing medicine.

The anti-tumor drug was suspended in water to make a suspension with the content of the anti-tumor drug at 0, 0.5, 1.0, 1.5 and 2.0 g/ml respectively, and the concentration of the aqueous suspension was analyzed by graphite furnace atomic absorption spectrometry (Perkin-Elmer Corp. , USA) for determination.

B16 melanoma cells (purchased from Shanghai Institute of Cells, Chinese Academy of Sciences) were seeded into 96-well plates at a density of 1×10 ⁵ cells/ml, and after 24 hours, the above-mentioned concentrations of antitumor drug aqueous suspensions were added to expose B16 cells to 0, 0.5, 1.0, 1.5 and 2.0 g/ml anti-tumor drug aqueous suspension for 48 hours. Then the cell growth rate was analyzed by MTT assay (cell growth rate=OD value of anti-tumor drug at each concentration÷OD value without anti-tumor drug (0 concentration)*100%).

The results of the MTT experiment are shown in Figure 9, indicating that the proliferation of cells is significantly inhibited in a concentration-dependent manner, and the inhibitory effect on proliferation will be significantly weakened when the concentration of the antitumor drug aqueous suspension is less than 0.2g/ml; when the concentration is greater than 1.0g /ml, it can directly kill tumor cells.

Example 9, Analgesic and antitumor drugs and autophagy

According to the method of Example 1, realgar and ginger are made into nanoparticles (particle diameter is 60-200nm). The realgar and ginger nanoparticles are mixed according to the mass ratio of 1:1 to obtain the antitumor drug.

The antitumor drug was suspended in water to prepare a suspension with a content of antitumor drug of 0.2 g/ml, and the concentration of the aqueous suspension was determined by graphite furnace atomic absorption spectrometry (Perkin-Elmer Corp., USA).

Add the above-mentioned antitumor drug aqueous suspension to the Hela-LC3 cells, expose the Hela-LC3 cells to 0.2 g/ml of the above-mentioned anti-tumor drug aqueous suspension for 8 hours, and observe under a fluorescence microscope.

Sterile water was added to Hela-LC3 cells as a control.

The experimental results showed that phagocytic vesicles aggregated in Hela-LC3 cells (Fig. 10A), but not in the control group (Fig. 10B). Autophagy.

Embodiment 10, pharmacokinetic analysis and tissue distribution

1×10 ⁷ B16 melanoma cells were implanted subcutaneously in the dorsal side of male C57BL/6 mice (6-8 weeks, body weight 20-24 grams), and a small tumor mass formed after about a week.

Male C57BL/6 mice implanted with B16 melanoma cells were randomly divided into 3 groups, a group directly applied to the tumor skin, a group not directly applied to the tumor skin, and a control group, with 6 mice in each group. Apply directly to the tumor skin of the tumor skin group the analgesic and antitumor ointment of embodiment 2 on the tumor skin of male C57BL/6 mice 9 days after tumor planting; The non-tumor skin of C57BL/6 mice was coated with the analgesic and tumor-reducing ointment of Example 2; in the control group, male C57BL/6 mice were treated with cream matrix after tumor implantation. The analgesic and antitumor ointment of Example 2 was transdermally administered at a dose of 100 microliters/(20g body weight 2 square centimeters) per day for 3 consecutive days. Tail vein blood of male C57BL/6 mice was used to determine blood arsenic content. The experiment was repeated three times.

The arsenic content was determined by graphite furnace atomic absorption spectrometry (Perkin-Elmer Corp., USA).

20 days after the tumor was planted, the male C57BL/6 mice in the above groups were sacrificed, and the liver, kidney and tumor tissues were separated to determine the content of arsenic in the tissues, and the determination method was the same as above.

The experimental results showed that the distribution of arsenic in the viscera of male C57BL/6 mice directly applied to the tumor skin group was higher than that of other groups, and the distribution in the tumor was the highest, which was related to the anti-tumor relationship (Figure 13). There was no significant difference in blood arsenic between the male C57BL/6 mice that were directly applied to the tumor skin group and those that were not directly applied to the tumor skin group ( FIG. 14 ).

