CN107468670A - A kind of nanometer formulation for diabetes B treatment and preparation method thereof - Google Patents

Abstract

The invention discloses a nano-preparation for the treatment of type 2 diabetes, which is composed of the following effective components in terms of mass percentage: pluronic 35%-45%, SRT1720 7%-12%, lyoprotectant 40% ‑60%, the sum of the mass percentages of the above components is 100%, and the present invention also discloses a preparation method for nano-preparations for the treatment of type 2 diabetes: using dimethyl sulfoxide that can be miscible with water in any proportion SRT1720 is dissolved together with the wrapping material Pluronic, then the mixed solution is added to normal saline, and then the stable nano-drug-loaded particle dispersion is directly obtained by ultrasonic treatment, and then the dimethyl sulfoxide and soluble impurities in the dispersion are removed by dialysis , then add a protective agent, and finally remove water, so as to obtain dry nano-preparation powder. The present invention solves the problems in the prior art that the particle size of the nano-preparation does not meet the requirements and the drug-loading performance is relatively poor.

Description

A nano-preparation for the treatment of type 2 diabetes and its preparation method

technical field

The invention belongs to the field of nanobiology technology, and in particular relates to a nano preparation for treating type 2 diabetes, and also relates to a preparation method of the nano preparation for treating type 2 diabetes.

Background technique

Sirt1 (Sirtuin 1) is a sirtuin homolog existing in mammals. It is a nicotinamide adenine dinucleotide-dependent histone deacetylase that can regulate a variety of cellular functions, such as gene silencing, cell cycle, apoptosis and energy metabolism disorders. Sirt1 is generally considered a longevity gene. Sirt1 plays an important regulatory role in the balance of glucose metabolism and insulin sensitivity. SRT1720 is a specific small molecule agonist of Sirt1, and its agonistic effect is 1000 times that of the commonly used Sirt1 non-specific agonist resveratrol. A study reported in the world-renowned academic journal "Nature" that SRT1720 can significantly improve the symptoms of type 2 diabetes. However, due to the poor solubility and high toxicity of SRT1720 in the aqueous phase, the bioavailability of the compound is low and the prospect of clinical application is bleak. Therefore, reducing the toxicity of SRT1720, improving its water solubility, and enhancing its bioavailability are necessary for the development of SRT1720 as a drug for treating diabetes.

Nano-drug carriers can effectively disperse water-insoluble compounds in aqueous solution, increase their stability in vivo, prolong the circulation time in vivo, thereby effectively improving the bioavailability of drugs, and can also play a role in sustained and controlled release. Keep the blood or lesion site at a high drug concentration. Amphiphilic materials can self-assemble to form nanomicelles with a core-shell structure. The inner core is a hydrophobic ultrastructure, and hydrophobic drugs can be firmly encapsulated in it, while the outer shell is composed of a hydrophilic skeleton, making the whole The system has very good dispersion in aqueous solution. This kind of nano drug carrier can be prepared relatively easily by self-assembly method.

Planick It is a non-ionic amphiphilic polymer compound composed of polyoxyethylene and polyoxypropylene ether. It has been approved by the FDA as a conventional drug excipient. It has good biocompatibility, low toxicity, no irritation and allergies, etc. Advantages, it can be used in the preparation of various dosage forms. It is especially suitable for the preparation of various insoluble drugs or compounds. The patent of pluronic drug-loaded micelles has been reported, and the method described in the patent number: US2012032115A1 cannot be used for large-scale production, and there are hidden dangers in quality control in different batches. In China, Jin Yi and others used Pluronic to modify PAMAM to obtain a new type of drug assembly material, which effectively reduces the toxicity of PAMAM, and used it in the construction of anti-tumor nano-drug carriers, and applied for a patent (CN103289097A).

The small molecular compound STR1720 has extremely poor solubility in water, which makes it unable to further play its role in the treatment of type 2 diabetes, which can be solved by the nano-medicine prepared by pluronic. Although Pluronic can be used for the encapsulation of non-water-soluble drugs, the available nano-preparations must include good encapsulation properties; small enough particle size; maintain a certain stability in the body circulation; slow and effective release of encapsulating drug molecules, with long-term storage ability. It is very difficult to meet the above conditions at the same time, and other related technical methods cannot directly serve as a reference. For example, the applicant previously disclosed a preparation method of gossypol nano-preparation (publication number: CN104523606A), but the nano-drug particles prepared after changing the drug can not reach the ideal particle size at all, and the drug-loading performance is also relatively poor, so it cannot be obtained at all. for further application. Therefore, it is necessary to continuously explore and test in combination with practical experience in the new construction process, and optimize and change key nodes.

