KR20200020403A - Method for Manufacturing Nanosuspension Comprising Insoluble Drug Using Bottom-up Method, and Nanosuspension Made thereby - Google Patents

Abstract

The present invention relates to a method for manufacturing a nanosuspension comprising a step of dissolving a poorly soluble drug in transcutol, which is a solvent, and the nanosuspension of the poorly soluble drug manufactured by the same. By using transcutol having low toxicity and being able to be applied for oral administration or the skin instead of an existing organic solvent, it is possible to manufacture the nanosuspension without going through an additional solvent removal process, and an average diameter of a particle size of the nanosuspension is less than 300 nm, allowing long-term storage for 6 months or longer. Therefore, the nanosuspension can be usefully utilized as medicinal products.

Description

Method for Manufacturing Nanosuspension Comprising Insoluble Drug Using Bottom-up Method, and Nanosuspension Made by the Bottom-up Method

The present invention relates to a method for preparing a nanosuspension comprising a poorly soluble drug and a nanosuspension prepared by the bottom-up method.

Resveratrol (3,5,4'-trihydroxystilbene) is a non-flavonoid polyphenolic substance found primarily in grapes, peanuts, mulberries, cranberries, blueberries, and red wines and is generally a part of people's diet. Consumed as part. Resveratrol has been reported to prevent coronary heart disease by inhibiting myocardial infarction, arrhythmia, hypertension, hypertrophy, fibrosis, atherosclerosis and thrombosis, and has been reported in a very wide range, including heart protection, cancer prevention and prolonged lifespan Has a beneficial biological effect. Resveratrol is considered a class II compound in the BCS (Biopharmaceutical Classification System) because it exhibits high permeability (Log P = 3.1) and low water solubility. The physical and chemical properties of resveratrol are as follows: molecular weight is 228.25 g / mol, melting point is 253 to 255 ° C, pK _a value is 9.22, insoluble in water and soluble in methanol, ethanol and DMSO.

The therapeutic application of resveratrol is very limited because of its instability, low water solubility, short biological half-life, and fast metabolic and elimination properties. To overcome this limitation of resveratrol, a number of strategies have been evaluated, such as the use of polymeric nanoparticles, solid lipid nanoparticles, self-emulsifying drug delivery systems, nanoemulsions, liposomes, nanosuspensions and nanofibers.

Dual nanosuspensions refer to colloidal dispersions of stabilized drug particles under conditions with polymers, surfactants, or both. Nanosuspensions are used to deliver drug substances that exhibit low water solubility and lipolysis. Small particles in the nanosuspension provide very large drug surface areas, increasing the rate of dissolution of insoluble drugs. As a result, BCS class II and IV compounds exhibit improved bioavailability, fast activity, and other desirable biopharmaceutical effects. The nanosuspension technology can be largely divided into a top-down method and a bottom-up method.

Top-down methods reduce the particle size of the drug through milling (jet mill and ball mill) and high pressure homogenization. However, this top-down method is very difficult to apply to materials that exhibit thermal instability because it requires high energy to generate heat. In addition, large amounts of energy may produce amorphous particles and modified crystal structures.

The bottom up method involves inducing precipitation of particles from the supersaturated drug solution. Bottom-up methods include a variety of techniques such as solvent evaporation, supercritical fluids, antisolvent precipitation, and chemical precipitation. These methods require lower energy pressures than top-down methods and can be applied to thermodynamically unstable materials. Therefore, the present invention prepared a nanosuspension in a bottom-up method, by using this technique, it can be said that it is important to control the residual solvent and particle growth. Inadequate control of particle growth is due to an incomplete understanding of formulation composition and manufacturing process. Therefore, the manufacturing process and formulation composition should be understood using scientific and systematic methods. In the present invention, drug design based quality by design (QbD) was applied to the development of nanosuspensions.

The US Food and Drug Administration (FDA) recommends applying the QbD principles in risk-based approaches and new drug development, new drug manufacturing and regulation. The FDA's focus on QbD is that an increase on the test does not necessarily improve the quality of the product. In the last few years, in the pharmaceutical sector, QbD has issued ICH Q8 (pharmaceutical development), ICH Q9 (quality risk management), and ICH Q10 (pharmaceutical quality system). They provide a high level of guidance regarding the scope and definition of QbD for the pharmaceutical industry.

In the ICH Q8 guidelines, QbD is defined as a systematic approach to development that begins with a predefined goal, and is also based on sound science and quality risk management (ICH Q8, 2009), as well as understanding of products and processes as well as process. Highlight the control of In order to implement QbD, the quality target product profile (QTPP) and critical quality attributes (CQAs) of the finished product must be defined. Based on risk assessments (RAs), critical material attributes (CMAs) and critical process variables (CPPs) and prior knowledge that affect CQAs should also be identified. After the design of experiment (DoE) and risk analysis, a design space should be established. The most important goal in product design and understanding is to develop a robust product that can deliver the desired QTPP during its life cycle. Products produced through QbD can improve stability, reduce production costs, increase patient efficacy and minimize side effects.

Currently, research, development, manufacture, storage and clinical process development of nanosuspensions are still useful and necessary to apply QbD in infancy. The major barriers in the manufacture and clinical application of nanosuspensions include structural instability and incomplete understanding of the manufacturing process. Therefore, there is a need for the development of technologies that can overcome these barriers.

Korean Patent Registration No. 10-1171375 (registered on July 31, 2012).

It is an object of the present invention to provide a method for preparing a nanosuspension of a poorly soluble drug while eliminating the solvent removal process without using an organic solvent.

Another object of the present invention is to provide a nanosuspension of poorly soluble drugs that can be stored for a long time and excellent in bioavailability and drug release rate.

In order to achieve the above object, the present invention provides a method for producing a nanosuspension comprising the step of dissolving a poorly soluble drug in a transcutol solvent.

In order to achieve the above another object, the present invention provides a nanosuspension prepared by the above method.

The present invention relates to a method for preparing a nanosuspension comprising dissolving a poorly soluble drug in a transcutol as a solvent, and to a nanosuspension of a poorly soluble drug prepared therefrom. By using the applicable transcutol, it is possible to prepare nanosuspension without additional solvent removal process. The nanosuspension thus prepared contains particles with an average diameter of less than 300 nm and is stored for a long time more than 6 months. This can be usefully used as a drug product.

