WO2018008986A1 - In vitro release testing method and evaluation method of polymer micelle preparation containing poorly water-soluble drug - Google Patents

Abstract

The present invention relates to: a method for testing the in vitro release of a polymer micelle preparation containing a poorly water-soluble drug; a method for evaluating a polymer micelle preparation containing a poorly water-soluble drug; and a polymer micelle composition containing a poorly water-soluble drug.

Description

In vitro release test and evaluation method of polymer micelle formulations containing poorly water soluble drugs

The present invention relates to an in vitro release test method of a polymer micelle formulation comprising a poorly water-soluble drug, a method for evaluating a polymer micelle formulation comprising a poorly water-soluble drug, and a polymer micelle composition comprising a poorly water-soluble drug.

Release test methods for poorly water-soluble drugs should be biorelevant and measure drug release in vitro ( in vitro ) by the same mechanism as drug release from in vivo ( in vivo ) dosage forms. It should be. This test method should also be able to distinguish the difference between "good" and "bad" batches, which means that it should be possible to detect changes in the product that could affect drug release.

Much attention is paid to the development of suitable devices for measuring in vitro release from micelles. At this time, additional considerations such as the choice of agitation medium and agitation are also not to be overlooked. In contrast to the release media of oral dosage forms, which typically mimic the pH of the gastrointestinal tract, the choice of release media for micelle dosage forms is not easy to simulate because of the in vivo conditions depending on the site of administration of the formulation as well as the site of action. . In general, the release medium is selected based on the solubility and stability of the drug, the sensitivity of the assay and the method used. Although maintenance of sink conditions is desirable, non-sink conditions have been adopted. Agitation is also an important process condition that is frequently used to prevent aggregation of the dispersed phase in in vitro release studies. However, the dynamic conditions resulting from the agitation depend on the device used. Moreover, sampling and buffer replacement (full or partial) techniques are also based on the type of in vitro method used.

Accordingly, there is a need for in vitro release testing or quality assessment of poorly water-soluble drugs from novel micelle formulations that can detect differences in product quality that can affect drug release while reproducing in vivo conditions.

It is an object of the present invention to provide a method for in vitro release testing of polymeric micelle formulations comprising poorly water soluble drugs, which can reflect the in vivo release mechanism as well as evaluate the quality of the drug product.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The first aspect of the present invention comprises the steps of: 1) adding an organic solution of a sequentially diluted poorly water-soluble drug to the albumin-containing aqueous medium, and removing the poorly water-soluble drug and albumin precipitated therefrom to prepare a standard solution; 2) preparing a release solution by adding an aqueous solution of the polymer micelle formulation including a poorly water-soluble drug to an albumin-containing aqueous medium, and then removing the poorly water-soluble drug and albumin precipitated therefrom to prepare a sample solution; 3) analyzing the standard solution of step 1) and the sample solution of step 2) by high performance liquid chromatography (HPLC), respectively; And 4) obtaining a release rate (%) of the poorly water-soluble drug by the following Equation 1, wherein the polymer micelle formulation has an amphipathy in which the poorly water-soluble drug includes a hydrophilic block (A) and a hydrophobic block (B). It is possible to provide an in vitro release test method of a polymer micelle formulation containing a poorly water-soluble drug, which is encapsulated in a polymer micelle formed from a block copolymer.

[Equation 1]

Release rate of poorly water-soluble drugs (%) = Precipitation rate of poorly water-soluble drugs (%) = (Water-soluble drug concentration of the initial sample-water-soluble drug concentration of the sample at time t) / Water-soluble drug concentration of the initial sample × 100 = ( A ₀ -A _t ) / A ₀ × 100

A ₀ = peak area of poorly soluble drug in the initial sample solution / peak area of poorly soluble drug in the standard solution × poorly soluble drug concentration in the standard solution × dilution factor × volume of the release solution

A _t = peak area of poorly soluble drug in the sample solution / peak area of poorly soluble drug in the standard solution × poorly soluble drug concentration in the standard solution × dilution factor × volume of release solution

A second aspect of the present invention provides a method for irradiating a poorly water-soluble drug according to the method according to the first aspect of the present invention; Evaluating the quality or efficacy of the polymer micelle formulation from the irradiated release pattern, wherein the poorly water-soluble drug is encapsulated in a polymer micelle formed from an amphiphilic block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B) A method for evaluating a polymer micelle formulation containing a poorly water-soluble drug can be provided.

The third aspect of the invention is characterized in that the poorly water-soluble drug release rate in the test according to the method according to the first aspect of the present invention is more than 90% in 3 hours, wherein the poorly water-soluble drug is a hydrophilic block (A) and a hydrophobic block ( A polymer micelle composition comprising a poorly water-soluble drug, which is enclosed in a polymer micelle formed from an amphiphilic block copolymer comprising B), can be provided.

The above-mentioned means for solving the problems are merely exemplary, and should not be construed to limit the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

According to the present invention, a novel release assay for polymer micelle formulations containing poorly water-soluble drugs can be provided. The method of the present invention can be used as an indicator of the quality and performance of polymer micelle formulations containing poorly water-soluble drugs, and also reproduces in vivo conditions and provides an in vitro-in vivo correlation with the release mechanism. IVIVC) can be used to establish.

1 is a graph showing the concentration of paclitaxel with shaking time in mPEG-PLA polymer solution of various concentrations.

2 is a graph showing the concentration of paclitaxel with shaking time in mPEG-PLA polymer solution at 25 ℃ and 37 ℃.

Figure 3 is a graph showing the supersaturation solubility (Fig. 3a) and high solubility (Fig. 3b) of paclitaxel according to the polymer concentration in distilled water and 4% albumin phosphate buffer.

Figure 4 is a graph showing the kinetic stability of paclitaxel supersaturated polymer micelles.

Figure 5 is a graph showing paclitaxel precipitation of Genexol PEM in 37 ℃, 4% albumin phosphate buffer.