Contents of the invention

The purpose of the present invention is to provide a nano-preparation for the treatment of type 2 diabetes, which solves the problems in the prior art that the particle size of the nano-preparation does not meet the requirements and the drug-loading performance is relatively poor.

Another object of the present invention is to provide a preparation method of nano-preparation for the treatment of type 2 diabetes.

The first technical solution adopted in the present invention is a nano-preparation for the treatment of type 2 diabetes, which consists of the following effective components in terms of mass percentage: Pluronic 35%-45%, SRT17207%-12%, 40%-60% of the lyoprotectant, the sum of the mass percentages of the above components is 100%.

The feature of the first technical solution of the present invention is also that,

Pluronic specifications include: F68, F87, F108, F127.

The second technical solution adopted in the present invention is a method for preparing a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1. Weighing raw materials: Weigh 35%-45% of Pluronic, 7%-12% of SRT17207% and 40%-60% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic and SRT1720 at a ratio of 4:1, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution;

Step 3. Slowly drop the mixed solution obtained in step 2 into the physiological saline under high-speed stirring, and continue stirring after dropping the mixed solution to obtain a nano drug-loaded particle dispersion;

Step 4. Use a probe-type ultrasonic breaker to sonicate the nano drug-loaded particle dispersion obtained in step 3, and keep an ice bath during the sonication process;

Step 5. Dialyzing the nano drug-carrying particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml of nano formulation solution.

Step 7. Divide the nano-preparation solution into equal volumes, and remove the moisture to obtain the final nano-preparation dry powder.

The second technical solution of the present invention is characterized in that,

In step 2, filter a fat-soluble membrane with a practical pore size of 0.22 microns.

In step 3, the stirring speed of dropping the mixed solution into the physiological saline is controlled at 3-5ml/min.

The mass-volume ratio of SRT1720 and normal saline input in step 3 is 10mg:80ml, and the stirring speed is 1000-1500 rpm.

In step 4, each ultrasonic treatment time is 6-8 seconds, the interval time is 3-4 seconds, the total treatment time is 10-15 minutes, and the ultrasonic power is 300-500W.

The dialysis in Step 5 is specifically to put the dispersion of nano drug-loaded particles into a dialysis bag with a molecular weight cut-off of 3.5 kDa, and use normal saline for dialysis.

In step 6, the stirring speed of the nano-drug-loaded particle dispersion is 200-400 rpm, and the filter uses a sterile water-soluble filter membrane with a pore size of 0.22 microns.

The water removal in step 7 is carried out by pre-freezing in a -70-80°C ultra-low temperature refrigerator for 24-48 hours, and then using a freeze dryer at a pressure lower than 0.1 Pascal.

The beneficial effect of the present invention is a nano-preparation for the treatment of type 2 diabetes and a preparation method thereof, which uses the amphiphilic compound pluronic to SRT1720 to prepare nano-scale drug carrier particles through a self-assembly method. Specifically, SRT1720 and the wrapping material Pluronic are co-dissolved with dimethyl sulfoxide, which can be miscible with water in any proportion, and then the mixed solution is added to physiological saline, and then the stable nano-drug-loaded particle dispersion is directly obtained by ultrasonic treatment , eliminating the intermediate film formation, redispersion and other steps that cause quality differences or affect the yield, and then use dialysis to remove dimethyl sulfoxide and soluble impurities in the dispersion, then add a protective agent, and finally freeze-dry The moisture in the solution is removed, so as to obtain the final product—dry powder of nano-preparation.

