A nanoemulsion comprising tamoxifen and ginsenoside and a preparation method thereof

Abstract

The present application relates to the technical field of pharmaceutical preparation, in particular to a nanoemulsion containing tamoxifen and ginsenoside and a preparation method thereof. The present application prepares the nanoemulsion by citric acid tamoxifen, ginsenoside, glyceryl tributyrate, isopropyl myristate, tragacanth, poloxamer and glycerol, the prepared nanoemulsion has smaller particle size, good stability, can significantly improve the solubility, and further improve the targeting and therapeutic effect of the nanoemulsion.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of preparation, and particularly relates to a nanoemulsion containing tamoxifen and ginsenosides and a preparation method thereof. BACKGROUND

[0002] Breast cancer is the second most common malignant tumor after lung cancer, and the estrogen drug tamoxifen has been widely used in the treatment of advanced breast cancer and ovarian cancer. Its anticancer mechanism is the inhibition of estrogen receptors and cytotoxicity, and the curative effect can be as high as 49%. However, with the extensive use of tamoxifen, breast cancer cells show multi-drug resistance, which greatly limits the anticancer efficacy of tamoxifen.

[0003] Ginsenosides are natural anticancer ingredients that scientists have high expectations for. Studies have shown that ginsenosides exert anticancer effects through multiple mechanisms, including inhibition of tumor cell proliferation, induction of tumor cell apoptosis, inhibition of tumor cell migration and invasion, inhibition of tumor angiogenesis, enhancement of immune function, and antioxidant stress. These mechanisms work together to make ginsenosides show significant potential in cancer treatment.

[0004] However, not only is tamoxifen poorly soluble and resistant, but ginsenosides also have low solubility in water and are almost insoluble in water, which directly affects the absorption and utilization of the drug and greatly limits the exertion of drug efficacy.

[0005] Therefore, how to improve the drug resistance of tamoxifen and increase the solubility of the drug is the key to improving the efficacy.

[0006] Currently, tamoxifen and ginsenosides are only available in oral formulations, and the ordinary tablets of tamoxifen citrate have a dissolution of only 75% of the labeled amount in 30 minutes according to the dissolution test standard of the 2020 edition of the Chinese Pharmacopoeia, and the dissolution problem has not been effectively solved.

[0007] CN111925295A discloses a co-amorphous tamoxifen citrate-ginsenoside, in which the release of tamoxifen reaches 90% in 60 minutes and the release of ginsenoside Rg reaches 88% in 60 minutes using 0.1M hydrochloric acid medium in the dissolution test, but the problem of poor stability of the co-amorphous tamoxifen citrate-ginsenoside prepared has not been solved. SUMMARY

[0008] In view of the deficiencies in the prior art, the present application aims to solve the technical problem of low solubility and dissolution rate of the above-mentioned drugs by providing a nanoemulsion of tamoxifen citrate-ginsenoside and a preparation method and use thereof.

[0009] The present application solves its technical problems by adopting the technical scheme of preparing a kind of heparin complex nanoemulsion, preferably, the heparin nanoemulsion further comprises ginsenoside.

[0010] Further, the weight parts of each component of the heparin complex nanoemulsion are as follows:

[0011] 10-15 parts by weight of citric acid heparin;

[0012] 8-10 parts by weight of ginsenoside;

[0013] 25-50 parts by weight of tributyrin;

[0014] 30-60 parts by weight of isopropyl myristate;

[0015] 60-100 parts by weight of tragacanth;

[0016] 20-50 parts by weight of poloxamer;

[0017] 60-100 parts by weight of glycerol.

[0018] Further, the weight parts of each component of the heparin nanoemulsion are as follows:

[0019] 10-15 parts by weight of citric acid heparin;

[0020] 8-10 parts by weight of ginsenoside;

[0021] 25-35 parts by weight of tributyrin;

[0022] 30-50 parts by weight of isopropyl myristate;

[0023] 60-80 parts by weight of tragacanth;

[0024] 20-35 parts by weight of poloxamer;

[0025] 60-100 parts by weight of glycerol.