6 is a graph showing the calibration curve of paclitaxel standard solution.

Figure 7 is a graph showing the paclitaxel release behavior of Genexol PM in 37 ℃, 4% albumin phosphate buffer using the precipitation method according to the concentration of paclitaxel.

Figure 8 is a graph showing the paclitaxel release behavior in 37 ℃, 4% albumin phosphate buffer for Genexol PEM release test reproducibility confirmation test.

Figure 9 is a graph showing the paclitaxel release behavior of Genexol PM in 37 ℃, 4% albumin phosphate buffer solution according to various product numbers.

FIG. 10 is a graph showing paclitaxel release behavior in 37 ° C., 4% albumin phosphate buffer of Genexol PM defective product (50: 500) and normal product (100: 500, GP11511).

Figure 11 is a graph showing the paclitaxel release mechanism of Genexol PEM in vivo.

Hereinafter, embodiments and implementations of an in vitro release test method of a polymer micelle formulation containing a poorly water-soluble drug of the present invention, an evaluation method of a polymer micelle formulation containing a poorly water-soluble drug, and a polymer micelle composition comprising a poorly water-soluble drug It will be described in detail with reference to examples and drawings. However, the present invention is not limited to these embodiments, embodiments, and drawings.

The first aspect of the present invention comprises the steps of: 1) adding an organic solution of a sequentially diluted poorly water-soluble drug to the albumin-containing aqueous medium, and removing the poorly water-soluble drug and albumin precipitated therefrom to prepare a standard solution; 2) preparing a release solution by adding an aqueous solution of the polymer micelle formulation including a poorly water-soluble drug to an albumin-containing aqueous medium, and then removing the poorly water-soluble drug and albumin precipitated therefrom to prepare a sample solution; 3) analyzing the standard solution of step 1) and the sample solution of step 2) by high performance liquid chromatography (HPLC), respectively; And 4) obtaining a release rate (%) of the poorly water-soluble drug by the following Equation 1, wherein the polymer micelle formulation has an amphipathy in which the poorly water-soluble drug includes a hydrophilic block (A) and a hydrophobic block (B). It is possible to provide an in vitro release test method of a polymer micelle formulation containing a poorly water-soluble drug, which is encapsulated in a polymer micelle formed from a block copolymer.

[Equation 1]

Release rate of poorly water-soluble drugs (%) = Precipitation rate of poorly water-soluble drugs (%) = (Water-soluble drug concentration of the initial sample-water-soluble drug concentration of the sample at time t) / Water-soluble drug concentration of the initial sample × 100 = ( A ₀ -A _t ) / A ₀ × 100

A ₀ = peak area of poorly soluble drug in the initial sample solution / peak area of poorly soluble drug in the standard solution × poorly soluble drug concentration in the standard solution × dilution factor × volume of the release solution

A _t = peak area of poorly soluble drug in sample solution / peak area of poorly soluble drug in standard solution × poorly soluble drug concentration in standard solution × dilution factor × volume of release solution.

For example, the amount of the hydrophilic block of the amphiphilic block copolymer may be about 20 to 95 wt%, or about 40 to 95 wt% based on the total weight of the copolymer, but may not be limited thereto. In addition, the content of the hydrophobic block of the amphiphilic block copolymer may be about 5 to 80% by weight, or about 5 to 60% by weight based on the total weight of the copolymer, but may not be limited thereto.

For example, the number average molecular weight of the amphiphilic block copolymer may be 1,000 to 50,000 Daltons, more specifically 1,500 to 20,000 Daltons, but may not be limited thereto. The polymer micelle may be formed by a method commonly used in the art to which the present invention pertains.

For example, the organic solution may include an organic solvent selected from the group consisting of water miscible organic solvents such as alcohol, acetone, tetrahydrofuran, acetic acid, acetonitrile and dioxane, and combinations thereof, but is not limited thereto. It may not be. In addition, the aqueous solution may include one selected from the group consisting of conventional water, distilled water, distilled water for injection, saline solution, glucose solution (eg, 5% glucose solution), buffer, and combinations thereof, but is not limited thereto. It may not be.

For example, the poorly water-soluble drug may be selected from drugs having a solubility in water (25 ° C.) of 100 mg / mL or less, for example, anticancer agents, antifungal agents, and immunosuppressants. , Analgesics, anti-inflammatory agents, antiviral agents, anxiolytic sedatives, contrasting agents, corticosteroids, diagnostic agents, diagnostic contrast agents ( diagnostic imaging agents, diuretics, prostaglandins, radiopharmaceuticals, sex hormones, including steroids, and combinations thereof, but may not be limited thereto. have.

According to one embodiment of the invention, the albumin-containing aqueous medium of steps 1) and 2) may be distilled water or buffer containing albumin at a concentration of greater than about 0 and up to 20%, but may not be limited thereto. For example, the albumin-containing aqueous medium may contain about 1 to 10% (w / v), about 1 to 5% (w / v) or about 4% (w / v) of albumin, but It may not be limited.

According to one embodiment of the present invention, the poorly water-soluble drug precipitated in step 1) and step 2) may be removed by filtration, but may not be limited thereto.

According to one embodiment of the present invention, filtration may be performed using a filter having a pore size of about 0.8 μm or less, but may not be limited thereto. For example, the filtration may be performed using a filter having a pore size of about 0.1 μm or more but less than 0.8 μm, about 0.2 μm or more and less than 0.8 μm, or about 0.2 μm or more and less than 0.5 μm, but may not be limited thereto.

According to one embodiment of the present invention, albumin may be precipitated by adding an organic solvent in step 1) and step 2) and removed by centrifugation, but may not be limited thereto.

According to one embodiment of the present invention, the initial poorly water-soluble drug concentration in the release solution of step 2) may be about 0.5 mg / ml or more, for example, 0.5 mg / ml or more and 30 mg / ml or less, but may not be limited thereto. have.