Description of drawings

Fig. 1 is a kind of preparation method flowchart of the nano-preparation that is used for the treatment of type 2 diabetes of the present invention;

Fig. 2 is a kind of preparation method of the nano-preparation that is used for the treatment of type 2 diabetes of the present invention Example 1 prepared pluronic package SRT1720 nano-preparation dispersion situation comparative figure, wherein A is the nano-preparation solution before freeze-drying, B is SRT1720 aqueous dispersion control group, C is the nano-preparation solution after redissolving the lyophilized powder;

Fig. 3 is a transmission electron microscope photo of Example 1 pluronic-coated SRT1720 nano-preparation redissolved in a preparation method of a nano-preparation for the treatment of type 2 diabetes of the present invention;

Fig. 4 is a particle size analysis result figure after redissolving of the nano-preparation of Pluronic coating SRT1720 in Example 1 in the preparation method of the nano-preparation for the treatment of type 2 diabetes of the present invention;

Fig. 5 is a graph of the particle size stability test results of the pluronic-coated SRT1720 nano-preparation solution prepared in Example 1 in a method for preparing a nano-preparation for the treatment of type 2 diabetes of the present invention;

Fig. 6 is the experimental result of the influence of the SRT1720 nano-preparation prepared in Example 1 on the blood sugar of the spontaneous hyperglycemia Zuckerfa/fa rat model in a preparation method of the nano-preparation for the treatment of type 2 diabetes of the present invention;

Fig. 7 is the experimental result of the influence of the SRT1720 nano-preparation prepared in Example 1 on the glucose tolerance of the Zuckerfa/fa rat model of spontaneous hyperglycemia in a preparation method of the nano-preparation for the treatment of type 2 diabetes of the present invention.

detailed description

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

The invention discloses a nano-preparation for treating type 2 diabetes and a preparation method thereof. The amphiphilic compound pluronic is used to self-assemble SRT1720 to prepare nano-scale drug carrier particles. Specifically, SRT1720 and the wrapping material Pluronic are co-dissolved with dimethyl sulfoxide, which can be miscible with water in any proportion, and then the mixed solution is added to physiological saline, and then the stable nano-drug-loaded particle dispersion is directly obtained by ultrasonic treatment , eliminating the intermediate film formation, redispersion and other steps that cause quality differences or affect the yield, and then use dialysis to remove dimethyl sulfoxide and soluble impurities in the dispersion, then add a protective agent, and finally freeze-dry The moisture in the solution is removed to obtain the final product—nano-preparation dry powder, as shown in Figure 1, specifically as follows:

A nano-preparation for the treatment of type 2 diabetes according to the present invention is composed of the following effective components in terms of mass percentage: 35%-45% of pluronic, 7%-12% of SRT1720, 40%-60% of lyoprotectant %, the sum of the mass percentages of the above components is 100%, wherein the Pluronic specification includes: F68, F87, F108, F127, and the lyoprotectant includes one or both of mannitol and hydroxyethyl starch mixture.

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1. Weighing raw materials: Weigh 35%-45% of Pluronic, 7%-12% of SRT17207% and 40%-60% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic and SRT1720 at a ratio of 4:1, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution. Fat-soluble filter membrane with a pore size of 0.22 microns;

Step 3. Slowly drop the mixed solution obtained in step 2 into the normal saline under high-speed stirring. The stirring speed of the normal saline is controlled at 3-5ml/min, and the stirring speed after dripping into the mixed solution is 1000-1500 rpm , to obtain nano drug-loaded particle dispersion;

Step 4. Use a probe-type ultrasonic breaker to sonicate the nano drug-carrying particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time of each sonication is 6-8 seconds, and the interval is 3-4 seconds. The total processing time is 10-15min, and the ultrasonic power is 300-500W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a 3.5kDa dialysis bag, use normal saline for dialysis, the dialysis time is 12-24 hours, during which the external fluid is replaced with normal saline 3-5 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano drug-carrying particle dispersion is 200-400 revolutions/min, and the filter utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, remove the water to obtain the final dry powder of the nano-preparation, wherein, to remove the water, pre-freeze in a -70-80°C ultra-low temperature refrigerator for 24-48 hours, and then use a freeze dryer with a pressure lower than 0.1 under Pascal conditions.