Figure 1 shows an embodiment of the nanosuspension prepared by the anti-solvent precipitation method in an embodiment of the present invention.
2 shows the solubility of resveratrol in various solvents at 298.15K.
Figure 3 shows the solubility of resveratrol for the trans kyutol ^® HP and water mixture at 288.15 to 313.15K.
4 shows the value T _hm of ΔH ° against ΔG ° Results of enthalpy-entropy correction analysis of the solubility of resveratrol for a mixture of various Transcutol ^® HP and water at the value (300.40 K) are shown.
Figure 5 is the value of the ΔH TΔS ° ° for the T _hm Results of enthalpy-entropy correction analysis of the solubility of resveratrol for a mixture of various Transcutol ^® HP and water at the value (300.40 K) are shown.
Figure 6 shows the inhibitory effect of povidone on resveratrol precipitation.
Figure 7 shows the inhibitory effect of HPMC and HPC on resveratrol precipitation.
8 shows a response curve plot showing the effect of various formulation parameters on reaction values Y ₁ , Y ₄ and Y ₉ (X ₁ : concentration of PVP VA64 (mg / mL), X ₂ : concentration of PVP K12) (mg / mL), concentration of SLS (mg / mL), Y ₁ : particle size (z-mean, first day), day), Y ₄ : particle size (z-mean, day 7), Y ₉ : zeta Potential).
FIG. 9 shows overlay plots associated with PVP VA64 (X ₁ ) and PVP K12 (X ₂ ) at the lowest (A), medium (B), and high (C) concentrations of SLS (X ₃ ).
10 shows target response values for response values Y ₁ -Y ₉ at SLS concentrations of 0.5 mg / mL (A), 1.0 mg / mL (B) and 1.5 mg / mL (C) by Monte Carlo simulations. The probability map satisfies (Y ₁ : particle size (z-mean, initial), Y ₂ : particle size (z-mean, day 1), Y ₃ : particle size (z-mean, day 3), Y ₄ : particle size (z-average, day 7), Y ₅ : particle size (d90, initial), Y ₆ : particle size (d90, day 1), Y ₇ : particle size (d90, day 3), Y ₈ : particle size (d90, day 7), Y ₉ : zeta potential).
FIG. 11 is a probability map satisfying target response values for response values Y ₁ -Y ₉ when the mixing speeds are 500 rpm (A), 750 rpm (B), and 1,000 rpm (C) by Monte Carlo simulation. Y ₁ : particle size (z-mean, initial), Y ₂ : particle size (z-mean, day 1), Y ₃ : particle size (z-mean, day 3), Y ₄ : particle size ( z-means, day 7), Y ₅ : particle size (d90, initial), Y ₆ : particle size (d90, day 1), Y ₇ : particle size (d90, day 3), Y ₈ : particle size (d90 , Day 7), Y ₉ : zeta potential).
Figure 12 shows the trend of particle growth of the nanosuspension prepared by complete factor experiments (the formulations and process conditions of Run-1 to Run-9 are as shown in Table 16).
FIG. 13 shows the resveratrol profile dissolved from the optimal nanosuspension formulation and resveratrol feedstock.
Figure 14 shows the plasma concentration-time profile of resveratrol in rats after oral administration of resveratrol nanosuspension and resveratrol raw material according to the present invention (data are expressed as mean ± standard deviation [n = 4]).
FIG. 15 shows TEM image and particle size distribution of resveratrol nanosuspension (A: initial (day 1) TEM image), B: TEM image after 6 months, C: initial (day 1) particle size distribution, D: 6 Particle size distribution after months).
Figure 16 shows the results of confirming the long-term stability of the optimized resveratrol nanosuspension (A: particle size [z-average and d90], B: drug content).

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention, as shown in Figure 1, poorly soluble drugs such as resveratrol or MHY498 shows high solubility in transcutol, when using a specific surfactant and water-soluble polymer combination as a surface stabilizer, the average particle is 300 The present invention was completed by confirming that nanosuspensions containing nanoparticles less than nm and retaining this form for at least 6 months can be prepared.

Accordingly, the present invention provides a method for preparing a nanosuspension comprising dissolving a poorly soluble drug in a transcutol as a solvent.

The poorly soluble drugs include resveratrol, MHY498 ((Z) -5- (2,4-dihydroxybenzylidene) thiazolidine-2,4-dione), aprepitant, cyclosporine, celecoxib, coenzyme Q10, It may be selected from the group consisting of valsartan, dabigatran etexilate, sorafenib, and silymarin, but is not limited thereto.

In addition, the poorly soluble drug may be included in 1 to 100 parts by weight with respect to 100 parts by weight of the solvent.

More specifically, the nanosuspension of the poorly soluble drug can be prepared by using a solvent / antisolvent precipitation method. As the solvent, transcutol and water as the antisolvent can be used. Specifically, the transcutol and water Silver may be used in a weight ratio of 1: 5 to 1: 100, and more particularly, in a weight ratio of 1:10 to 1:20.

In one embodiment of the present invention, after dissolving the poorly soluble drug, dissolving a water-soluble polymer in water to prepare a polymer aqueous solution; And it may further comprise the step of injecting a solution in which the drug is dissolved in the aqueous polymer solution, the water-soluble polymer can stabilize the particles by forming a layer on the surface of the drug particles to prevent aggregation of drug particles.

Specifically, the water-soluble polymer is selected from the group consisting of polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, polyvinylpyrrolidone K30 and polyvinylpyrrolidone vinyl acetate VA64 It may be one or more, preferably polyvinylpyrrolidone K12, polyvinylpyrrolidone vinyl acetate VA64 or mixtures thereof.

In one embodiment of the present invention, after dissolving the poorly soluble drug, dissolving the surfactant in water to prepare a surfactant solution; And injecting a solution in which the drug is dissolved into the aqueous solution of the surfactant, wherein the surfactant is adsorbed at the interface of the solid-liquid to suppress particle growth and aggregation by reducing hydrophobic interaction and coagulation. Can be.

Specifically, the surfactant may be sodium lauryl sulfate (SLS), sucrose fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene glycolated natural or hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester (e.g., twin (Tweens, etc.), polyethylene glycol, polyoxyethylene stearate, polyoxyethylene-polyoxypropylene copolymers (e.g., poloxamer, poloxamine, etc.), polyethylene glycolated phospholipids, and alpha-tocopherols It may be selected from the group consisting of polyethylene glycol succinate, preferably sodium lauryl sulfate.

The nanosuspension prepared by the above method may include poorly soluble drug nanoparticles having an average diameter of 1 nm to 300 nm, and thus the nanoparticles have a larger surface area and are more easily dissolved, thereby maximizing bioavailability. .

On the other hand, in all the above process, the process of mixing and mixing a poorly soluble drug and a solvent, a water-soluble polymer or a surfactant is included, wherein the nanosuspension can be prepared at a mixing speed of 500 rpm to 1,000 rpm.

In addition, the present invention produces a nanosuspension prepared by the above method.

