Pharmaceutical composition containing solanesol as well as preparation method and application of pharmaceutical composition

Abstract

The invention relates to a pharmaceutical composition containing solanesol as well as a preparation method and application of the pharmaceutical composition. The solanesol-containing pharmaceutical composition provided by the invention comprises solanesol, a cosolvent, a dispersant and water. According to the pharmaceutical composition, the solubility of solanesol can be improved, the medicine can enter blood circulation more easily, the bioavailability is improved, and the treatment effect of the medicine is improved. In addition, the pharmaceutical composition can improve the physical and chemical stability of the drug itself, reduces the use of organic solvents, and is more environment-friendly.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a pharmaceutical composition containing solanesol, a preparation method and use thereof. Background Art

[0002] Solanesol, also known as nonaprenyl alcohol, tripterpene alcohol, is abundant in Solanaceae plants such as tobacco, tomatoes, potatoes, eggplants and peppers. It is a tripterpene alcohol composed of nine isoprene units. It is currently mainly used as a key drug intermediate for the synthesis of vitamin K side chains, coenzyme Q10, anti-ulcer and anti-cancer drugs. It has a variety of biological activities, such as antibacterial, anti-ulcer, antioxidant, and neuroprotective.

[0003] Although solanesol has many biological activities, it is difficult to make solanesol into pharmaceutical preparations because it is insoluble in water. Solanesol is a non-polar or weakly polar compound with poor solubility in water. Low solubility limits its application in aqueous pharmaceutical preparations, especially when water solubility is required to improve bioavailability; solanesol-derived micelles can be used for the delivery of hydrophobic drugs, but the stability of solanesol derivatives is still affected by pH and other physiological conditions; the use of drug carriers will bring metabolic problems related to exogenous biological toxicity.

[0004] Due to these problems, no formulations of solanesol currently exist. Summary of the invention

[0005] In order to overcome the defects of the prior art, the present invention provides a pharmaceutical composition containing solanesol and its preparation method and use. The pharmaceutical composition of the present invention can improve the solubility of solanesol, which is insoluble in water. The pharmaceutical composition of the present invention can improve the solubility of solanesol (for example, 1.8g of solanesol can be dispersed and dissolved in 100mL water), which is conducive to the drug entering the blood circulation more easily, improving bioavailability and improving drug treatment effects. In addition, the pharmaceutical composition can improve the physical and chemical stability of the drug itself, reduce the use of organic solvents, and be more environmentally friendly. Compared with dispersing solanesol directly in water, or dissolving it in ethanol and then dispersing it in water, the pharmaceutical composition of the present invention can be stored for a long time after successful preparation, and there will be no solanesol precipitation. The pharmaceutical composition experiment after being placed for 3 months is still effective, which is conducive to medication stability. It has been verified that the pharmaceutical composition of the present invention can treat atherosclerosis and reduce weight.

[0006] The present invention is achieved by adopting the following technical solutions.

[0007] In one aspect, the present invention provides a pharmaceutical composition comprising solanesol, the pharmaceutical composition comprising solanesol, a co-solvent, a dispersant and water.

[0008] Preferably, the co-solvent is selected from C2-C4 lower alkanols, preferably ethanol.

[0009] Preferably, the dispersant is selected from hydroxymethyl cellulose.

[0010] Preferably, the pharmaceutical composition comprises 1-3 parts by weight of solanesol, 1-3 parts by weight of a co-solvent, 0.1-1 parts by weight of a dispersant and 100 parts by weight of water.

[0011] Preferably, the pharmaceutical composition comprises 1.5 parts by weight of solanesol, 2 parts by weight of a cosolvent, 0.5 parts by weight of a dispersant and 100 parts by weight of water.

[0012] Preferably, the pharmaceutical composition is an oral liquid preparation.

[0013] In a specific embodiment, the pharmaceutical composition of the present invention is formulated as follows:

[0014] Solanesol 1.5g

[0015] 2 mL of ethanol

[0016] Hydroxymethylcellulose 0.5g

[0017] 100mL water.