A nano-preparation used for the treatment of type 2 diabetes of the present invention is dissolved in sterilized pure water, its particle size is measured by a Malvern particle size analyzer, and its performance is analyzed: SRT1720 is constructed by wrapping non-ionic amphiphilic material Pluronic Nano preparations to overcome the shortcomings of SRT1720 such as poor water solubility and low bioavailability. The obtained nano drug-loaded particles have the advantages of good stability, high encapsulation efficiency, and uniform particle size, which can effectively ensure the long-term circulation of drugs in the body, significantly reduce the dosage of drugs, and can be used for the treatment of type 2 diabetes. Its core is to solve the problems of complex components and cumbersome preparation process of most pluronic preparations, directly use pluronic to construct a SRT1720 nano-preparation that can be highly dispersed in water environment, and improve its activity in aqueous solution without affecting the activity of SRT1720. Dispersion in . The established process system is simple and practical, and precise quality control can be carried out in each step. The obtained nano-drug-loaded particles have a very high encapsulation rate and drug-loading capacity, and the dry powder of the lyophilized preparation can be stored stably for more than one year.

Example 1

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1, weighing raw materials: Weigh 35% of Pluronic (F68), 8.75% of SRT1720, and 56.25% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drop the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 3ml/min, and the stirring speed after dripping into the mixed solution is 1500 rpm to obtain the nano-medicine particle-loaded dispersion;

Step 4. Use the probe type ultrasonic breaker to sonicate the nano-medicine-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time for each sonication is 8 seconds, and the interval is 4 seconds. The total treatment time is 10min, ultrasonic power is 300W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a 3.5kDa dialysis bag, use normal saline for dialysis, the dialysis time is 12 hours, during which the external fluid is replaced with normal saline 5 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano-drug-carrying particle dispersion is 200 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, and remove the water to obtain the final nano-preparation dry powder. The water removal is carried out in a -70°C ultra-low temperature refrigerator for 24 hours, and then use a freeze dryer at a pressure lower than 0.1 Pascal. .

Part of the nano-preparation dry powder obtained in this example was redissolved in sterilized pure water, and its particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle diameter of 153±4.5nm, and an encapsulation rate of 93±3.6%.

Referring to Figure 2, it can be seen that the label A in Figure 2 is the nano-preparation solution before freeze-drying; the label B is the SRT1720 aqueous dispersion control group, because the compound is insoluble in water, and its dispersion is turbid, and there is a significant difference after standing. Precipitation; the one labeled C is the dissolved SRT1720 nano-preparation, which is basically the same as the nano-preparation before freeze-drying, and the solution is clear and uniform as a whole; it shows that the preparation of nano-drug-loaded particles by pluronic coating in this example can significantly increase the non-water-soluble Dissolution of sexual drug SRT1720 in water. Particle size also meets therapeutic needs.

Example 2

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1, weighing raw materials: Weigh 45% of Pluronic (F87), 1.25% of SRT17201, and 43.75% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drip the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 5ml/min, and the stirring speed after dripping into the mixed solution is 1000 rpm to obtain the nano medicine particle-loaded dispersion;

Step 4. Use the probe-type ultrasonic breaker to sonicate the nano-medicine-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time of each sonication is 6 seconds, and the interval is 3 seconds. The total treatment time is 15min, ultrasonic power is 500W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a 3.5kDa dialysis bag, use normal saline for dialysis, the dialysis time is 24 hours, during which the external fluid is replaced with normal saline 3 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano drug-carrying particle dispersion is 400 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, and remove the water to obtain the final dry powder of the nano-preparation. The water removal is performed by pre-freezing in an ultra-low temperature refrigerator at 80°C for 48 hours, and then using a freeze dryer at a pressure lower than 0.1 Pascal.

Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized fresh water, and the particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle diameter of 160±5.3nm, and an encapsulation rate of 94±2.7%.

Example 3

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1. Weighing raw materials: Weigh 38% of Pluronic (F108), 9.5% of SRT1720, and 52.5% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drip the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 3ml/min, and the stirring speed after dripping into the mixed solution is 1100 rev/min, to obtain nano medicine particle-loaded dispersion;

Step 4. Use the probe-type ultrasonic breaker to sonicate the nano-medicine-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time of each sonication is 6 seconds, and the interval is 3 seconds. The total treatment time is 12min, ultrasonic power is 320W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a dialysis bag of 3.5kDa, normal saline is used for dialysis, and the dialysis time is 12 hours, during which the external fluid saline is replaced 3 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano-drug-carrying particle dispersion is 200 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, and remove the water to obtain the final dry powder of the nano-preparation. The water removal is pre-frozen in a -90°C ultra-low temperature refrigerator for 24 hours, and then a freeze dryer is used at a pressure lower than 0.1 Pascal. .

Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle size of 154±5.7nm and an encapsulation rate of 93±4.9%.

Example 4

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1, weighing raw materials: Weigh 39% of Pluronic (F68), 9.7% of SRT1720, and 51.3% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drip the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 5ml/min, and the stirring speed after dripping into the mixed solution is 1200 rev/min, to obtain nano medicine particle-loaded dispersion;

Step 4. Use the probe-type ultrasonic breaker to sonicate the nano drug-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time of each sonication is 7 seconds, and the interval is 4 seconds. The total treatment time is 15min, ultrasonic power is 400W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a 3.5kDa dialysis bag, use normal saline for dialysis, the dialysis time is 18 hours, during which the external fluid is replaced with normal saline 5 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano drug-carrying particle dispersion is 280 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, and remove the water to obtain the final nano-preparation dry powder. The water removal is performed by pre-freezing in an ultra-low temperature refrigerator at 10°C for 28 hours, and then using a freeze dryer at a pressure lower than 0.1 Pascal.

Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle diameter of 155±7.1nm and an encapsulation rate of 92±3.8%.

Example 5

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1, weighing raw materials: Weigh 40% of Pluronic (F87), 10% of SRT1720, and 50% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drip the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 4ml/min, and the stirring speed after dripping into the mixed solution is 1300 rev/min, to obtain nano medicine particle-loaded dispersion;

Step 4. Use the probe-type ultrasonic breaker to sonicate the nano-medicine-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time of each sonication is 6 seconds, and the interval is 3.5 seconds. The total treatment time is 13min, ultrasonic power is 380W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a dialysis bag of 3.5kDa, normal saline is used for dialysis, and the dialysis time is 20 hours, during which the external fluid normal saline is replaced 4 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano drug-carrying particle dispersion is 300 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, and remove the water to obtain the final dry powder of the nano-preparation. The water removal is performed by pre-freezing in a 30°C ultra-low temperature refrigerator for 30 hours, and then using a freeze dryer at a pressure lower than 0.1 Pascal.

Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle size of 150±7.1nm and an encapsulation rate of 91±4.3%.

Example 6

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1, weighing raw materials: Weigh 41% of Pluronic (F108), 10.25% of SRT1720, and 48.75% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drip the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 5ml/min, and the stirring speed after dripping into the mixed solution is 1500 rev/min, to obtain nano medicine particle-loaded dispersion;

Step 4. Use the probe type ultrasonic breaker to sonicate the nano-medicine-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time for each sonication is 8 seconds, and the interval is 4 seconds. The total treatment time is 15min, ultrasonic power is 400W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a 3.5kDa dialysis bag, use normal saline for dialysis, the dialysis time is 21 hours, during which the external fluid is replaced with normal saline 5 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano drug-carrying particle dispersion is 350 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, and remove moisture to obtain the final nano-preparation dry powder. The moisture removal is performed by pre-freezing in a 40°C ultra-low temperature refrigerator for 40 hours, and then using a freeze dryer at a pressure lower than 0.1 Pascal.

Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle size of 161±5.0nm and an encapsulation rate of 91±4.9%.

Example 7

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1, weighing raw materials: Weigh 42% of Pluronic (F127), 10.5% of SRT1720, and 47.5% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drip the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 4ml/min, and the stirring speed after dripping into the mixed solution is 1400 rev/min, to obtain nano medicine particle-loaded dispersion;

Step 4. Use the probe-type ultrasonic breaker to sonicate the nano drug-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time for each sonication is 7.5 seconds, and the interval is 3.5 seconds. The total treatment time is 14min, ultrasonic power is 450W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a 3.5kDa dialysis bag, use normal saline for dialysis, the dialysis time is 22 hours, during which the external fluid is replaced with normal saline 4 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano drug-carrying particle dispersion is 380 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, and remove the water to obtain the final nano-preparation dry powder. The water removal is performed by pre-freezing in a 60°C ultra-low temperature refrigerator for 44 hours, and then using a freeze dryer at a pressure lower than 0.1 Pascal.

Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle diameter of 151±5.2nm, and an encapsulation rate of 92±6.6%.