[0026] The preparation process of the heparin nanoemulsion comprises the following steps:

[0027] (1) Dissolve heparin and ginsenoside in an appropriate amount of ethanol, mix tributyrin and isopropyl myristate in proportion and stir uniformly, slowly add the dissolved drug solution to the oil phase, and ultrasonic to prepare an oil phase mixture;

[0028] (2) Mix tragacanth and poloxamer uniformly, then add glycerol, and stir uniformly under heating to prepare an emulsifier mixture;

[0029] (3) Slowly add the oil phase mixture of step (1) into the emulsifier mixture of step (2), stir evenly, form the primary emulsion, and further refine by ultrasound;

[0030] (4) Add the primary emulsion of step (3) into appropriate amount of water, stir evenly, form the coarse emulsion, and prepare the tamoxifen complex nanoemulsion by homogenization treatment.

[0031] Preferably, the ultrasonic power in step (1) is 30% to 35%.

[0032] Preferably, a surfactant, specifically polyvinyl alcohol aqueous solution with a concentration of 1% to 2%, is further added in step (2).

[0033] Preferably, the pH value of the emulsifier mixture is adjusted to 5.0-6.5 and the heating temperature is adjusted to 40-60℃ in step (2).

[0034] Preferably, the volume ratio of the organic phase to the aqueous phase is 3:1 to 6:1 when the organic phase and the aqueous phase are mixed in step (3).

[0035] Preferably, the pressure of the homogenization is set to 18000-29000 psi, the homogenization is performed 2-3 times, and the temperature is maintained at 20±3℃ during the homogenization process in step (3).

[0036] Specifically, the preparation process of the tamoxifen nanoemulsion includes the following steps:

[0037] (1) Take appropriate amount of glyceryl tributyrate and isopropyl myristate, add tamoxifen and ginsenoside, and prepare an oil phase mixture by ultrasonic treatment with an ultrasonic power of 30% to 35%, an ultrasonic temperature setting of 55-65℃, and an ultrasonic treatment time of 0.5-1h;

[0038] (2) Mix tragacanth and poloxamer evenly, then add glycerol and polyvinyl alcohol aqueous solution with a concentration of 1% to 2%, adjust the pH value to 5.0-6.5, heat at 40-60℃, and stir evenly to prepare an emulsifier mixture;

[0039] (3) Slowly add the oil phase mixture of step (1) into the emulsifier mixture of step (2), wherein the volume ratio of the organic phase to the aqueous phase is 3:1 to 6:1, stir evenly, form the primary emulsion, and further refine by ultrasound;

[0040] (4) Add the primary emulsion of step (3) into appropriate amount of water, stir evenly, form the coarse emulsion, and prepare the tamoxifen complex nanoemulsion by homogenization treatment with a pressure setting of 18000-29000 psi, 2-3 times of homogenization, and a temperature maintenance of 20±3℃ during the homogenization process.

[0041] Further, the step (4) further comprises a centrifugation step, specifically: adding the colostrum after ultrasonic refinement in step (3) into an appropriate amount of water, stirring uniformly to form a rough emulsion, homogenizing treatment, discarding the supernatant, high-speed centrifugation, washing the precipitate, drying, and adding excipients to prepare a pharmaceutically acceptable preparation.

[0042] Preferably, the pharmaceutically acceptable preparation is one or more of nanoemulsion gel, soft capsule, tablet, capsule, injection.

[0043] Further, the present application also provides the pharmaceutical use of the tamoxifen complex nanoemulsion, specifically the application of the tamoxifen complex nanoemulsion in preparing a drug for preventing and treating breast cancer.