According to one embodiment of the invention, the high performance liquid chromatography in step 3) may preferably be using the conditions of (a) and (b): (a) pentafluor having a particle size of 4 μm or less Rophenyl porous particle stationary phase; And (b) a column having an inner diameter of 5 mm or less and a length of 50 mm or more. In the present invention, the analysis of step 3) can be performed according to the disclosure of Korean Patent Application No. 2017-0014057, in which case Korean Patent Application No. 2017-0014057 is incorporated herein by reference in its entirety.

According to one embodiment of the present invention, the aqueous solution of the polymer micelle formulation including the poorly water-soluble drug of step 2) may be a reconstitution of the lyophilized formulation into an aqueous solution, but may not be limited thereto.

According to one embodiment of the invention, the hydrophilic block (A) is selected from the group consisting of polyethylene glycol, monomethoxypolyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol and polyacrylamide, and the hydrophobic block (B) is Polylactide, polyglycolide, polydioxan-2-one, polycaprolactone, polylactic-co-glycolide, polylactic-co-caprolactone, polylactic-co-dioxan-2-one and polyglycolic- It may be selected from the group consisting of co-caprolactone, and derivatives thereof whose carboxylic acid ends are substituted with fatty acid groups, but may not be limited thereto.

According to one embodiment of the invention, the poorly water-soluble drug may be an anticancer agent, but may not be limited thereto.

According to one embodiment of the invention, the anticancer agent may be a taxane anticancer agent, but may not be limited thereto. For example, the taxane anticancer agent may include paclitaxel, docetaxel, 7-epipaclitaxel, t-acetylpaclitaxel, 10-desacetyl paclitaxel, and 10-desacetylpaclitaxel. 10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel, 10-desacetyl-7-glutarylpaclitaxel ), 7-N, N-dimethylglycylpaclitaxel (7-N, N-dimethylglycylpaclitaxel), 7-L-alanylpaclitaxel (7-L-alanylpaclitaxel) and cabazitaxel may be one or more selected from the group consisting of , More specifically, it may be paclitaxel, but may not be limited thereto.

A second aspect of the present invention provides a method for irradiating a poorly soluble drug release pattern according to the method according to the first aspect of the present invention; Evaluating the quality or efficacy of the polymer micelle formulation from the irradiated release pattern, wherein the poorly water-soluble drug is encapsulated in a polymer micelle formed from an amphiphilic block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B) A method for evaluating a polymer micelle formulation containing a poorly water-soluble drug can be provided.

For example, the method for evaluating the polymer micelle formulation containing the poorly water-soluble drug may include evaluating that the poorly water-soluble drug release rate has a desirable quality when the release rate of the poorly water-soluble drug is 10% or more in 1 hour and 90% or more in 3 hours. However, this may not be limited. For example, when a poorly water-soluble drug release rate is 10% or more and 30% or less in one hour, and 90% or more in three hours, the poorly water-soluble drug polymer micelle formulation can be evaluated to have a desirable quality, or the water-soluble drug release rate is 1 It may be evaluated that the poorly water-soluble drug polymer micelle formulation has a desirable quality when it is 10% or more and 30% or less in time, more than 30% and 70% or less in 1.25 hours and 90% or more in 3 hours, but the present invention may not be limited thereto. .

The third aspect of the invention is characterized in that the poorly water-soluble drug release rate in the test according to the method according to the first aspect of the present invention is more than 90% in 3 hours, wherein the poorly water-soluble drug is a hydrophilic block (A) and a hydrophobic block ( A polymer micelle composition comprising a poorly water-soluble drug, which is enclosed in a polymer micelle formed from an amphiphilic block copolymer comprising B), can be provided.

According to an embodiment of the present invention, the composition may have a poorly water-soluble drug release rate of 10% or more in one hour, 90% or more in 3 hours, for example, 90% or more and 99.5% or less, but may not be limited thereto. .

According to an embodiment of the present invention, the composition may have a poorly water-soluble drug release rate of 10% or more and 30% or less in one hour, 90% or more, for example, 90% or more and 99.5% or less in three hours, but is not limited thereto. You may not.

According to one embodiment of the invention, the composition has a poorly water-soluble drug release rate of 10% or more and 30% or less in 1 hour, more than 30% and 70% or less in 1.25 hours, 90% or more, for example 90 in 3 hours It may be greater than or equal to 99.5% but may not be limited thereto.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

Drug products

Genexol-PM for injection administration is a formulation using low molecular weight, amphiphilic diblock copolymers to form polymeric micelles that dissolve paclitaxel. Each 100 mg Genexol PME vial for injection administration contains the components shown in Table 1 below.

TABLE 1

In Vitro ( in vitro Release Experiment Method

1. Reagents and Materials

Paclitaxel Standard: Lot G479 (Samyang Biofarm)

MPEG-PDLLA: lot PM1403 (Samyang Biofarm)

-Jengensol PM: Part Number (Lot) GP11511, GP114A1, GP114B1, GP114C1 (Samyang Biofarm)

20% Human Serum Albumin 100 mL: Lot 161D14240 (Green Cross)

0.9% physiological saline solution (CJ CheilJedang)

Acetonitrile (Burdick & Jackson.ACS / HPLC Grade)

2. Appliances and Equipment

-Shaking bath: Maxturdy 30, DAIHAN Scientific.

Shake shake incubator: Vision Sci. Co.

High Performance Liquid Chromatography (HPLC): HP 1200 series, Agilent Technologies.

-Centrifuge: Mini spin plus, Eppendorf.

3. In copolymer solution Paclitaxel  Solubility Measurement

1) Paclitaxel Solubility in Distilled Water Containing mPEG-PDLLA Copolymer

Instrument: shake incubator, Vision Sci. Co.

Medium: distilled water

Preparation of Copolymer Solution

5 g of mPEG-PDLLA copolymer was dissolved in 50 mL of distilled water (100.0 mg / mL). The aqueous copolymer solution was sequentially diluted with distilled water to a concentration of 2.5, 5, 10, 25, 50, 100 mg / mL.