[0018] In another aspect, the present invention provides a method for preparing a pharmaceutical composition comprising solanesol, the method comprising:

[0019] (1) dissolving solanesol with a co-solvent to obtain an ethanol solution of solanesol;

[0020] (2) dissolving the dispersant in water to obtain an aqueous solution of the dispersant;

[0021] (3) mixing the aqueous solution of the dispersant obtained in step (2) with the ethanol solution of solanesol obtained in step (1) to obtain a product.

[0022] Preferably, in step (1), the co-solvent is selected from C2-C4 lower alkanols, preferably ethanol.

[0023] Preferably, in step (2), the dispersant is selected from hydroxymethyl cellulose.

[0024] Preferably, in step (3), the temperature is 30-50 degrees, preferably 30-40 degrees, more preferably 37 degrees.

[0025] Preferably, in step (3), the aqueous solution of the dispersant obtained in step (2) is slowly added to the ethanol solution of solanesol obtained in step (1) to mix.

[0026] In yet another aspect, the present invention provides use of the above pharmaceutical composition in preparing a medicament for preventing and / or treating atherosclerosis.

[0027] In another aspect, the present invention provides use of the above pharmaceutical composition in preparing a medicament for preventing and / or treating obesity.

[0028] The dosage of the pharmaceutical composition of the present invention is 1 to 200 mg / kg, preferably 25 to 100 mg / kg.

[0029] Compared with the prior art, the present invention has at least the following beneficial technical effects:

[0030] The present invention adopts ethanol as a cosolvent, which can not only increase the solubility of the drug, reduce the shortcomings of the original drug and improve the quality of the preparation, but also increase the stability of the preparation, improve absorption and increase the pharmacological effect.

[0031] The dispersant hydroxymethyl cellulose of the present invention can not only help solanesol dissolve in water, but also shorten the dissolution time, improve the stability of the drug itself, prolong the effective period, and make the drug in a high-energy state, accelerate the dissolution rate in the body, improve the bioavailability, and exert better drug efficacy.

[0032] In the present invention, the co-solvent and the dispersant must be used simultaneously. When only the co-solvent is used, the solanesol will re-precipitate when dispersed in water.

[0033] In the present invention, the aqueous solution of the dispersant obtained in step (2) is slowly added to the ethanol solution of solanesol obtained in step (1) to mix them, thereby achieving a better dispersion effect.

[0034] The present invention controls the temperature of the system during the mixing stage of step (3), thereby achieving an optimal dispersion rate without affecting the activity, and can improve the stability of the drug and enhance the bioavailability.

[0035] The present invention first discovered that solanesol can be used to prepare a drug for preventing and / or treating atherosclerosis. Specifically, the inventors found that solanesol has an excellent therapeutic effect on vascular endothelial damage in atherosclerosis. In a cell model experiment, it was shown that solanesol can effectively remove ROS in HUVECs cells induced by H2O2 and reduce oxidative stress. Solanesol can reduce IFN-γ and IL-1β secreted by HUVECs cells after oxidative damage. In a mouse model, it was shown that solanesol can improve the pathological changes of aortic H&E stained sections, and the concentrations of factors IFN-γ and IL-1β in mouse serum are reduced.

[0036] In addition, the weight changes of mice were recorded during the experiment, and it was found that solanesol could reduce the weight of mice with atherosclerosis.

[0037] The pharmaceutical composition of the present invention still has a therapeutic effect after being placed for 3 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Solanesol reduces oxidative stress and inhibits endothelial cell inflammation: (A) ROS fluorescence changes of HUVECs cells; SOD and GPx pseudoenzyme activities of Solanesol (B, C); (D) IFN-γ concentration secreted by HUVECs cells; (E) IL-1β concentration secreted by HUVECs cells; (F) Solanesol inhibits HUVECs cells from taking up oxidized low-density lipoprotein; (G) Solanesol inhibits macrophages from being induced into foam cells by oxidized low-density lipoprotein;

[0039] Figure 2 Oral administration of solanesol to treat atherosclerosis in mice: (A) Analysis of aortic lesions by H&E staining of the aorta after mouse modeling; (B) Detection of interferon IFN-γ levels in the blood of mice after treatment; (C) Detection of inflammatory factor IL-1β levels in the blood of mice after treatment.