The nanosuspension according to the present invention may include poorly soluble drug nanoparticles having an average diameter of 1 nm to 300 nm, thereby maximizing bioavailability of poorly soluble drugs.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are provided to more fully explain the present invention to those skilled in the art, and are merely illustrative of the contents of the present invention, the scope of the present invention is limited to the following examples no.

< Example  1> Resveratrol  Preparation of Nano Suspension

One. Resveratrol  Study before formulation of nanosuspension ( Preformulation )

One) Resveratrol  variety Solubility in Solvents

The solubility of resveratrol in various organic solvents was measured. Samples were prepared by adding excess resveratrol to a glass bottle containing 10 mL of organic solvent. At this time, each experiment was repeated three times. The sample was vortexed for 5 minutes, mixed well, and sonicated for 1 hour in an ultrasonic bath (model 5800, Branson, Danbury, USA). Thereafter, the glass bottle was placed in a shaking constant temperature bath (BS-21, Jeiotech Co Ltd, Daejeon, Korea) at 24 ° C. for 24 hours. The saturated solution was then filtered through a 45 μm RC syringe filter, then transferred to a volumetric flask and diluted with methanol. Next, the concentration of resveratrol is adjusted for UV / VIS detector SPD-20A, Communications Bus Module CBM-20A, AUTO Sampler SIL-20AC, Liquid Chromatograph LC-20AT, Degassing Unit It was measured using a Shimadzu HPLC system with DGU-20A 5R, and column oven CTO-20A (Shimadzu, Tokyo, Japan). Chromatographic separation was performed through a Gemini C18 reversed-phase column (Phenomenex, 150 mm 4.6 mm, 5 μm). Injection volume was 10 μL. The mobile phase consisted of water and acetone (60:40, v / v). The flow rate was 0.8 mL / min at 30 ° C, and was detected in the ultraviolet region at a wavelength of 303 nm. A calibration curve was prepared by measuring the peak area of the known resveratrol reference at concentrations from 1 μg / mL to 100 μg / mL.

2) Resveratrol  menstruum Solubility in mixtures

The mole fraction solubility of the resveratrol in the pure water (w = 0) and pure trans kyutol ^® HP (w = 1.0) trans kyutol ^® HP and co-solvent mixture of water (mass fraction w = 0.1-0.9) including measured. The solubility was measured using a shake constant temperature bath at a temperature of 288.15 K to 313.15 K. Samples were prepared by adding excess resveratrol to a glass jar containing a mixture of Transcutol ^® HP and water containing 15 mL of pure solvent. At this time, each experiment was repeated four times. The sample was vortexed for 5 minutes, mixed well, and sonicated for 1 hour in an ultrasonic bath (model 5800, Branson, Danbury, USA). The glass bottle was then 288.15 K to 313.15 K in a shaker bath. The mixture was shaken for 48 hours at a temperature of 50. Preliminary experiments indicated that this time period was sufficient to achieve complete saturation. Finally, the resveratrol concentration was analyzed by performing the HPLC method under the same conditions as above at 303 nm.

Next, the molar fraction solubility ( χ _e ) of the experimental resveratrol was calculated using the following formulas (1) and (2):

In Equations 1 and 2, m ₁ represents the mass of resveratrol in a saturated solution, m ₂ represents the mass of Transcutol ^® HP, m ₃ represents the mass of water, and M ₁ , M ₂ and M ₃ represent resveratrol and trans, respectively. The molecular weight of Cutol ^® HP and water. Equation (1) is the χ _e in pure solvent (Transcutol ^® HP and water) Values, equation (2) of trans-resveratrol from kyutol ^® HP and mixtures χ _e Applied to calculate the value.

 In addition, the Jouyban-Acree model was applied to the mixed solvent system to determine the correlation between physicochemical properties. A general equation for predicting solute solubility in water co-solvent mixtures at various temperatures is given by equation (3):

,

, 및

은 각각 온도 T(켈빈)의 혼합 용매, 트랜스큐톨^® HP 및 물에서의 레스베라트롤의 몰분율이며, w ₁ 및 w ₂는 트랜스큐톨^® HP 및 물의 순수한 질량 분율이고, J _i 는 다음 식

을 다음 식(

,

) 및(

)에 대하여 회귀함으로써 계산된 상수이다.Where

,

, And

Is the mole fraction of resveratrol in the mixed solvent, Transcutol ^® HP and water at temperature T (Kelvin), respectively, w ₁ and w ₂ are the pure mass fractions of Transcutol ^® HP and water, and J _i is

With the following expression (

,

) And (

Constant calculated by regression for

 Regarding the logarithm of the mole fraction of resveratrol over temperature, the Worthof equation [Equation (4)], which is the inverse linear function of absolute temperature T, was used:

은 레스베라트롤의 몰 분율이고, 반트호프 모델 패러미터(A 및 B)는 최소제곱법분석(least squares analysis)을 통해 계산되었다. 사실 대부분의 진짜 해들(real solutions)은 비이상적인 거동을 나타내었다. 그러므로, 표준 엔탈피 및 엔트로피가 용액 과정에서 고려되어야 한다. 결과적으로, 하기 식 (5)가 얻어졌다:Where

Is the mole fraction of resveratrol and the Wandhof model parameters (A and B) were calculated through least squares analysis. In fact, most real solutions have exhibited non-ideal behavior. Therefore, standard enthalpy and entropy should be considered in the solution process. As a result, the following formula (5) was obtained:

Wherein ΔH ° and ΔS ° are the enthalpy and entropy of the solution, respectively.

Applying the Bandt Hope equation of large amounts of experimental mole fraction data to the Jouyban-Acree model can provide a more predictable model. Thus, the formula thus combined was as follows:

In the above formula, A ₁ , B ₁ , A ₂ , and B ₂ are constants of the Banthoff equation calculated by regression analysis of the mole fraction of resveratrol in pure solvent at various temperatures.

 Mean relative deviation (MRD) was used to confirm the accuracy of the prediction model of the method and was calculated by the following equation (7):

Where N is the number of data points in each set.

The thermodynamic properties of resveratrol dissolved in the mixed solution were determined by analysis of solubility enthalpy (ΔH ° ), Gibbs free energy (ΔG ° ), and dissolution entropy (ΔS ° ). The thermodynamic change was calculated according to the following equation (8) at the average harmonic temperature ( T _hm ):

Where n is the number of temperatures analyzed. Δ H ° was calculated using a modified Worthof equation, such as the following equation (9):

Where X is the mole fraction solubility in the mixed solution, T is the absolute temperature (K), and R is the universal ideal gas constant (8.314 J · K ⁻¹ · mol ^{− 1} ). Derived enthalpy value (ΔH ° ) was calculated using the Banthoff equation such as the following equation (10).