Example 8

A preparation method for a nano-preparation for the treatment of type 2 diabetes, specifically implemented according to the following steps:

Step 1, weighing raw materials: Weigh 44% of Pluronic (F127), SRT172011%, and 45% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%;

Step 2. Mix Pluronic with SRT1720, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution, wherein the lipid with a practical pore size of 0.22 microns is filtered Soluble membrane;

Step 3, slowly drip the mixed solution obtained in step 2 into the normal saline under high-speed stirring state, the stirring speed of the normal saline is controlled at 5ml/min, and the stirring speed after dripping into the mixed solution is 1100 rev/min, to obtain nano medicine particle-loaded dispersion;

Step 4. Use the probe-type ultrasonic breaker to sonicate the nano-medicine-loaded particle dispersion obtained in step 3. During the sonication process, keep an ice bath. The time of each sonication is 6 seconds, and the interval is 3 seconds. The total treatment time is 14min, ultrasonic power is 450W;

Step 5. Dialyze the nano-drug-loaded particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion. Specifically, the dialysis is to load the nano-drug-loaded particle dispersion In a 3.5kDa dialysis bag, use normal saline for dialysis, the dialysis time is 12 hours, during which the external fluid is replaced with normal saline 5 times;

Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml nano-preparation solution, wherein, the stirring speed of the nano drug-carrying particle dispersion is 280 revolutions/min, and the filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns.

Step 7. Divide the nano-preparation solution into equal parts, remove moisture to obtain the final nano-preparation dry powder, wherein, remove moisture by pre-freezing in a 70°C ultra-low temperature refrigerator for 46 hours, and then use a freeze dryer at a pressure lower than 0.1 Pascal.

Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a Malvern particle size analyzer.

The SRT1720 nano-preparation obtained in this example has an average particle diameter of 157±5.1nm and an encapsulation rate of 91±5.6%.

The particle size distribution and stability of the prepared SRT1720 nano-preparation were measured, and its morphological characteristics were observed through a transmission electron microscope. The results showed that the nano-preparation has high stability, strong ability to wrap drugs, and uniform morphological characteristics. Good monodispersity, see Figure 2, Figure 3, Figure 4, and Figure 5 for details. The in vitro SIRT1 pathway activation ability of the SRT1720 nano-preparation was tested, and it was found that the activation ability of the nano-preparation was more obvious than that of the free drug. By intervening in the rat diabetes model, it was found that the nano-preparation can enhance the glucose metabolism ability of the model rats and reduce the blood sugar concentration at a dosage of only one-fifth of the free drug. In contrast, SRT1720 directly administered The drug group still cannot reach the therapeutic effect of the nano-preparation group at a very high dosage, see Fig. 6 and Fig. 7, wherein, Fig. 6 is the prepared SRT1720 nano-preparation (20mg/kg, tail vein administration, 4 weeks) Experimental results on the blood sugar effect of the spontaneous hyperglycemia Zuckerfa/fa rat model (Note: Since SRT1720 is insoluble in normal saline, it cannot be injected intravenously, therefore, the dose of SRT1720 in the free SRT1720 group is 100 mg/kg, administered by intragastric administration, 4 week), Fig. 7 is the experimental result of the prepared SRT1720 nano-preparation (20mg/kg, tail vein administration, 4 weeks) on the glucose tolerance of spontaneous hyperglycemia Zuckerfa/fa rat model (note: because SRT1720 is insoluble in normal saline , unable to carry out intravenous injection, therefore, the dose of SRT1720 in the SRT1720 group was 100mg/kg, intragastric administration, 4 weeks), in addition, high-dose SRT1720 also has cardiovascular toxicity.

Preliminary experiments have found that the use of commonly used Tween and Span series of non-ionic surfactants for medical or food use cannot form stable SRT1720 nano-preparations, and there are still problems such as short storage time and inability to prepare lyophilizates. More importantly, the quality control of preparations cannot meet the standards. However, the pluronic packaging material used in the present invention can effectively overcome the shortcomings in the preparation of SRT1720 nano preparations. Although many relevant technical solutions have been published, a series of preliminary experiments found that the required nano-preparation cannot be obtained by directly referring to the process parameters in other related documents. Relying on previous experience, the present invention repeatedly tests and modifies key parameters such as reagent ratio, preparation process and use of auxiliary materials through a large number of experiments, overcomes the shortcomings of insufficient stability and poor repeatability in the preparation process, and finally obtains the most Excellent preparation process. The quality control of the preparation is guaranteed.