[0040] Figure 3 The figure shows the weight changes of mice after they were treated with the pharmaceutical composition of the present invention for 3 months. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0042] Example 1: Pharmaceutical composition of the present invention

[0043] formula:

[0044] Solanesol 1.5g

[0045] 2 mL of ethanol

[0046] Hydroxymethylcellulose 0.5g

[0047] 100mL water.

[0048] Preparation method:

[0049] (1) dissolving solanesol in ethanol to obtain an ethanol solution of solanesol;

[0050] (2) dissolving hydroxymethyl cellulose in water to obtain an aqueous solution of hydroxymethyl cellulose;

[0051] (3) In a water bath at 37 degrees, slowly add the aqueous solution of hydroxymethyl cellulose obtained in step (2) to the ethanol solution of solanesol obtained in step (1) to obtain a product.

[0052] It can be seen that the solubility of solanesol in the pharmaceutical composition is 1.8 g.

[0053] After being placed for three months, the pharmaceutical composition was still stable without precipitation of solanesol.

[0054] Example 2: Pharmaceutical composition of the present invention

[0055] formula:

[0056] Solanesol 1g

[0057] Ethanol 2mL

[0058] Hydroxymethylcellulose 0.5g

[0059] 100mL water.

[0060] Preparation method:

[0061] (1) dissolving solanesol in ethanol to obtain an ethanol solution of solanesol;

[0062] (2) dissolving hydroxymethyl cellulose in water to obtain an aqueous solution of hydroxymethyl cellulose;

[0063] (3) In a water bath at 37 degrees, slowly add the aqueous solution of hydroxymethyl cellulose obtained in step (2) to the ethanol solution of solanesol obtained in step (1) to obtain a product.

[0064] It can be seen that the solubility of solanesol in the pharmaceutical composition is 1.8 g.

[0065] After being placed for three months, the pharmaceutical composition was still stable without precipitation of solanesol.

[0066] Example 3: Pharmaceutical composition of the present invention

[0067] formula:

[0068] Solanesol 1.5g

[0069] Ethanol 2mL

[0070] Hydroxymethylcellulose 1g

[0071] 100mL water.

[0072] Preparation method:

[0073] (1) dissolving solanesol in ethanol to obtain an ethanol solution of solanesol;

[0074] (2) dissolving hydroxymethyl cellulose in water to obtain an aqueous solution of hydroxymethyl cellulose;

[0075] (3) In a water bath at 37 degrees, slowly add the aqueous solution of hydroxymethyl cellulose obtained in step (2) to the ethanol solution of solanesol obtained in step (1) to obtain a product.

[0076] It can be seen that the solubility of solanesol in the pharmaceutical composition is 2.7 g.

[0077] After being placed for three months, the pharmaceutical composition was still stable without precipitation of solanesol.

[0078] Example 4: Selection of cosolvent type

[0079] There are many types of cosolvents, but not all cosolvents are suitable for all active ingredients. This example is to select a cosolvent suitable for the solanesol of the present invention. This example selects the following cosolvents: dimethyl sulfoxide, sodium benzoate, p-aminobenzoic acid, acetamide, Tween80, and ethanol. The results show that dimethyl sulfoxide, sodium benzoate, p-aminobenzoic acid, acetamide, and Tween80 will bring toxicity risks. For example, dimethyl sulfoxide will cause protein denaturation, and have vascular toxicity and hepato-renal toxicity. Ethanol has multiple advantages as a cosolvent: increase drug solubility; reduce the shortcomings of the original drug and improve the quality of the preparation; increase the stability of the preparation, improve absorption and increase pharmacological effects. Therefore, in order to improve the solubility of solanesol and improve the quality of the preparation, this system selects ethanol dissolution.