The Gibbs energy △ G ° of resveratrol generated during the dissolution process is T _hm It was calculated through the following formula (11).

ΔS ° of resveratrol for the dissolution process was calculated as shown in the following formulas (12) and (13) by combining the values of ΔH ° and ΔG ° .

The relative contribution of enthalpy (% ζ _H ) and entropy of relative contribution (% ζ _TS ) to Gibbs energy during the dissolution process of resveratrol in the mixed solution was calculated as shown in the following equations (14) and (15).

3) Polymer Resveratrol  Confirmation of precipitation inhibitory effect

Precipitation of resveratrol in 500 mL of distilled water (containing 0.5% (w / v) of polymer) was carried out at 37 ° C. and 50 rpm using a USP rotating paddle apparatus (Electlab, Mumbai, India). Was measured. Samples (3 mL) were removed at specific times, filtered using a 0.45 μm RC syringe filter and then diluted with methanol. Then, as described above, resveratrol concentration was analyzed using HPLC.

2. Resveratrol  Preparation of Nanosuspension

Resveratrol nanosuspension was prepared using an antisolvent precipitation method. In other words, the trans-resveratrol was dissolved kyutol ^® HP. In addition, an aqueous solution was prepared by dispersing a specific concentration of polymer and surfactant in distilled water. The resulting Transcutol ^® HP solution was then quickly added to the aqueous solution to be used as the solvent being self stirring at 750 rpm. The blue suspension was then produced immediately, resulting in the resveratrol nanosuspension through stirring.

3. Resveratrol  Experimental Design for Preparation of Nanosuspension

One) Formulation Parameter  Box for optimization Behnken  design

Box-Behnken design (BBD) and response surface methodology were used to optimize the composition of the resveratrol nanosuspension. In this design, the experimental area was assumed to be one cube. Experiments were performed at the points corresponding to the center points of each axis, and repeated experiments were performed at the center of the multidimensional cube. A total of 17 experiments were performed at three levels of three elements. The center point was verified five times. Formulation parameters and parameter levels were selected based on this preliminary study. That is, PVP VA64 (X ₁ ), PVP K12 (X ₂ ), and SLS (X ₃ ) were selected as formulation parameters (elements) at three levels (-1, 0, +1). Particle size (z-mean, d90) and zeta potential (ζ) were chosen as response. Formulation parameters and reaction values were as shown in Table 1 below.

This experimental design was generated and evaluated using Design Expert ^® 11.0 (Stat-Ease, Inc. Minneapolis, USA).

2) process variables Robustness  Full factorial to establish design; FD )

In this experiment, complete factor placement was performed to ensure the manufacturing process robustness of the optimized configuration. A total of nine experiments were performed with three elements at two levels, with a central point once. Injection rate, temperature, and mixing rates were selected as process variables at two levels (-1, +1) and particle size (z-mean, d90) and zeta potential (ζ) were used as reaction values. Process variables and reaction values were as shown in Table 2 below.

This experimental design was generated and evaluated using Design Expert ^® 11.0 (Stat-Ease, Inc. Minneapolis, USA).

< Example  2> Resveratrol  Of nanosuspension Characterization

1.particle size and zeta Potential  Measure

For characterization of the particle surface, the particle size distribution and zeta potential of the resveratrol nanosuspension were measured at 25 ° C. using a dynamic light scattering (DLS) instrument (ELSZ-1000, Photal Otsuka Electronics, Osaka, Japan). Particle size was specified three times and expressed as z-average diameter. DLS was also used to determine the hydrodynamic diameter and particle distribution of nanoparticles in suspension. DLS is a fast and sensitive method, especially in the small nanometer range, requiring very small amounts of particles. Therefore, when very few active pharmaceutical ingredients are used, they are very suitable methods for confirming regular measurements and early formation development. Zeta potential is a measure of the electrical charge on a particle surface, indicating the physical stability of the colloidal system. Zeta potential was measured using the Laser-Doppler method.

2. Morphological Characterization

Morphological analysis was performed at 80 kV using transmission electron microscopy (TEM, Hitachi H-7600, Tokyo, Japan). The suspension was first dropped on a carbon film coated with a copper grid, dried in a fume hood at room temperature for 24 hours, and then observed by TEM.

3. In vitro dissolution assay

Dissolution was performed in 500 mL of distilled water at 37 ± 0.5 ° C. using USP Dissolution Apparatus II (paddle method, Electrolab, Mumbai, India) at a paddle speed of 50 rpm. Resveratrol suspension (5 mg / mL, 20 mL) and resveratrol raw material (100 mg) were placed in a dissolution vessel. Samples (5 mL) were then drawn at different time intervals and replaced with fresh dissolution medium maintained at 37 ± 0.5 ° C. to maintain a constant volume. The extracted samples were filtered using a 0.45 μm RC syringe filter and then diluted with methanol. The final concentration of resveratrol was measured using HPLC as described above.

4. In rats  drug Dynamics  Research

Eight male Sprague-Dawley rats (200 ± 10 g; Orient Bio Inc., Seongnam, Korea) were divided into two treatment groups of four each. Prior to the experiment, rats were fasted for 18 hours. Resveratrol raw material or resveratrol suspension was administered orally to each of the two groups so that the dose of resveratrol was 10 mg / kg. Resveratrol raw material was dispersed immediately in 1 mL of water immediately before oral administration. Thereafter, blood samples (approximately 0.25 mL) were drawn from the neck veins of the rats 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 hours after dosing and collected in heparinized tubes. The blood sample was then centrifuged at 8,000 rpm for 10 minutes at 4 ° C. The resulting plasma was transferred to individual centrifuge tubes and stored at -70 ° C. The amount of resveratrol was determined by HPLC using the method disclosed in the Das et al., 2011, Journal of Liquid Chromatography & Related Technologies. 2011; 34: 1399-1414.