[0044] The present application has the following beneficial effects:

[0045] (1) The same nano-preparation is prepared for the two drugs of tamoxifen citrate and ginsenoside, the preparation process is simple, and the solubility and bioavailability of the two drugs are significantly improved;

[0046] (2) The prepared nanoemulsion has a small particle size and good stability;

[0047] (3) The pharmacological experiment proves that the drug irritation and side effects can be significantly reduced, and the targeting and therapeutic effect of the nanoemulsion are further improved. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Total impurity content in the tamoxifen nanoemulsion prepared by the present application in stability experiment

[0049] Figure 2 Cumulative dissolution of tamoxifen in the tamoxifen nanoemulsion prepared by the present application

[0050] Figure 3 Cumulative dissolution of ginsenoside in the tamoxifen nanoemulsion prepared by the present application DETAILED DESCRIPTION

[0051] The present application will be further described below by examples. It should be correctly understood that: the examples of the present application are only used to illustrate the present application, and are not limited to the present application, and the person skilled in the art should know that the described examples do not limit the present application in any way.

[0052] Example 1:

[0053] Component allocation ratio: 10 parts by weight of tamoxifen citrate;

[0054] 10 parts by weight of ginsenoside;

[0055] 35 parts by weight of glyceryl tributyrate;

[0056] Myristic acid isopropyl ester 50 parts by weight;

[0057] Tragacanth gum 60 parts by weight;

[0058] Poloxamer 20 parts by weight;

[0059] Glycerol 60 parts by weight;

[0060] Preparation method:

[0061] (1) Take an appropriate amount of glyceryl tributyrate and myristic acid isopropyl ester, add tamoxifen and ginsenosides, ultrasonic, ultrasonic power is 30% ~ 35%, ultrasonic temperature setting is 60℃, ultrasonic time is 0.5h, preparation of oil phase mixture;

[0062] (2) Take tragacanth gum, poloxamer mixed evenly, then add glycerol, 1% polyvinyl alcohol aqueous solution, pH to 5.5, 50℃ heating, stirring evenly, preparation of emulsifier mixture;

[0063] (3) Slowly add the oil phase mixture of step (1) to the emulsifier mixture of step (2), the volume ratio of organic phase to aqueous phase is 5:1, stirring evenly, forming the primary emulsion further ultrasonic refinement;

[0064] (4) Add an appropriate amount of water to the primary emulsion after ultrasonic refinement in step (3), stir evenly, form a coarse emulsion, homogenization treatment, the homogenization process is set to 18000-29000psi, homogenization 2 times, the homogenization process keeps the temperature at 202℃, preparation of tamoxifen complex nanoemulsion.

[0065] Example 2:

[0066] Component ratio: tamoxifen citrate 15g;

[0067] Ginsenosides 8g;

[0068] Glyceryl tributyrate 30g;

[0069] Myristic acid isopropyl ester 50g;

[0070] Tragacanth gum 60g;

[0071] Poloxamer 35g;

[0072] Glycerol 100g;

[0073] Preparation method:

[0074] (1) Take an appropriate amount of glyceryl tributyrate and myristic acid isopropyl ester, add tamoxifen and ginsenosides, ultrasonic, ultrasonic power is 30% ~ 35%, ultrasonic temperature setting is 55℃, ultrasonic time is 1h, preparation of oil phase mixture;

[0075] (2) Mix the tragacanth and poloxamer uniformly, then add glycerol and 2% polyvinyl alcohol solution, adjust the pH to 6.5, heat at 60°C, and stir uniformly to prepare the emulsifier mixture;

[0076] (3) Slowly add the oil phase mixture of step (1) to the emulsifier mixture of step (2), with the volume ratio of organic phase to aqueous phase being 6:1, stir uniformly, and further refine by ultrasound after forming the primary emulsion;

[0077] (4) Add the primary emulsion after ultrasound refinement of step (3) to an appropriate amount of water, stir uniformly to form a coarse emulsion, and homogenize, with the pressure being set to 29000 psi during homogenization, homogenized twice, and the temperature being maintained at 202°C during homogenization, to prepare the tamoxifen complex nanoemulsion.

[0078] Example 3

[0079] Ingredients: tamoxifen citrate 15 g;

[0080] ginsenoside 10 g;

[0081] glycerol tributyrate 35 g;

[0082] isopropyl myristate 30 g;

[0083] tragacanth 80 g;

[0084] poloxamer 35 g;

[0085] glycerol 80 g

[0086] Preparation method same as example 2.