Addition of paclitaxel to the copolymer solution

100 mg of amorphous paclitaxel was placed in 20 mL of an aqueous solution of mPEG-PDLLA copolymer. Paclitaxel and the polymer suspension were placed in a shaking constant temperature bath and stirred at 25 ° C., 37 ° C. and 200 rpm.

-Sampling

At each sampling time (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24 hours) 1 mL of the suspension was taken and filtered through a 0.45 μm membrane filter. 0.6 mL of the filtrate and 0.6 mL of acetonitrile were mixed and analyzed by HPLC.

- analysis

HPLC was used to analyze the concentration of paclitaxel. The concentration of paclitaxel was calculated using the following formula: a = (paclitaxel peak area-y fragment) / slope x 100 (mg), and the slope was calculated by the calibration curve of the standard solution. (2.5, 5, 25, 50, 250, 500 μg / mL)

[HPLC]

Column: Poroshell 120 PFP column (2.7 μm, 4.6 x 150 mm, Agilent, USA)

Detector: UV (λ = 227 nm)

Flow rate: 0.6 mL / min

Injection volume: 10 μl

-Column temperature: 25 ℃

Mobile phase:

0-25 minutes: water / acetonitrile = 65/35 (v / v) 45/55 (v / v)

25-28 minutes: Water / acetonitrile = 45/55 (v / v)

25 ~ 30 minutes: water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

30 ~ 35 minutes: water / acetonitrile = 65/35 (v / v)

2) Paclitaxel Solubility in 4% Albumin Phosphate Buffer Containing -mPEG-PDLLA Copolymer

Instrument: shake incubator: Vision Sci. Co.

Medium: 4% albumin phosphate buffer

Preparation of Copolymer Solution

5 g of mPEG-PDLLA copolymer was dissolved in 50 mL of 4% albumin buffer. (100.0 mg / mL) The copolymer solution was diluted sequentially with 4% albumin phosphate buffer to a concentration of 2.5, 5, 10, 25, 50, 100 mg / mL.

Addition of paclitaxel to the copolymer solution

100 mg of amorphous paclitaxel was added to 20 mL of mPEG-PDLLA copolymer solution. Paclitaxel and the polymer suspension were placed in a shaking constant temperature bath and stirred at 25 ° C, 37 ° C, and 200 rpm.

-Sampling

At each sampling time (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24 hours) 1 mL of the suspension was taken and filtered through a 0.45 μm membrane filter. Albumin was precipitated by mixing 0.2 mL of the filtrate and 0.8 mL of acetonitrile. The solution was centrifuged at 14,000 rpm for 5 minutes, 0.5 mL of the supernatant and 0.5 mL of 80% acetonitrile solution were mixed, and this solution was used as the sample solution.

Preparation of Standard Solution

12.5 mg of paclitaxel was precisely weighed into a 100 mL volumetric flask and dissolved in acetonitrile (125 μg / mL). This solution was diluted sequentially with acetonitrile to make standard stock solutions of 62.5, 12.5, 6.25, 1.25, and 0.625 μg / mL. 0.8 mL of each standard stock solution was added, 0.2 mL of 4% albumin phosphate buffer solution was added to precipitate albumin, and the mixture was centrifuged at 14,000 rpm for 5 minutes. 0.5 mL of this supernatant was taken and 0.5 mL of 80% acetonitrile aqueous solution was mixed to prepare a standard solution (100, 50, 10, 5, 1, 0.5 μg / mL).

TABLE 2

- analysis

HPLC was used to analyze the concentration of paclitaxel. The concentration of paclitaxel was calculated using the following formula: a = (paclitaxel peak area-y-intercept) / slope x 100 (mg), and the slope was calculated from the calibration curve of the standard solution (2.5, 5, 25, 50, 250). , 500 μg / mL),

4. in albumin solution Genexol  PM Paclitaxel  Release test

-Steam equipment: shake bath: Maxturdy 30, DAIHAN Scientific.

Medium: 4% albumin phosphate buffer

Drug concentration: 1.0 mg / mL

-Release of paclitaxel in 4% albumin phosphate buffer

20 mL of physiological saline was added to a 100 mg vial of Genexol PEM to reconstitute a solution of 5.0 mg / mL. 4 mL of the gastric solution was taken and placed in 16 mL of 37 ° C, 4% albumin phosphate buffer. This solution was stirred at 37 ° C. and 200 rpm.

Sample Collection

Take 1 mL of gastric solution and filter with 0.45 μm PVDF syringe filter at each sampling time (0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, 24 hours) It was. 0.2 mL of the filtrate was taken and mixed with 0.8 mL of acetonitrile. The mixture was centrifuged at 14,000 rpm for 5 minutes, 0.5 mL of the supernatant was mixed with 0.5 mL of 80% acetonitrile aqueous solution. This solution was used as the sample solution for HPLC analysis.

Preparation of standard solution

3. Prepared in the same manner as in 2).

- analysis

3. The concentration of paclitaxel was analyzed using HPLC in the same manner as in 1).

5. Genexol PM Emission Test

Preparation of Emission Solution

20 mL of physiological saline was added to a 100 mg vial of Genexol PEM to reconstitute a solution of 5.0 mg / mL. 4 mL of this solution was taken and added to 16 mL of 4% albumin phosphate buffer solution at 37 ° C. The solution was stirred at 37 ° C. and 200 rpm to obtain a release solution.

- Release

The release solution was stirred at 200 ° C. at 37 ° C. in a shaking constant temperature bath.

Sample Collection

Take 1 mL of gastric solution and filter with 0.45 μm PVDF syringe filter at each sampling time (0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, 24 hours) It was. 0.2 mL of the filtrate was taken and mixed with 0.8 mL of acetonitrile. The mixture was centrifuged at 14,000 rpm for 5 minutes, 0.5 mL of the supernatant was mixed with 0.5 mL of 80% acetonitrile aqueous solution. This solution was used as the sample solution for HPLC analysis.