[0080] Example 5: Selection of dispersant type

[0081] After studying the co-solvent, the inventor found that even if the co-solvent is used to dissolve the solanesol to form a solanesol solution, when the solution is dispersed in water, the solanesol will re-precipitate. Therefore, the present invention considers further adding a dispersant thereto to facilitate dispersion. This embodiment screens the types of dispersants, and the specific dispersants are as follows:

[0082] Ethylcellulose, copovidone, hydroxypropylcellulose, hydroxymethylcellulose.

[0083] The specific preparation method is as follows:

[0084] Method 1: Using ethyl cellulose as a dispersant. Ethyl cellulose is insoluble in water and cannot be further mixed with the solanesol solution.

[0085] Method 2: Using copovidone as a dispersant, dissolve 0.1 g in 2 mL of water, dissolve 0.03 g of solanesol in 40 μL of ethanol, mix the copovidone aqueous solution with the ethanol containing solanesol to obtain a 0.5% copovidone solution + solanesol;

[0086] Method 3: Use hydroxypropyl cellulose as dispersant, 400 μL water in a 60 degree water bath, take 0.1g and slowly add it, after adding, add the remaining 1.6mL water to ensure complete dissolution. Take 40 μL ethanol to dissolve 0.03g solanesol, mix the hydroxypropyl cellulose aqueous solution with the ethanol containing solanesol to obtain 0.5% hydroxypropyl cellulose solution + solanesol.

[0087] Method 4: Using hydroxymethyl cellulose as a dispersant, take 0.1 g and dissolve it in 2 mL of water, and place it in a water bath at 37 degrees until it is completely dissolved. Take 40 μL of ethanol and dissolve 0.03 g of solanesol. Mix the hydroxymethyl cellulose aqueous solution with the ethanol containing solanesol to obtain a 0.5% hydroxymethyl cellulose solution + solanesol.

[0088] Compare the dissolution effects of the above four methods.

[0089] Method 1 found that ethyl cellulose could not be dissolved in water and could not help solanesol dissolve in water.

[0090] Method 2 successfully obtained 0.5% copovidone solution + solanesol solution.

[0091] Compared with method 4, the 0.5% hydroxypropyl cellulose solution + solanesol solution is relatively turbid, some solanesol is not completely dispersed in the 0.5% hydroxypropyl cellulose solution, and the overall solution preparation time is relatively long, requiring 30 minutes. The overall preparation time of method 4 is 15 minutes.

[0092] In addition, the reducing ability of 0.5% copovidone solution + solanesol solution and 0.5% hydroxymethyl cellulose solution + solanesol was compared by using the phenanthroline method, and it was found that the reducing ability of 0.5% copovidone solution + solanesol solution was lower than that of 0.5% hydroxymethyl cellulose solution + solanesol.

[0093] Therefore, hydroxymethyl cellulose was finally selected as the dispersant.

[0094] Example 6: Effect of the order of drug addition

[0095] The same technical scheme as in Example 1 is adopted, except that in step (3), the ethanol solution of solanesol obtained in step (1) is slowly added to the aqueous solution of hydroxymethyl cellulose obtained in step (2) at 37 degrees to mix them.

[0096] The results showed that the solanesol dissolution effect was poor, and some solanesol precipitated and floated on the surface of the solution after the solution was left for a period of time.

[0097] Example 7: Effect of Temperature

[0098] The same technical scheme as in Example 1 was adopted, except that in step (3), the temperatures were 4 degrees, room temperature, 37 degrees, and 60 degrees, respectively. The drug dispersion rate was investigated.

[0099] The results showed that without affecting the activity, the dissolution and dispersion rate in a 37-degree water bath was the best, which only took 5 minutes. At 4 degrees and room temperature, it took 19 minutes and 15 minutes. Although the dissolution rate in a 60-degree water bath was the fastest, its reduction activity was reduced. Therefore, 37 degrees was finally selected.

[0100] Experimental Example 1: Experiment on the inhibition of oxidative damage and inflammation of vascular endothelial cells by solanesol

[0101] 1. HUVECs cells were cultured at 2×10 5 The cells were inoculated at a density of 100 μg / ml in a 6-well plate and cultured in a carbon dioxide incubator for 24 hours. A blank control group, a H2O2 group, and a H2O2+solanesol group were set up; the concentration of solanesol was 50 μg / ml, the concentration of H2O2 was 300 μmol / L, and the cells were cultured in a carbon dioxide incubator for 24 hours.