[0087] Example 4

[0088] Ingredients: tamoxifen citrate 15 g;

[0089] ginsenoside 10 g;

[0090] glycerol tributyrate 25 g;

[0091] isopropyl myristate 60 g;

[0092] tragacanth 80 g;

[0093] poloxamer 50 g;

[0094] glycerol 100 g

[0095] Preparation method same as example 1.

[0096] Example 5

[0097] Ingredients: tamoxifen citrate 30 g;

[0098] Ginsenoside 16g;

[0099] Glycerin tributyrate 50g;

[0100] Isopropyl myristate 60g;

[0101] Tragacanth 100g;

[0102] Poloxamer 20g;

[0103] Glycerin 60g

[0104] The preparation method is the same as that of Example 2.

[0105] Example 6

[0106] Take the appropriate amount of citric acid tamoxifen, ginsenoside, other components according to the following table weight parts, the specific operation steps according to the method of Example 1 experiment.

[0107] Table 1 different proportion of tamoxifen nano emulsion

[0108]

[0109] Table 2 different components of tamoxifen nano emulsion

[0110]

[0111]

[0112] Take the above prepared nano emulsion for stability and nano emulsion particle size test, wherein the accelerated experiment stability test conditions are 40℃±2℃, relative humidity 75%±5% conditions for 6 months, respectively, record the precipitation, stratification, particle size, impurity situation.

[0113] Table 3 stability test results

[0114]

[0115] From the above table results can be seen, nano emulsion 4, nano emulsion 8 show excellent stability, while other groups appear different degree of precipitation, stratification or particle size change too big phenomenon. At the same time, the impurity situation as shown in table 1, can be seen, after 6 months of experiment, the total impurities of each group have different degrees of increase, among which the total impurities of nano emulsion 4, nano emulsion 8 is the lowest.

[0116] Example 7 dissolution experiment

[0117] The nanoemulsions 6-9 in Examples 1-3 and Example 6 were high-speed centrifuged, precipitated and washed with water, dried, and added with excipients to prepare tablets. The dissolution of the tablets was determined according to the quality standard for tablets of tamoxifen citrate in the 2020 edition of the Chinese Pharmacopoeia.

[0118] The tablets prepared from the nanoemulsions 6-9 in Examples 1-3 and Example 6 were used to determine the dissolution of ginsenoside Re in the dissolution medium of 900 mL of 0.1M hydrochloric acid at a rotation speed of 75 r / min and a temperature of (37±0.5) °C.

[0119] Specifically, the dissolution of tamoxifen is shown in Figure 2 The dissolution of ginsenoside Re is shown in Figure 3 Figure 2 The dissolution of tamoxifen in Examples 1-3 was higher than 88% at 30 min and almost all dissolved at 60 min. Figure 2 The dissolution of ginsenoside Re in Examples 1-3 was higher than 82% at 30 min and higher than 92% at 60 min.

[0120] Example 8 Effect of the composition of the application on a mouse model of breast cancer

[0121] 1 Experimental materials

[0122] 1.1 Experimental animals

[0123] Healthy BABL / c mice (provided by Lunan Pharmaceutical Group Pharmaceutical Center) with a body weight of 20-22 g were used for the experiment after adaptive feeding for 7 days.

[0124] 1.2 Experimental reagents

[0125] Commercially available tamoxifen citrate tablets and tamoxifen nanoemulsion in Example 3.

[0126] 2 Test method

[0127] 2.1 Modeling

[0128] Healthy BABL / c mice were inoculated with 4T1 breast cancer cell suspension under the left fourth mammary fat pad of the mice to establish a mouse breast cancer model, with a concentration of 2*10 6 / mL, 0.1 mL per mouse, and continuous inoculation for 7 days.