HPLC

1) Preparation of Standard Solution

12.5 mg of paclitaxel was precisely weighed into a 100 mL volumetric flask and dissolved in acetonitrile (125 μg / mL). This solution was diluted sequentially with acetonitrile to prepare standard stock solutions of 62.5, 12.5, 6.25, 1.25, and 0.625 μg / mL. 0.8 mL of this standard stock solution was added, 0.2 mL of 4% albumin phosphate buffer solution was added to precipitate albumin, and the mixture was centrifuged at 14,000 rpm for 5 minutes. 0.5 mL of this supernatant was taken and 0.5 mL of 80% acetonitrile aqueous solution was mixed to prepare a standard solution (100, 50, 10, 5, 1, 0.5 μg / mL).

2) Preparation of Initial Sample Solution

20 mL of saline was added to a 100 mg vial of Genexol PM and reconstructed. 4 mL was taken and added to 16 mL of 37 ° C., 4% albumin phosphate buffer. 1 mL of solution was taken and filtered with 0.45 μm PVDF filter. 0.2 mL of the filtrate was taken, 0.8 mL of acetonitrile was added, and albumin was precipitated. The solution was centrifuged at 14,000 rpm for 5 minutes, 0.5 mL of the supernatant was mixed with 0.5 mL of an aqueous 80% acetonitrile solution, and this solution was used as the initial sample solution.

The standard solution and the sample solution were injected into HPLC under the following conditions. Paclitaxel release was calculated from the peak area of paclitaxel in the sample and standard solutions.

[HPLC]

Column: Poroshell 120 PFP column (2.7 μm, 4.6 × 150 mm, Agilent, USA)

Detector: UV (λ = 227 nm)

Flow rate: 0.6 mL / min

Injection volume: 10 μl

-Column temperature: 25 ° C

Mobile phase

0-25 minutes: water / acetonitrile = 65/35 (v / v) 45/55 (v / v)

25-28 minutes: Water / acetonitrile = 45/55 (v / v)

25 ~ 30 minutes: water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

30 ~ 35 minutes: water / acetonitrile = 65/35 (v / v)

- Calculation

Paclitaxel Release (%) = Precipitated Paclitaxel (%) = (Paclitaxel Concentration of Initial Sample-Paclitaxel Concentration of Sample at Each Sampling Time (t) / Paclitaxel Concentration of Initial Sample x 100 = (A ₀ -A _t ) / A ₀ x 100

A ₀ = paclitaxel peak area of the initial sample solution / paclitaxel peak area of the standard solution x paclitaxel concentration of the standard solution x 10 x 20

-A _t = paclitaxel peak area of the sample solution at time t / paclitaxel peak area of the standard solution x paclitaxel concentration of the standard solution x 10 x 20

The concentration of paclitaxel in the sample solution (A _t ) is equal to the sum of the solubility in 4% albumin phosphate buffer and the amount of paclitaxel not released from micelles.

Results and Discussion

1. Solubility of Paclitaxel in Polymer Solutions

1) Paclitaxel Concentration According to Time of Polymer Solution of Various Concentrations

The solubility of paclitaxel was tested in distilled water and 4% albumin phosphate buffer. When an excess amount of amorphous paclitaxel is added to a polymer solution of various concentrations, the amorphous paclitaxel is spontaneously dissolved in the polymer micelle and supersaturated in the polymer solution for a period of time. 1 and Table 3 show the concentration of paclitaxel over time of mPEG-PLA polymer solution of various concentrations. As shown in Fig. 1 and Table 3, the concentration of paclitaxel was decreased in supersaturated solubility after the supersaturated state was maintained, resulting in high solubility. The reason why the concentration of paclitaxel drops from supersaturated solubility to high solubility concentration is that amorphous paclitaxel, which was temporarily trapped in the polymer micelle, becomes a form of dihydrate having low solubility.

TABLE 3

The concentration of paclitaxel over time varied with temperature. Figure 2 and Table 4 shows the concentration of paclitaxel over time at 25 ℃ and 37 ℃ of mPEG-PLA polymer. As shown in Fig. 2 and Table 4, the paclitaxel supersaturated solubility at 37 ° C was higher than the paclitaxel supersaturated solubility at 25 ° C, and the high solubility at 37 ° C was lower than that at 25 ° C.

Also, the supersaturation duration was shorter at 37 ° C than at 25 ° C. This is because at higher temperatures amorphous paclitaxel dissolves rapidly in polymeric micelles, but can crystallize more quickly in the form of dihydrate.

TABLE 4

2) Solubility of paclitaxel according to the polymer concentration

The supersaturation and high solubility (mPEG-PLA 25 mg / mL, 37 ° C) of paclitaxel according to the polymer concentration in distilled water and 4% albumin phosphate buffer solution are shown in FIGS. 3A, 3B and Table 5. Supersaturation and high solubility of paclitaxel increased linearly with increasing polymer concentration. The supersaturation and high solubility of paclitaxel in distilled water and 4% albumin phosphate buffer solution was not significantly different in mPEG-PLA polymer concentrations above 25 mg / mL (Fig. 3a), but at 4% albumin concentration below 4 Phosphate buffer was higher (Figure 3b).

TABLE 5

2. Genexol  Of aqueous solution Paclitaxel  Sedimentation

The mPEG-PLA polymer micelles may contain excessive amounts of hydrophobic drugs because they are kinetically more stable than low molecular weight surfactant micelles. In the case of low molecular weight micelles, excess drug is easily precipitated when in contact with water and therefore is not suitable for injections that must contain excess drug for extended periods of time.