The preparation method of a nano-preparation for the treatment of type 2 diabetes is simple and efficient, and the quality of the prepared nano-preparation for the treatment of type 2 diabetes is stable, which can effectively overcome the poor water solubility and low bioavailability of SRT1720 itself To further promote its application level, it has a very good application prospect.

Claims (10)

1. A nano-preparation for the treatment of type 2 diabetes, characterized in that it is made up of the active components of the following raw materials by mass percentage: Pluronic 35%-45%, SRT1720 7%-12%, lyoprotectant 40%-60%, the sum of the mass percentages of the above components is 100%. 2. A nano-preparation for the treatment of type 2 diabetes according to claim 1, wherein the pluronic specifications include: F68, F87, F108, F127. 3. A preparation method for a nano-preparation for the treatment of type 2 diabetes, characterized in that, it is specifically implemented according to the following steps: Step 1. Weighing raw materials: Weigh 35%-45% of Pluronic, 7%-12% of SRT17207% and 40%-60% of lyoprotectant by mass percentage, and the sum of the mass percentages of the above components is 100%; Step 2. Mix Pluronic and SRT1720 at a ratio of 4:1, then mix the mixture with dimethyl sulfoxide at a ratio of 10mg:1ml, and filter to remove impurities to make a mixed solution; Step 3. Slowly drop the mixed solution obtained in step 2 into the physiological saline under high-speed stirring, and continue stirring after dropping the mixed solution to obtain a nano drug-loaded particle dispersion; Step 4. Use a probe-type ultrasonic breaker to sonicate the nano drug-loaded particle dispersion obtained in step 3, and keep an ice bath during the sonication process; Step 5. Dialyzing the nano drug-carrying particle dispersion after the ultrasonic treatment in step 4 to remove the dimethyl sulfoxide solvent and other soluble impurities in the dispersion; Step 6. Slowly add the lyoprotectant to the dispersion of nano-drug-loaded particles under rapid stirring. After it is completely dissolved, filter to remove other insoluble impurities and microorganisms. Use physiological saline to finally make up the volume to obtain 0.1 mg of SRT1720 /ml of nano-preparation solution; Step 7. Divide the nano-preparation solution into equal volumes, and remove the moisture to obtain the final nano-preparation dry powder. 4. a kind of preparation method for the nano-preparation that is used for the treatment of type 2 diabetes according to claim 3, is characterized in that, in described step 2, filter the fat-soluble filter membrane that practical aperture is 0.22 micron. 5. a kind of preparation method that is used for the nano preparation of type 2 diabetes treatment according to claim 3, is characterized in that, in described step 3, the mixing speed that mixed solution is dripped into normal saline is controlled at 3-5ml/min . 6. the preparation method of a kind of nano-preparation that is used for the treatment of type 2 diabetes according to claim 3, is characterized in that, the mass volume ratio of the SRT1720 that drops into in the described step 3 and normal saline is 10mg: 80ml, stirring speed 1000-1500 rpm. 7. a kind of preparation method for the nano-preparation that is used for the treatment of type 2 diabetes according to claim 3, is characterized in that, in described step 4, each time of ultrasonic treatment is 6-8 seconds, interval time 3-4 seconds, The total processing time is 10-15min, and the ultrasonic power is 300-500W. 8. A kind of preparation method for the nano-preparation that is used for the treatment of type 2 diabetes according to claim 3, it is characterized in that, the dialysis in the described step 5 is specifically that the nano drug-carrying particle dispersion liquid is loaded into and has a molecular weight cut-off of 3.5kDa In the dialysis bag, use normal saline for dialysis, the dialysis time is 12-24 hours, during which the external fluid is replaced with normal saline 3-5 times. 9. a kind of preparation method for the nano-preparation that is used for the treatment of type 2 diabetes according to claim 3, it is characterized in that, the stirring speed of nano drug-carrying particle dispersion liquid in the described step 6 is 200-400 rev/min, Filtration utilizes a sterile water-soluble filter membrane with a pore size of 0.22 microns. 10. The preparation method of a nano-preparation for the treatment of type 2 diabetes according to claim 3, characterized in that, removing moisture in the step 7 is pre-freezing in a -70-80°C ultra-low temperature refrigerator for 24-48 hours , and then use a freeze dryer to carry out under the condition of a pressure lower than 0.1 Pascal.