[0102] 2. The DCFH-DA probe was used to detect the ROS level in HUVECs cells. After the DCFH-DA probe was incubated with the cells for 40 minutes, the DCFH generated by esterase hydrolysis after entering the cells would be oxidized into fluorescent DCF by ROS. The DCF green fluorescence was photographed under a fluorescence microscope to compare the fluorescence changes of different groups.

[0103] 3. The SOD and GPx pseudoenzyme activities of solanesol at concentrations of 50μg / ml and 100μg / ml were detected using the pyrogallol oxidation method and GR coupling method to characterize the ability of solanesol to scavenge O2·- and H2O2.

[0104] 4. Collect the supernatants of different groups and store them in a -20°C refrigerator. Detect the concentration in the cell supernatant according to the instructions of the IFN-γ and IL-1β ELISA kits.

[0105] 5. HUVECs cells were cultured at 10 6 The cells were seeded at a density of 10 cells / well in each well of a 12-well plate overnight, and then stimulated with LPS (1 μg / mL) for 24 hours, and then solanesol was added for 2 hours. Then, Dil-oxLDL at a concentration of 40 μg / mL was incubated for 3 hours. The normal control group was treated with fresh culture medium, and the model group was stimulated with Dil-oxLDL only. After 3 hours, the cells were stained with Dil, Dio and DAPI and fluorescent images were taken with a fluorescence microscope.

[0106] 6. Macrophages were cultured at 10 6 Cells were seeded in each well of a 12-well plate at a density of 10 cells / well overnight, and then stimulated with LPS (1 μg / mL) for 24 hours, followed by incubation with solanesol for 2 hours. Then, oxLDL at a concentration of 50 μg / mL was incubated for 48 hours, the normal control group was treated with fresh culture medium, and the model group was stimulated with oxLDL only. After 48 hours, cells were stained with Oil Red O staining kit and observed under an optical microscope.

[0107] 7. Experimental results

[0108] Human umbilical vein endothelial cells (HUVECs) were used to treat with solanesol. The H2O2-induced ROS level in HUVECs cells was changed by Figure 1 A showed that solanesol could significantly reduce the DCF fluorescence caused by ROS in HUVECs cells and alleviate oxidative stress. The SOD and GPx enzyme simulation experiments showed that solanesol could effectively remove O2·- and H2O2( Figure 1 B, 1C), relieve endothelial oxidative stress and avoid endothelial dysfunction caused by oxidative stress damage of endothelial cells. The supernatant of HUVECs cells treated with solanesol was collected and the changes of interferon IFN-γ and inflammatory factor IL-1β were analyzed by ELISA kit. Figure 1 D, 1E show that after solanesol treatment, IFN-γ and IL-1β were significantly reduced, and solanesol can inhibit vascular wall inflammation. In addition, solanesol can inhibit HUVECs cells from taking up oxLDL ( Figure 1 F) to prevent oxLDL from inhibiting HUVECs cell viability, proliferation and angiogenesis. Figure 1 As shown in F and 1G, solanesol can also inhibit the formation of foam cells induced by oxidized low-density lipoprotein, preventing foam cell rupture from aggravating vascular wall inflammation caused by oxidative damage.

[0109] Experimental Example 2: Solanesol improves atherosclerosis

[0110] 1. Experimental Animals

[0111] ApoE in male C57BL / 6 - / - Fifteen mice were purchased from Vital River, 7 weeks old, SPF grade, weighing 18-20 g. Five male C57BL / 6 mice of the same strain and age were purchased, weighing 18-20 g. The mice were randomly divided into groups and housed in standardized independent ventilation cages (temperature 22±2°C, relative humidity 45-65%, light / dark cycle every 12 hours).