4 is a graph showing the kinetic stability of polymer micelles supersaturated with paclitaxel. Genexol PM contains an excess of paclitaxel in the polymer micelle, and the micelle solution in the aqueous phase is stable for a long time at room temperature, which is convenient for clinical use. However, at high temperatures such as body temperature, it becomes dynamically unstable, meaning that paclitaxel can be easily released from the polymer micelles at high temperatures. Paclitaxel of Genexol PEM precipitates as crystalline paclitaxel dihydrate from micelles in a limited amount of aqueous solution. In this regard, Figures 5 and 6 are graphs showing paclitaxel precipitation of Genexol PEM in 37 ° C, 4% albumin phosphate buffer. Using this feature of Genexol PM, it was possible to develop precipitation method, in vitro release test. The solubility of paclitaxel in distilled water and 4% albumin phosphate buffer at body temperature is close to zero (Table 6). Therefore, the precipitated amount of paclitaxel in this release test can be regarded as the concentration of paclitaxel released from Genexol PM. In fact, 0.9 mg / mL Genexol PM in 4% albumin phosphate buffer drops to 0.08 mg / mL after 2 hours. In fact, more than 90 and 97% of paclitaxel in micelles was released and precipitated after 2 and 6 hours.

TABLE 6

3. Genexol  Establishment of In Vitro Emission Test Method

1) Bio-like medium

The biological media in which Genexol PM is used are blood, plasma and serum, which are not readily available in sufficient quantities for in vitro release testing and also have difficulty in drug analysis. Therefore, phosphate buffer (PBS, pH 7.4) has been used as a bio-like medium, the phosphate buffer containing albumin was used in the in vitro release test method of Genexol PM of this embodiment.

2) Separation of Paclitaxel Precipitate in Bio-like Media

There is a method for separating paclitaxel precipitate from bio-like media. One is centrifugation and the other is filtration.

In centrifugation, choosing the proper centrifugal force and time is not easy. Table 7 shows the amount of paclitaxel released from Genexol PM after 24 hours in 37 ° C., 4% albumin phosphate buffer with centrifugation time. According to Table 7, the amount of paclitaxel released increased with longer centrifugation time. During centrifugation (more than 5 minutes) paclitaxel continues to settle, so the amount of paclitaxel released can be overestimated.

However, when the filtration method is used, the filtration time will not affect the experimental results because the filtration time is less than 1 minute. In the filtration method, the release medium containing Genexol PEM is taken at each sampling time and filtered with a PVDF syringe filter. The filter removes precipitated paclitaxel (crystallized paclitaxel) but leaves paclitaxel and albumin in micelles in the filtrate. The filtrate may comprise paclitaxel dissolved in pure water but is negligibly small. Thus, filtration is more reliable than centrifugation.

Since filters of various pore sizes can be used for filtration, the amount of paclitaxel released from Genexol PM after 24 hours in 37 ° C., 4% albumin phosphate buffer, depending on the size of the filter pores was evaluated (Table 8). The amount of paclitaxel in the filtrate (unreleased paclitaxel and negligible albumin-bound paclitaxel) when using a 0.22 μm filter was slightly less than when a 0.45 μm filter was used. However, the difference was negligible as can be seen in Table 8.

However, the 0.8 μm filter was found to be significantly higher than the 0.45 and 0.22 μm filters because relatively large precipitated paclitaxel penetrated the filter pores. Therefore 0.45 μm filter was used for the in vitro release test of Genexol PM.

TABLE 7

TABLE 8

3) Analysis of Paclitaxel in Bio-like Media

Validated HPLC assays were used to quantify the release of paclitaxel in the biolike medium. Prior to HPLC analysis, pretreatment was required to remove albumin from the release medium of Genexol PM. The albumin removal step is an essential pretreatment for HPLC analysis because albumin removal does not damage HPLC and columns during HPLC analysis. It has been reported that proteins can be denatured (precipitated) by adding organic solvents such as acetonitrile and methanol. Precipitated albumin can be removed by centrifugation or filtration, in this example a centrifugation method was used. Since 80% acetonitrile dissolves both paclitaxel and mPEG-PLA polymers, polymer micelles are absent and only paclitaxel and polymers are included in the albumin-free 80% acetonitrile solution.

Validated HPLC Method

[HPLC]

Column: Poroshell 120 PFP column (2.7 μm, 4.6 × 150 mm, Agilent, USA)

Detector: UV (λ = 227 nm)

Flow rate: 0.6 mL / min

Injection volume: 10 μl

-Column temperature: 25 ℃

Mobile phase:

0-25 minutes: water / acetonitrile = 65/35 (v / v) 45/55 (v / v)

25-28 minutes: Water / acetonitrile = 45/55 (v / v)

25 ~ 30 minutes: water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

30 ~ 35 minutes: water / acetonitrile = 65/35 (v / v)

The calibration curve and results of paclitaxel standard solution are shown in FIG. 6 and Table 9. FIG.

TABLE 9

4) Initial concentration of paclitaxel in the release medium

In order to measure the initial concentration of paclitaxel in the release medium, the effect of the concentration of paclitaxel in the release medium on the release behavior of Genexol PM was used. Figure 7 is a graph showing the paclitaxel release behavior of Genexol PM in 37 ℃, 4% albumin phosphate buffer. As can be seen in Figure 7, the lower the concentration of paclitaxel, the faster the release rate. This is because the lower the concentration of the polymer, the faster the critical micelle concentration is reached. This result means that Genexol PM can be easily dissociated from the blood so that paclitaxel can be easily released from the blood. It is therefore expected that Genexol PM will be released shortly after injection into the body.

Early low paclitaxel concentrations indicate a slow rate of intravenous infusion. However, paclitaxel released in this precipitation method includes precipitates that do not bind albumin and is negligibly low at about 8 μg / mL in a 4% albumin solution at 37 ° C. as shown in Table 5. However, lowering the concentration of Genexol PM (1.0 mg / mL-> 0.1 mg / mL) would result in an insignificant proportion of albumin-bound paclitaxel. Therefore, the initial paclitaxel concentration of the release medium was chosen to be 1.0 g / mL.