[0129] 2.2 Grouping

[0130] ​The mice with successful modeling were divided into a model group, an example 3 group, and a citric acid tamoxifen tablet group, each group having 12 mice, and dosing was started when tumor nodules appeared at the site of inoculation of 4T1 breast cancer cells, wherein the example 3 tamoxifen group was given example 3 nanoemulsion (4 mg / kg) by gavage, the citric acid tamoxifen tablet group was given citric acid tamoxifen tablets (6 mg / kg) by gavage, and the model group was given the same volume of normal saline, and dosing was continuously performed for 30 days.

[0131] 2.3 Measurement of tumor volume and mouse weight

[0132] 2.4 Data processing

[0133] Statistical analysis was performed by using SPSS 22.0 software, and data were represented by "mean ± standard deviation" , analysis among multiple groups was performed by using one-way analysis of variance, and P < 0.05 was considered to have statistical significance.

[0134] 3 Test results

[0135] 3.1 Daily observation of mice

[0136] After 30 days of daily observation, it was found that, as the tumor volume of the mice in the model group increased, the mice showed reduced activity and reduced appetite. The mice in the example 3 group and the commercially available citric acid tamoxifen group showed transient reduced activity at the beginning of dosing, but recovered to normal after 1-2 days, and had good diet and mental state.

[0137] 3.2 Observation of mouse weight and tumor volume

[0138] After 30 days of dosing treatment, it can be seen from Table 4 that the weight loss trend of the mice in the example 3 group was significantly slower than that of the mice in the model group and the commercially available citric acid tamoxifen group. After dosing, the tumor volume of the mice in the example 3 group was significantly smaller than that of the mice in the other groups.

[0139] Table 4: Observation results of mouse weight and tumor volume

[0140]

[0141] It is proved by the above experiments that the tamoxifen nanoemulsion prepared in the present application has a significant inhibitory effect on tumor growth in a breast cancer mouse model, can slow down the growth of tumor volume, and can significantly improve the survival state of mice.

Claims

1. A tamoxifen-containing complex nanoemulsion, characterized in that, The tamoxifen nanoemulsion contains ginsenosides, and the weight parts of each component are as follows: Tamoxifen citrate 10-15 parts by weight; 8-10 parts by weight of ginsenosides; 25-35 parts by weight of glyceryl tribanilate; 30-50 parts by weight of isopropyl myristate; 60-80 parts by weight of tragacanth gum; Poloxamer 20-35 parts by weight; 60-100 parts by weight of glycerol; The preparation method of the tamoxifen composite nanoemulsion includes the following steps: (1) Take appropriate amounts of glyceryl tartrate and isopropyl myristate, add tamoxifen and ginsenosides, sonicate, and prepare an oil phase mixture; (2) Mix tragacanth gum and poloxamer evenly, then add glycerol, heat and stir evenly to prepare an emulsifier mixture; (3) Slowly add the drug solution from step (2) to the oil phase mixture from step (1), stir evenly, and then further refine it by ultrasonication after forming the initial emulsion; (4) Add an appropriate amount of water to the coarse emulsion after ultrasonic refinement in step (3), stir evenly to form a coarse emulsion, homogenize it, and prepare tamoxifen composite nanoemulsion.

2. The tamoxifen composite nanoemulsion according to claim 1, characterized in that, In step (2), the ultrasonic power is 15% to 30%.

3. The tamoxifen composite nanoemulsion according to claim 1, characterized in that, In step (2), the pH of the emulsifier mixture is adjusted to 5.0-6.5, and the temperature is heated to 40-60°C.

4. The tamoxifen composite nanoemulsion according to claim 1, characterized in that, In step (3), when the organic phase is mixed with the aqueous phase, the volume ratio of the organic phase to the aqueous phase is 3:1 to 6:

1.

5. The tamoxifen composite nanoemulsion according to claim 1, characterized in that, In step (3), the homogenization pressure is set at 18000-29000 psi, and homogenization is performed 2-3 times. The temperature is maintained at 20±3℃ during the homogenization process.

6. The application of the tamoxifen composite nanoemulsion according to claim 1 in the preparation of drugs for the prevention and treatment of breast cancer.

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