[0112] 2. Grouping and Dosing

[0113] 1) Drug preparation: Solanesol is dissolved in ethanol and dispersed in a 0.5% hydroxymethylcellulose solution to obtain a 100 mg / kg solvent solution.

[0114] 2) Study on oral administration of solanesol to treat ApoE in rats fed a high-fat diet - / -Mouse atherosclerosis model: 15 model mice were randomly divided into three groups: the model group was fed with high-fat diet + 0.5% hydroxymethylcellulose solution, the experimental group was fed with high-fat diet + 100 mg / kg solanesol, and the control group was fed with high-fat diet + rosuvastatin 1.55 mg / kg; the blank group C57BL / 6J mice were fed with ordinary diet + 0.5% hydroxymethylcellulose solution. The mice in each experimental group were intragastrically administered with solanesol solution according to the weight of the mice at the same time every day.

[0115] 3. Experimental results

[0116] Oral administration of solanesol improves atherosclerosis Figure 2 After modeling, compared with the blank group, H&E stained sections of the aorta of the atherosclerosis modeling mice showed obvious lesions, aortic intima damage, endothelial cell shedding, and foamy accumulation of foam cells and fibroblasts ( Figure 2 A); while H&E staining of the aorta of the mice in the experimental group only showed intimal damage. The serum of mice in each group was collected and the concentrations of IFN-γ and IL-1β were analyzed by ELISA kit ( Figure 2 B, 2C), compared with the blank group, the IFN-γ and IL-1β in the model group were significantly higher; compared with the model group, solanesol in the experimental group significantly reduced the levels of IFN-γ and IL-1β in the serum of atherosclerotic ApoE- / - mice. At the end of the experiment, the weight of the mice in the model group was higher than that of the blank control, and the weight of the mice in the experimental group was significantly reduced compared with the model group (Table 1). The drug composition that had been placed for 3 months was re-administered to the mice, and it was observed that the drug composition still had a therapeutic effect, and the weight of the mice in the experimental group was significantly reduced compared with the model ( Figure 3 )

[0117] Table 1 Body weight changes of mice in each group

[0118]

[0119] Note: Compared with the blank group during the same period, △p<0.05, △△p<0.01; compared with the model group during the same period, *p<0.05, **p<0.01.

Claims

1. A pharmaceutical composition comprising solanesol, the pharmaceutical composition comprising solanesol, a solubilizing agent, a dispersant and water.

2. The pharmaceutical composition according to claim 1, wherein The cosolvent is selected from C2-C4 lower alkanols, preferably ethanol; Preferably, the dispersant is selected from hydroxymethyl cellulose.

3. The pharmaceutical composition according to claim 1 or 2, wherein The pharmaceutical composition comprises 1-3 parts by weight of solanesol, 1-3 parts by weight of a cosolvent, 0.1-1 parts by weight of a dispersant and 100 parts by weight of water; Preferably, the pharmaceutical composition comprises 1.5 parts by weight of solanesol, 2 parts by weight of a cosolvent, 0.5 parts by weight of a dispersant and 100 parts by weight of water.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The pharmaceutical composition is an oral liquid preparation.

5. A method for preparing a pharmaceutical composition containing solanesol according to any one of claims 1 to 4, the method comprising: (1) dissolving solanesol with a co-solvent to obtain an ethanol solution of solanesol; (2) dissolving the dispersant in water to obtain an aqueous solution of the dispersant; (3) mixing the aqueous solution of the dispersant obtained in step (2) with the ethanol solution of solanesol obtained in step (1) to obtain a product.

6. The method according to claim 5, wherein: In step (1), the co-solvent is selected from C2-C4 lower alkanols, preferably ethanol.

7. The method according to claim 5 or 6, wherein: In step (2), the dispersant is selected from hydroxymethyl cellulose.

8. The method according to any one of claims 5 to 7, wherein: In step (3), the temperature is 30-50 degrees, preferably 30-40 degrees, more preferably 37 degrees; Preferably, in step (3), the aqueous solution of the dispersant obtained in step (2) is slowly added to the ethanol solution of solanesol obtained in step (1) to mix.

9. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating atherosclerosis.

10. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating obesity.

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