3.4. Paclitaxel Release of Genexol PM in Bio-like Media

1) Reproducibility of Release Test

Three replicates were used to evaluate the reproducibility of the precipitation method using Genexol PM (Lot GP11511) of the same product number. Reproducibility test results are shown in FIG. 8 and Table 10 and this method is very reproducible.

TABLE 10

2) Release of Genexol PM according to various product numbers

The results of evaluating Genexol PEM release behavior of four product numbers using the precipitation method are shown in FIG. 9 and Table 11. FIG.

TABLE 11

3) Evaluation of defective product

Since the in vitro release method should be able to distinguish between defective products, two defective products were produced by the precipitation method to evaluate the behavior of the defective batches. The configuration of the defective arrangement is shown in Table 12. A release product with paclitaxel and mPEG-PLA ratio of 100: 300 could not be released because it was opaque when reconstructed with physiological saline. The release behavior of 50: 500 and 100: 500 (GP11511) is shown in Figure 10 and Table 13.

TABLE 12

TABLE 13

4. Emission test methods and standards

4.1. Release test method

1) Preparation of Standard Solution

 12.5 mg of paclitaxel was precisely weighed into a 100 mL volumetric flask and dissolved in acetonitrile (125 μg / mL). This solution was diluted sequentially with acetonitrile to make standard stock solutions of 62.5, 12.5, 6.25, 1.25, and 0.625 μg / mL. 0.8 mL of this standard stock solution was added, 0.2 mL of 4% albumin phosphate buffer solution was added to precipitate albumin, and the mixture was centrifuged at 14,000 rpm for 5 minutes. 0.5 mL of this supernatant was taken and 0.5 mL of 80% acetonitrile aqueous solution was mixed to prepare a standard solution (100, 50, 10, 5, 1, 0.5 μg / mL).

2) Preparation of Sample Solution

20 mL of saline was added to a 100 mg vial of Genexol PM and completely dissolved (5.0 mg / mL). 4 mL of this solution was taken and placed in 16 mL of 4% albumin phosphate buffer solution at 37 ° C., and this solution was used as a release solution. 1 mL of this release solution was taken and filtered using a 0.45 μm PVDF syringe filter. 0.2 mL of this filtrate was taken, 0.8 mL of acetonitrile was added, and albumin was precipitated. The solution was centrifuged at 14,000 rpm for 5 minutes and 0.5 mL of supernatant was taken and mixed with 0.5 mL of 80% acetonitrile. This solution was used as the initial release solution. Thereafter, the release solution was stirred (precipitated) at 200 rpm at 37 ° C. for a specific time according to the release conditions, and then 1 mL of this solution was taken and filtered using a 0.45 μm syringe filter. 0.2 mL of this filtrate was taken and poured into 0.8 mL of acetonitrile to precipitate albumin. The solution was centrifuged at 14,000 rpm for 5 minutes and 0.5 mL of supernatant was mixed with 0.5 mL of 80% acetonitrile. At this time, the solution was used as the elution solution at t time.

3) release and analysis

A water bath for stirring the discharge solution at 37 ° C. and 200 rpm was used.

[Emission condition]

Release Method: Precipitation

Release medium: 4% human albumin phosphate buffer (pH 7.4)

Capacity: 20 mL

Stirring Speed: 200 rpm

-Discharge temperature: 37.0 ± 0.5 ℃

Standard solution and sample solution were injected into HPLC to calculate the released paclitaxel using the peak area of paclitaxel according to the following conditions.

[HPLC]

Column: Poroshell 120 PFP column (2.7 μm, 4.6 × 150 mm, Agilent, USA)

Detector: UV (λ = 227 nm)

Flow rate: 0.6 mL / min

Injection volume: 10 μl

-Column temperature: 25 ° C

Mobile phase

0-25 minutes: water / acetonitrile = 65/35 (v / v) 45/55 (v / v)

25-28 minutes: Water / acetonitrile = 45/55 (v / v)

25 ~ 30 minutes: water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

30 ~ 35 minutes: water / acetonitrile = 65/35 (v / v)

- Calculation

Paclitaxel Release (%) = Precipitated Paclitaxel (%) = (Paclitaxel Concentration of Initial Sample-Paclitaxel Concentration of Sample at Each Sampling Time (t)) / Paclitaxel Concentration of Initial Sample x 100 = (A ₀ -A _t ) / A ₀ x 100

A ₀ = paclitaxel peak area of the initial sample solution / paclitaxel peak area of the standard solution x paclitaxel concentration of the standard solution x 10 x 20

-A _t = paclitaxel peak area of the sample solution at time t / paclitaxel peak area of the standard solution x paclitaxel concentration of the standard solution x 10 x 20

5. Possible mechanisms of paclitaxel release in vivo

Genexol PM is diluted very quickly when injected into the body, so there is no possibility of crystallization and precipitation. Instead, it quickly combines with other proteins and distributes throughout the body as well as blood. However, it is not easy to reproduce in vivo conditions that are diluted to infinity by precipitation method. Therefore, precipitation of paclitaxel in vitro was considered protein binding in vivo. That is, crystallization of paclitaxel in polymer micelles in vitro can be regarded as binding of paclitaxel and protein in vivo due to the presence of many proteins and blood components and rapid dilution. From the in vitro release test results using the precipitation method, the possible release mechanism of paclitaxel release from Genexol PM is shown in FIG. 11.

First step:

Supersaturated paclitaxel in polymer micelles is released from polymer micelles and immediately binds to plasma proteins. This stage accounts for more than 95% of the total emissions.

2nd step

Paclitaxel remaining in the polymer micelle is released slowly due to dissociation of the polymer micelle below CMC or degradation by enzymes in the blood. This stage accounts for less than 5% of the total emissions.