Embodiment 11, the analgesic effect of analgesic and eliminating tumor ointment

Select 3 patients with advanced liver cancer and severe pain from volunteers, give the analgesic and antitumor ointment of the present invention, apply it to the skin of the liver, and halve the dose of analgesic after 3 days, and the NRS scores of the 3 cases are recorded as follows:

*NRS: Numeral rating scale (NRS): 0-10 numbers represent different degrees of pain. 0 is no pain and 10 is the worst pain. Ask the patient to circle a number that best represents the pain level. Compare the difference in pain scores before and after treatment and the difference between groups

0 1 2 3 4 5 6 7 8 9 10

                   

painless most painful

Degree grading standard: 0: no pain 1-3: mild pain

                  4-6: moderate pain   7-10: severe pain

The above experimental results show that the patient's subjective symptoms are significantly improved and the pain is alleviated after administration of the analgesic and antitumor ointment of the present invention.

Claims (10)

1. An analgesic and tumor-eliminating drug, its active ingredients are realgar and ginger, and the mass ratio of the realgar and the ginger is (1-3): (1-3); the realgar and the ginger are The particle size is 60-500nm. 2. The medicine according to claim 1, characterized in that: the medicine also contains a carrier, and the mass ratio of the realgar, the ginger and the carrier is (1-3):(1-3): (4-10). 3. The medicine according to claim 2, characterized in that the dosage form of the analgesic and antitumor ointment is ointment or patch. 4. The drug according to claim 2 or 3, characterized in that: the carrier is composed of at least one of the following seven substances: glycerol monostearate, hydroxypropyl methylcellulose, nutmeg Isopropyl paraben, methyl paraben, propyl paraben, polyoxyl-40-stearate or normal saline. 5. The drug according to claim 4, characterized in that: the carrier is composed of the following substances: 8-12% of the glycerol monostearate, 0.5-1.5% of the hydroxypropyl methylcellulose, The isopropyl myristate 8-12%, the methylparaben 0.5-1.5%, the propylparaben 0.5-1.5%, the polyoxyl-40-stearate 9 -12% and 60-70% of the physiological saline; the percentages are mass percentages. 6. The drug according to claim 5, characterized in that: the carrier is composed of the following substances: 8% of the glycerol monostearate, 1.5% of the hydroxypropyl methylcellulose, and the nutmeg 8% of isopropyl paraben, 1.5% of said methylparaben, 1.5% of said propylparaben, 12% of said polyoxyl-40-stearate and 67.5% of said normal saline. 7. The drug according to claim 5, characterized in that: the carrier is composed of the following substances: 8% of the glycerol monostearate, 1% of the hydroxypropyl methylcellulose, and the nutmeg 10% of isopropyl paraben, 1% of said methylparaben, 1% of said propylparaben, 12% of said polyoxyl-40-stearate and 67% of said normal saline. 8. The drug according to claim 5, characterized in that: the carrier is composed of the following substances: the glycerol monostearate 9%, the hydroxypropyl methylcellulose 1%, the nutmeg 10% of isopropyl paraben, 0.5% of said methylparaben, 0.5% of said propylparaben, 12% of said polyoxyl-40-stearate and 67% of said normal saline. 9. The method for preparing the analgesic and antitumor drug according to claim 1 is: after baking the realgar and ginger into powder, processing it into 60-500nm particles, and then mixing the realgar and the ginger according to the mass parts Mix to obtain analgesic and antitumor drugs. 10. The method for preparing the analgesic and antitumor drug according to any one of claims 2 to 8, is to bake the realgar and ginger into powder, process them into 60-500nm granules, and then mix the realgar and ginger The mixture is mixed with the carrier according to the mass parts and then emulsified to obtain an analgesic and antitumor drug.

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