Resveratrol stock solution (1 mg / mL) and internal standard carbamazepine (1 mg / mL) were prepared by dissolving in methanol. Reference working solution of resveratrol (50 μg / mL) and carbamazepine (50 μg / mL) in a mixture of water and methanol (1: 1) were prepared immediately before the experiment. The reference working solution (50 μg / mL) of the resveratrol was serially diluted with a water-methanol (1: 1) mixture, and a known amount (10 μL) of the solution was placed inside the plaster plasma (100 μL) as a reference internal working solution. 5 μL (50 μg / mL) was added. Plasma samples were then extracted via the following liquid-liquid extraction method. Seven reference calibration solutions were prepared to obtain final concentrations (5 ng / mL to 5,000 ng / mL). Resveratrol and carbamazepine (internal criteria) were extracted from plasma using liquid-liquid extraction. That is, 40 μL of phosphate-buffered saline (PBS; 30 mM, pH 6) was added to the plasma sample in a 2 mL centrifuge tube, and the contents were mixed well by vortexing for 15 seconds. Finally, ethyl acetate (300 μL) was added and mixed for 30 seconds. After ethyl acetate extraction, the sample was centrifuged at 8,000 rpm for 10 minutes and the upper organic layer was transferred to a clean tube. This extraction process was carried out twice and evaporated using a heating block (Eyela MG-2200, Tokyo, Japan) at 35 ° C. to dry the mixed organic layer. The residue was returned to its original state with 75 μL of mobile phase and centrifuged for 10 minutes at 13,000 rpm. Thereafter, the supernatant was transferred to a glass insert pre-installed in a glass bottle for autosampler. During each assay, 20 μL of supernatant was injected into the HPLC system.

All the above processes were carried out in the absence of direct light. Chromatographic separation was done through a reversed phase HPLC column (Phenomenex, EVO 5 μιη C18 250 Х 4.6 mm, 100 A), which was protected by a guard column (Phenomenex, Gemini C18, 4 Х 3.0 mm). The assay was performed by isocratic elution of a mobile phase consisting of acetonitrile and 30 mM PBS (pH 7.0; 30:70 v / v) at a flow rate of 1 mL / min at a temperature of 35 ° C. The mobile phase was then filtered through a 0.2 mm nylon membrane and all gas was removed by sonication at 25 ° C. for 20 minutes before use. UV absorbance at 306 nm was also recorded.

5. Stabilization Research

Stabilization studies of resveratrol nanosuspensions were performed at room temperature (25 ± 5 ° C., 60% RH) for a period of 6 months. Every month, it was characterized in terms of particle size and drug content of resveratrol nanosuspension.

< Experimental Example  1> Resveratrol  Pre-Formulation Studies of Nanosuspensions

1. in various solvents Resveratrol  Solubility

The results of confirming the solubility of resveratrol in various solvents are shown in FIG. 2. According to FIG. 2, the solubility for tetrahydrofuran and Transcutol ^® HP was 412.7 mg / g and 332.9 mg / g, respectively. Despite the high solubility of resveratrol in tetrahydrofuran, rapid particle aggregation occurred during the preparation of the resveratrol nanosuspension using tetrahydrofuran. Therefore, tetrahydrofuran was not a suitable solvent for nanosuspension preparation. Transcutol ^® HP has low toxicity and high solubilization effect. In addition, Transcutol ^® HP has the advantage that it does not require solvent removal during the nanosuspension preparation because it can be absorbed in small amounts. Therefore, Transcutol ^® HP was chosen as the optimal solvent of the present invention.

2. Transcutol ^®  In a mixed solvent of HP and water Resveratrol  Solubility

At a temperature of 288.15 K to 313.15 K, χ _e value of resveratrol in the trans kyutol ^® HP and water mixtures were as shown in Table 3 and Fig.

(w is the weight fraction of Transcutol ^® HP)

In other words, the mole fraction solubility of the trans kyutol ^® HP and water mixture was increased with increasing the temperature of the mixture, or trans kyutol ^® HP is increased fraction of the. The highest mole fraction solubility (1.62 Х 10 ^-1 ) of resveratrol was observed in pure Transcutol ^® HP of 313.15 K, and the lowest value (1.29 Х 10 ^-6 ) was observed in 288.15 K of pure water. Because molar fraction solubility depends on many factors, including chemical structure, dielectric constant, solvent polarity, temperature and cosolvent ratio, the high χ _e value of resveratrol results in a lower transcutol ^® HP dielectric constant than water. It is thought to be due to the polarity. In view of these results, Transcutol ^® HP can be used as a potential physiologically compatible cosolvent that can improve the solubility of resveratrol in water. Thus, it was confirmed that the nanosuspension can be effectively prepared from a mixture of Transcutol ^® HP (mass fraction 0.1-0.2) and water.

Then, to confirm the molar solubility relationship of the resveratrol saturated solution, experimental data was applied to the Jouyban-Acree model. As a result, the mole fraction data obtained from the experiments fit the Jouyban-Acree model well, and the trained model was as shown in the following equations (16) and (17):

[Equation 16]

=> R ² = 0.986 → F = 4632.3 → p <0.0005 → MRD = 2.12%.

및

의 실험적인 값은 제한되었고, 추가적인 실험 수행이 요구되었다. 이러한 한계를 다루기 위해, Jouyban-Acree 및 반트호프 모델을 조합하여 사용하였다. 결과적으로 다음과 같은 예측 모델이 얻어졌다:The equation was used to predict the experimental mole fraction with an MRD of 2.12%.

And

The experimental value of was limited and further experimentation was required. To address this limitation, a combination of the Jouyban-Acree and Wandhof models were used. As a result, the following prediction model was obtained:

Formula 17

=> R ² = 0.986 → F = 4631 → p <0.0005 → MRD = 2.12%.

Like the trained equations, the equations inverted the mole fraction values remaining at 2.12% (N = 66) MRD.

Meanwhile, Table 4 below summarizes the thermodynamic parameters of the studied Transcutol ^® HP + Water mixture. Trans kyutol ^® HP + melting enthalpy of the water mixture (△ H °), the Gibbs free energy (△ G °), melting entropy (△ S °) to (8 a thermodynamic parameter at the time of resveratrol dissolved, as disclosed in Example 1 Calculated as shown in) to (13). In order to confirm the contributions related to enthalpy and solubility of entropy, ζ _H (relative contribution of enthalpy) and ζ _TS (relative contribution of entropy) are respectively shown in Equations (14) and (15) disclosed in Example 1 above. Calculated.

Referring to Table 4 above, ΔH ° of the Transcutol ^® HP and water mixture was a positive value. This result means that the dissolution of resveratrol in the solvent in the temperature range of the experimental conditions is an endothermic reaction. That means that the resveratrol and solvent intermolecular interactions are stronger than the solvent-solvent intermolecular and resveratrol-resveratrol interactions.

In the dissolution of resveratrol in pure Transcutol ^® HP, pure water and a mixture of Transcutol ^® HP and water, a positive ΔG ° value was shown, which means spontaneous dissolution of resveratrol.

In addition, the mass fraction of the trans kyutol ^® HP was observed an amount △ S ° value of from 0.6 trans kyutol ^® HP and the mixture of water or less. A positive ΔS ° value means that the dissolution of resveratrol is due to entropy. These results show favorable entropy upon solubilization of resveratrol in the mixed solvent.