6. Release test for polymer micelle formulations containing existing and purified polymers

150 g of monomethoxy polyethylene glycol (mPEG, number average molecular weight = 2,000) was added to a 500 ml round bottom flask equipped with a stirrer, followed by stirring at 120 ° C. and vacuum for 2 hours to remove moisture. 0.15 g of Stanus octoate (Sn (Oct) ₂ ) dissolved in 200 μl of toluene was added into the reaction flask, and then toluene was distilled off while stirring under vacuum for 1 hour. Thereafter, 150 g of D, L-lactide was added thereto, followed by melting in a nitrogen atmosphere. After the D, L-lactide was completely dissolved, the reactor was sealed and polymerized at 120 ° C. for 10 hours.

30 g of the obtained mPEG-PDLLA was placed in a 1-neck flask and melted at 120 ° C. Lactide was removed for 7 hours by sublimation at 1 torr or less by connecting with a vacuum pump while stirring the magnetic bar (existing polymer).

Alternatively, 20 g of the obtained mPEG-PDLLA was placed in a 1-neck flask, and 120 ml of ethanol was added and dissolved at 50 ° C. for 2 hours. The polymer solution was transferred to a separatory funnel and layered at 25 ° C. for 2 hours. After layer separation was completed, the lower polymer solution was placed in a 1-neck flask. Ethanol remaining in the polymer solution was removed by vacuum distillation using a vacuum distiller (purified polymer).

Paclitaxel polymer micelle preparation was prepared using the existing polymer and the purified polymer obtained as described above, and the release test was performed on the prepared formulation. The results are shown in Table 14.

TABLE 14

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is shown by the following claims rather than the above description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

Claims (16)

1) preparing a standard solution by adding an organic solution of a sequentially diluted poorly water-soluble drug to an albumin-containing aqueous medium, and removing the poorly water-soluble drug and albumin precipitated therefrom;

2) preparing a release solution by adding an aqueous solution of the polymer micelle formulation including a poorly water-soluble drug to an albumin-containing aqueous medium, and then removing the poorly water-soluble drug and albumin precipitated therefrom to prepare a sample solution;

3) analyzing the standard solution of step 1) and the sample solution of step 2) by high performance liquid chromatography (HPLC), respectively; And,

4) calculating the release rate (%) of poorly water-soluble drugs by the following equation (1)

Including,

The polymer micelle formulation is a poorly water-soluble drug is encapsulated in a polymer micelle formed from an amphiphilic block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B),

In vitro release test method for polymer micelle formulations containing poorly water soluble drugs:

[Equation 1]

Release rate of poorly water-soluble drugs (%) = Precipitation rate of poorly water-soluble drugs (%) = (Water-soluble drug concentration of the initial sample-water-soluble drug concentration of the sample at time t) / Water-soluble drug concentration of the initial sample × 100 = ( A ₀ -A _t ) / A ₀ × 100

A ₀ = peak area of poorly soluble drug in the initial sample solution / peak area of poorly soluble drug in the standard solution × poorly soluble drug concentration in the standard solution × dilution factor × volume of the release solution

A _t = peak area of poorly soluble drug in sample solution / peak area of poorly soluble drug in standard solution × poorly soluble drug concentration in standard solution × dilution factor × volume of release solution.

The process of claim 1 wherein the albumin-containing aqueous medium of steps 1) and 2) is distilled water or a buffer containing albumin at a concentration of greater than 0 and up to 20%.

The method of claim 1, wherein the poorly water-soluble drug precipitated in steps 1) and 2) is removed by filtration.

The method of claim 3, wherein the filtration is performed using a filter having a pore size of 0.8 μm or less.

The method of claim 1, wherein in step 1) and step 2) albumin is precipitated by addition of an organic solvent and removed by centrifugation.

The method of claim 1 wherein the initial poorly water-soluble drug concentration in the release solution of step 2) is at least 0.5 mg / ml.

The process of claim 1 wherein the high performance liquid chromatography in step 3) uses the conditions of (a) and (b):

(a) a pentafluorophenyl porous particle stationary phase having a particle size of 4 μm or less; And

(b) a column having an inner diameter of 5 mm or less and a length of 50 mm or more.

The method of claim 1, wherein the aqueous solution of the polymer micelle formulation comprising the poorly water-soluble drug of step 2) is a reconstituted lyophilized formulation into an aqueous solution.

The hydrophilic block (A) according to claim 1, wherein the hydrophilic block (A) is selected from the group consisting of polyethylene glycol, monomethoxy polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol and polyacrylamide, and the hydrophobic block (B) is polylactide , Polyglycolide, polydioxan-2-one, polycaprolactone, polylactic-co-glycolide, polylactic-co-caprolactone, polylactic-co-dioxan-2-one and polyglycolic-co-capro Lactone, and its carboxylic acid terminus is selected from the group consisting of derivatives thereof substituted with fatty acid groups.

The method of claim 1, wherein the poorly water-soluble drug is an anticancer agent.

The method of claim 10, wherein the anticancer agent is a taxane anticancer agent.

Investigating the release pattern of the poorly water-soluble drug according to the method according to any one of claims 1 to 11;

Evaluating the quality or efficacy of the polymer micelle formulation from the irradiated release pattern:

Including,

A method for evaluating a polymer micelle formulation comprising a poorly water-soluble drug, wherein the poorly water-soluble drug is enclosed in a polymer micelle formed from an amphiphilic block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B).

According to any one of claims 1 to 11 characterized in that the poorly water-soluble drug release rate of the test is more than 90% in 3 hours, wherein the poorly water-soluble drug is a hydrophilic block (A) and a hydrophobic block (B) A polymer micelle composition comprising a poorly water-soluble drug, which is enclosed in a polymer micelle formed from an amphiphilic block copolymer.

The composition of claim 13, wherein the poorly water-soluble drug release rate is at least 10% in one hour and at least 90% in three hours.

The composition according to claim 14, wherein the poorly water-soluble drug release rate is 10% or more and 30% or less in one hour, and 90% or more in three hours.

The composition according to claim 15, wherein the poorly water-soluble drug release rate is 10% or more and 30% or less in 1 hour, more than 30% and 70% or less in 1.25 hours, and 90% or more in 3 hours.

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