In contrast, when the mass fraction of Transcutol ^® HP was greater than 0.7, a negative ΔS ° value was observed. These results can be explained in Transcutol ^® HP that resveratrol dissolves rapidly without requiring substantial heat absorption. That is, as is due to the high solubility of the trans kyutol ^® HP of resveratrol, trans kyutol ^® HP is increased fraction of △ S ° the value is reduced.

Referring to Table 4, the% ζ _TS value was less than 41.4% and the% ζ _H value was greater than 58.6% in all cases. This means that enthalpy is a major contributor to the ΔG ° value, which is consistent with the above results that dissolution of resveratrol in the selected solvent is caused by enthalpy.

Enthalpy-entropy compensation was analyzed to determine the solvation behavior and co-solvent action of resveratrol in a mixture of Transcutol ^® HP and water containing pure solvent. The results of this analysis are shown in FIGS. 4 and 5.

Between 4 and 5, in any mixture, resveratrol is a R ² value of 0.9961 (both) and with a positive slope (> 1.0), △ H ° and, △ G ° or △ S ° A linear relationship is shown. Therefore, a mechanism that causes solvation of resveratrol can be suggested to be by enthalpy. This is compared to the thermodynamic behavior plum for resveratrol in the water molecules, it is judged to be due to very high solubilization in trans kyutol ^® HP molecule.

3) Resveratrol Inhibition of Stabilizers on Precipitation  Check the effect

Precipitation inhibitory effects of polyvinylpyrrolidone vinyl acetate (PVP VA64), polyvinylpyrrolidone (PVP), hydroxypropyl methyl cellulose (HPMC) and hydroxypropyl cellulose (HPC) are shown in FIGS. 6 and 7. . In the absence of stabilizers, precipitation of resveratrol occurred at a very rapid rate. PVP and PVP VA64 showed superior precipitation inhibiting activity compared to HPMC and HPC. For PVP K12, PVP K17 and PVP VA64 concentrations of at least 330 μg / mL were maintained for 120 minutes. On the other hand, when HPMC and HPC were used, particle aggregation occurred gradually over time. Therefore, it was confirmed that PVP and PVP VA64 were more effective than HPMC and HPC in controlling particle growth in preparing nanosuspensions.

< Experimental Example  2> Resveratrol  Optimization Study of Nanosuspension

1. Formulation Parameter  Box for optimization Behnken  design( BBD )

To determine the optimal composition of resveratrol nanosuspension, according to the matrix produced by BBD, 17 resveratrol nanosuspensions were prepared at different concentrations of PVP VA64 (X ₁ ), PVP K12 (X ₂ ), and SLS (X ₃ ). It was prepared using. Results obtained for particle size and zeta potential are shown in Table 5 below.

In Table 5, X ₁ is the concentration of PVP VA64 (mg / mL), X ₂ is the concentration of PVP K12 (mg / mL), and X ₃ is the concentration of SLS (mg / mL).

Referring to Table 5, for all the prepared nanosuspensions, the particle size (z-average, initial) was smaller than 200 nm, and particles grew over time. However, all zeta potential values were below -30 mV, which met the target value of the response.

Regression analysis, on the other hand, is used to identify functional relationships between agent composition variables and response values, so that not only a specific pattern, but most suitable regression functions can be found. Constants in the regression analysis were estimated based on the least squares method. The main effects, interactions, and curvature effects of each element were identified through ANOVA.

In ANOVA, data were analyzed based on F-values to determine whether the factor was significantly related to the response value. Only response values with a p-value less than or equal to 0.05 were considered statistically significant.

(Table ₁ above, Y ₁ : particle size (z-average, initial), Y ₂ : particle size (z-average, day 1), Y ₃ : particle size (z-average, day 3), Y ₄ : Particle size (z-average, day 7), Y ₅ : particle size (d90, initial), Y ₆ : particle size (d90, day 1), Y ₇ : particle size (d90, day 3), Y ₈ : particle Size (d90, day 7), Y _{9 is} zeta potential)

Table 6 shows the regression and ANOVA results for the response values. The p-values of all models were less than 0.05, which means that they are significant. The p-value of the lack of fit assay was greater than 0.05, meaning no significance. In the Y ₁ -Y ₉ model, the regression constant (R ² ) ranged from 0.9959 to 0.8629. In general, a regression constant of 0.8 or higher is considered to be a good model.

Positive coefficients indicate a beneficial effect in the optimization analysis, whereas negative values mean an inverse correlation between the element and the response. As shown in Table 6, the concentration of PVP VA64 (X ₁ ) and the concentration of SLS (X ₃ ) had a negative effect on particle size (Y ₁ -Y ₈ ), whereas PVP K12 (X ₂ ) showed a positive effect. These results indicate that the particle size increases with increasing concentration of PVP K12 (X ₂ ) or decreasing concentration of PVP VA64 (X ₁ ) and concentration of SLS (X ₃ ). In particular, the concentration effect of PVP K12 (X ₂ ) on particle size (z-mean, Y ₁ -Y ₈ ) increased over time. In the early stages of production, the concentration of PVP VA64 (X ₁ ) and the concentration of SLS (X ₃ ) had a greater effect than PVP K12 (X ₂ ). However, in the particle size maintenance step, all formulation composition variables showed a significant impact on the particle size.

For zeta potential (Y ₉ ), PVP VA64 concentration (X ₁ ) and PVP K12 concentration (X ₂ ) showed positive effects, and SLS concentration (X ₃ ) showed negative effects. PVP VA64 and PVP K12 are nonionic polymers and SLS is an anionic surfactant. As the concentration of SLS increases or the concentration of the polymer decreases, the charge on the particle surface becomes negative. If the surface is negatively charged, an ion barrier is formed between the particles to inhibit the growth of the particles.

On the other hand, the manufacturing process of the nanosuspension is thermodynamically unstable. Therefore, stabilizers such as polymers or surfactants are essential for maintaining a physically stable state. As is known, PVP, a nonionic polymer, can be fixed on the surface of drug particles to occupy adsorption sites and prevent the drug molecules from binding to the crystal lattice in solution, thus providing a mechanical barrier to crystallization. However, if the polymer concentration is not sufficient, the crystals grow rapidly and aggregate. As the concentration of polymer continues to increase, the size of the particles increases together due to the thick layer of the particle surface, and diffusion between the solvent and the antisolvent is suppressed during the aggregation process. In addition, as the concentration of the polymer increases, the osmotic pressure increases, resulting in an increase in attraction between the colloidal particles. This leads to the growth of the particles.

The surfactant is adsorbed at the interface of the solid-liquid to reduce the surface tension of the interface and increase the nucleation rate, which results in an initial decrease in particle size. Moreover, adsorption of surfactants reduces hydrophobic interactions and coagulation, making resveratrol particles less hydrophobic and reducing particle growth. In particular, when SLS, an ionic surfactant, is adsorbed on the particle surface, the particle surface becomes negatively charged. This increases the repulsive force between the particles, increasing the energy barrier, thereby preventing particle growth and aggregation. As a result, the addition of an appropriate concentration of stabilizing agent can lower the excessively high surface energy of the nanoparticles produced.

Next, the relationship between the formulation composition variables and the response value variables was determined via the response curve plot. To make a response curve plot, only two formulation composition variables were displayed at a time; Accordingly, one formulation composition variable must be fixed. Referring to FIG. 8, it can be seen that the particle size decreases as the concentrations of PVP VA64 and SLS increase. After 7 days, the particle size decreased with decreasing PVP K12 concentration or increasing SLS concentration. Similar trends were also seen in the response variables Y ₅ -Y ₈ (results not shown), ie the zeta potential decreased with decreasing PVP VA64 and PVP K12 concentrations. This result is also consistent with the negative contribution of the SLS shown in Table 6 and FIG. 8 and the positive contribution of PVP VA64 and PVP K12.

2. Establish the design space

ICH guideline Q8 defines the design space as "a multidimensional combination and interaction of proven input variables (e.g., material properties) and process variables to provide quality correction." Work in this design space does not account for change and a flexible and robust process can be established.

In the present invention, the target value was set to 250 nm or less, 1 μm (Y ₅ -Y ₈ ) or less, and -20 mV (Y ₉ ), and a 95% confidence interval was applied. An overlay plot consisting of all response values is shown in FIG. 9. The yellow part of the plot means the area that meets the target value. The established yellow areas met the target values in all areas when the concentrations of SLS were 1.0 mg / mL and 1.5 mg / mL. However, the yellow area decreased when the concentration of SLS was 0.5 mg / mL.

FIG. 10 shows a probability map drawn using MODDE software based on calculated particle size and zeta potential models and Monte Carlo simulation for risk analysis. Monte Carlo simulations were performed for the error propagation of the function from independent variables to dependent variables, indicating the distribution of dependent variables (ie resolution) for each operating condition of the RSM.

The design space established by Monte Carlo simulation was expected to be significantly smaller than that of the overlay plot. However, the area obtained in the Monte Carlo simulation was a robust area with few errors and meeting the set target. In this experiment, the design space is represented by the probability of failure for each combination of parameters. A failure probability of 1% was chosen, which identifies the design space as an area that satisfies a defined element of 99% or more probability in the predicted CQA. For each combination, 10,000 simulations were performed and a 95% confidence interval was applied to account for the variation of the Monte Carlo simulation. As a result, the design space of the green area shown in FIG. 10 was a robust area in which reliable experimental values are expected. The established green area met the target response in all areas when the SLS concentration was 1.0 mg / mL. In other words, the area was guaranteed to be robust. Therefore, the best spot was identified in the region where the concentration of SLS was 1.0 mg / mL. Based on the expected function, the optimal values of the parameters were confirmed by schematic and numerical analysis using Design Expert ^® 11.0, and the formulation to produce a robust product with target high quality characteristics was determined. That is, mixed formulations (PVP VA64, PVP K12 and SLS) that maximized the expected function were identified (10% / 5% / 1%, w / v). This combination is expected to represent the smallest particle size and zeta potential.

3. Robustness of the Process Variables in the Optimization Configuration

In the formulation of optimized resveratrol nanosuspension, nine experiments were performed using FD to establish the robustness of the process variables and the results are shown in Table 7 below.

Referring to Table 7, the measured particle size distribution and zeta potential satisfy the target value. As shown in FIG. 11, the probability map obtained by the Monte Carlo simulation showed a defect probability of 1% or less in all regions. Therefore, it was confirmed that the robustness of the process parameters for the optimized formulation was established.

4. Model Validation

As shown in Table 8 below, a 95% prediction interval for the response was calculated from the regression model generated in the present invention for the optimized formulation. The BBD that was used for the optimization of the formulation composition parameters was used to calculate the system suitability value of the selected response.

(Table ₁ above, Y ₁ : particle size (z-average, initial), Y ₂ : particle size (z-average, day 1), Y ₃ : particle size (z-average, day 3), Y ₄ : Particle size (z-average, day 7), Y ₅ : particle size (d90, initial), Y ₆ : particle size (d90, day 1), Y ₇ : particle size (d90, day 3), Y ₈ : particle Size (d90, day 7), Y _{9 is} zeta potential)

Referring to <Table 8>, the observed response value was within 95% of the prediction interval. Therefore, the validity of the model generated according to the present invention was confirmed. In addition, FD was used to establish the robustness of the process variables, and as a result, as shown in FIG. 12, it stabilized over time. In all the experiments shown in Table 7, the trend in particle size was similar.

< Experimental Example  3> optimized Resveratrol  Characterization of Nanosuspension

1. Check the dissolution characteristics

The in vitro drug dissolution ability of the above-described derivatized resveratrol nanosuspension preparation was compared with the drug dissolution ability of the raw materials. The dissolution profiles of resveratrol nanosuspension and resveratrol raw material are shown in FIG. 13.

Referring to FIG. 13, the resveratrol nanosuspension showed higher dissolution rate and drug release rate than the resveratrol raw material. Drug release rates after 5 minutes of resveratrol nanosuspension and raw materials were 90.2% and 3.5%, respectively, and drug release rates after 360 minutes were 90.5% and 21.2%, respectively. From these results, it was found that the resveratrol nanosuspension according to the present invention showed a much higher drug release rate than the resveratrol powder.

2. In rats  drug Dynamics  Experiment

To determine whether the nanosuspension drug delivery system can improve oral bioavailability, in vivo experiments were performed using Sprague-Dawley rats, and the pharmacokinetic parameters of the resveratrol nanosuspension and the raw material were compared. 14 shows the plasma concentration-time profile of resveratrol after oral administration of nanosuspension and raw material. In addition, pharmacokinetic parameters (AUC _{0 → 12 h} , C _max , and T _max ) are shown in Table 9 below.

(a: p <0.05 for the resveratrol raw material, and data are expressed as mean ± standard deviation [n = 4].)

Referring to FIG. 14, the plasma concentration and fast drug absorption rate of the resveratrol nanosuspension were dramatically higher than those of the resveratrol raw material. AUC _{0 → 12 h} , C _max , and T _max values were 387.0 ± 26.0 ng · h / mL, 301.4 ± 79.6 ng / mL, and 0.44 ± 0.13 h, respectively. The oral absorption of resveratrol nanosuspension was also higher than that of resveratrol raw material, and as shown in Table 9, the increase in AUC _{0 → 12 h} and C _max values was 1.6 and 5.7 times, respectively. For oral administration drugs, solubility is a critical rate decision step that is very important for absorption. As the nanosuspension enters the gastrointestinal tract, the nanoparticles provide a larger surface area for dissolution and the molecular diffusion of resveratrol obtained from the dissolved nanoparticles, which, according to the classical passive diffusion theory, increases the concentration at the surface of the nanocrystals. Leads to a gradient. These results indicate that the oral absorption of resveratrol was significantly increased by the nanosuspension form.

3. Long-term stability check

The stability of the resveratrol nanosuspensions was analyzed over a period of 6 months through identification of particle size, zeta potential and drug content. As a result, as shown in Figure 15, the particles formed by the anti-solvent precipitation method was mainly in the form of a spherical shape with a smooth surface and a diameter smaller than 200 nm in the initial stage of production. After 6 months, the particles were about 200 nm in size and also spherical in shape.

(N.D: No data because no data can be measured)

In addition, as shown in Table 10 and FIG. 16, the particle size was smaller than 250 nm, and the drug content was higher than 95%. Thus, the nanosuspension remained stable without causing drug aggregation or resveratrol degradation for 6 months.

< Experimental Example  4> Preparation and comparison of nano suspension of various poorly soluble drugs

Based on the experimental results obtained in the above Examples and Experimental Examples, nanosuspensions were prepared under different conditions such as using other poorly soluble drugs besides resveratrol, PEG400, which is known to be similar to transcutol, or ethanol. The comparison was made.

Thus, each was prepared according to the components as shown in Table 11 below. In other words, the drug solution was prepared by taking a certain amount of drug into a 10 mL volumetric flask and aligning the mark accurately with a solvent. Then, in order to prepare a stabilizer (polymer and surfactant) solution, the polymer and the surfactant were taken in a certain ratio, placed in a 100 mL volumetric flask, and the mark was adjusted using water as a solvent. Then, to prepare the nanoparticles, a certain amount of stabilizer solution was taken and drug solution was slowly injected while stirring at 700 rpm using a magnetic stirrer. Thereafter, after the solution was injected, the mixture was further stirred for 1 minute, and the particle size of each nanoparticle was measured by using dynamic light scattering.

As a result, as shown in Table 11 above, when PEG400 was used as the solvent for resveratrol, when resveratrol aggregated to form a large mass, a nanosuspension containing nanoparticles could not be prepared. Formation of nanoparticles having a size of nm showed a problem that the size is too large, but in the case of using the transcutol HP, the solvent selected in the present invention was prepared a nanosuspension containing nanoparticles of the appropriate particle size.

In addition, when aprepitant was used as a drug and transcutol P was used as a solvent, a nanosuspension was prepared with an average particle size of 291.4 nm when the composition of the stabilizer solution was a combination of PVP VA64 / PVPK12.

When cyclosporin and transcutol P were used, it was confirmed that the preferred suspension was prepared at 193.5 nm in the combination of PVP VA64 / PVP K12 / SLS.

When celecoxib and transcutol P were used, when the composition of the stabilizer solution was a combination of PVP VA64 / PVPK12, the average particle size was 234.1 nm, and when the combination of PVP VA64 / PVP K12 was 235.2 nm, At 274.3 nm when combined with PVP VA64 / PVP K12 / SLS, nanosuspensions were generally well prepared.

< Experimental Example  5> MHY498 ((Z) -5- (2,4- Dihydroxybenzylidene ) Thiazolidine -2,4- D Preparation and Comparison of Nano Suspensions

A nanosuspension using transcutol was prepared for MHY498, a poorly soluble compound, under the conditions shown in Table 12 below. Piperidine was added to 2,4-dihydroxybenzaldehyde and thiazolidine-2,4-dione solutions substituted with ethanol. . After several reactions, MHY498 was obtained as a solid yellow powder. Detailed synthesis method of MHY498 is described in Korean Patent No. 10-2013-0045887 (published on May 06, 2013).

In other words, the drug solution was prepared by taking a certain concentration of drug into a 10 mL volumetric flask and accurately matching the mark using a transcutol HP solvent. Then, in order to prepare a stabilizer solution, PVP K30 was taken to a certain ratio, placed in a 100 mL volumetric flask, and the mark was adjusted using water as a solvent. Then, to prepare the nanoparticles, a certain amount of stabilizer solution was taken and drug solution was slowly injected while stirring at 700 rpm using a magnetic stirrer. Thereafter, after the solution was injected, the mixture was further stirred for 1 minute, and the particle size of each nanoparticle was measured by using dynamic light scattering.

As a result, as shown in Table 12 above, in the configuration using Transcutol HP as the solvent and PVP K30 as the stabilizer, the average particle size was appropriate from 35.3 nm to 142.9 nm regardless of the concentration of the MHY498 solution or the proportion of the solvent. It was confirmed that the nanosuspension was prepared.

Having described the specific part of the present invention in detail, it is apparent to those of ordinary skill in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. Do. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

A method for preparing a nanosuspension comprising dissolving a poorly soluble drug in a transcutol as a solvent. The method of claim 1, wherein the poorly soluble drug is resveratrol, MHY498 ((Z) -5- (2,4-dihydroxybenzylidene) thiazolidine-2,4-dione), aprepitant, cyclosporine, sele A method for producing a nanosuspension, characterized in that selected from the group consisting of coxib, coenzyme Q10, valsartan, dabigatran etexilate, sorafenib and silymarin. The method of claim 1, wherein after dissolving the poorly soluble drug,
Dissolving a water-soluble polymer in water to prepare an aqueous polymer solution; And injecting a solution in which the drug is dissolved into the polymer aqueous solution. The method of claim 3, wherein the water-soluble polymer is polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, polyvinylpyrrolidone K30 and polyvinylpyrrolidone vinyl acetate VA64 Method for producing a nanosuspension, characterized in that selected. The method of claim 1, wherein after dissolving the poorly soluble drug,
Dissolving the surfactant in water to prepare an aqueous surfactant solution; And injecting a solution in which the drug is dissolved into the aqueous surfactant solution. The method of claim 5 wherein the surfactant is sodium lauryl sulfate (SLS), sucrose fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene glycolated natural or hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester [Tweens )], Polyethylene glycol, polyoxyethylene stearate, polyoxyethylene-polyoxypropylene copolymer (poloxamer or poloxamine), polyethylene glycolated phospholipid and alpha-tocopherol polyethylene glycol succinate Method for producing a nanosuspension, characterized in that. The method of claim 1, wherein the nanosuspension comprises poorly soluble drug nanoparticles having an average diameter of 1 nm to 300 nm. A nanosuspension prepared by the method according to any one of claims 1 to 7.

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