KR101906321B1 - Method of in vitro release test and evaluation for polymeric micelle formulation containing pooly water soluble drug - Google Patents

Abstract

The present invention relates to a test method for in vitro release test of a polymeric micelle preparation containing a water-soluble drug, a method for evaluating a polymeric micelle preparation containing a water-soluble drug, and a polymeric micelle composition comprising a water-soluble drug.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a polymeric micelle preparation containing a water-soluble drug,

The present invention relates to a test method for in vitro release test of a polymeric micelle preparation containing a water-soluble drug, a method for evaluating a polymeric micelle preparation containing a water-soluble drug, and a polymeric micelle composition comprising a water-soluble drug.

The release test method for water-soluble drugs should have a biorelevant and measure the drug release in vitro ( in vitro , in vitro ) by the same mechanism as drug release from the in vivo ( in vivo ) dosage form . This test method should also be able to distinguish the differences between "good" batches and "bad" batches, which means that they should be able to detect changes in the product that could affect drug release.

Much attention has been paid to the development of suitable devices for measuring in vitro release from micelles. At this time, additional considerations such as selection and agitation of the release medium can not be overlooked. In contrast to the release medium of the oral dosage form, which typically mimics the pH of the gastrointestinal tract, the selection of the release medium for the micelle dosage form depends on the site of administration as well as the site of administration of the formulation, . Generally, the release medium is selected based on the solubility and stability of the drug, the sensitivity of the assay and the method used. Although the maintenance of the sink condition is desirable, the non-sink condition has been adopted. Agitation is also an important process condition that is frequently used to prevent agglomeration of the dispersed phase in in vitro release studies. However, the dynamic conditions generated by stirring depend on the apparatus used. Moreover, the sampling and buffer replacement (full or partial) technique is also based on the type of in vitro method used.

There is therefore a need for a method for in vitro release testing or quality assessment of a water miscible drug from a novel micelle formulation 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 a poorly water soluble drug capable of reflecting the in vivo release mechanism as well as the quality of the drug product.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

A first aspect of the present invention is a method for preparing a standard solution, comprising the steps of: 1) adding an organic solution of a serially diluted poorly water-soluble drug to an albumin-containing aqueous medium, removing the precipitated drug and albumin precipitated therefrom; 2) adding an aqueous solution of a polymeric micelle preparation containing a water-insoluble drug to an albumin-containing aqueous medium to prepare a release solution, removing the water-soluble drug and albumin precipitated therefrom, and preparing a sample solution; 3) analyzing the standard solution of step 1) and the test solution of step 2) by high performance liquid chromatography (HPLC) respectively; And (4) obtaining a release rate (%) of the water-soluble drug by the following equation (1), wherein the water-miscible drug is an amphipathic drug comprising a hydrophilic block (A) and a hydrophobic block Wherein the polymeric micelle is encapsulated in a polymeric micelle formed from a block copolymer. The present invention also provides a method for in vitro release test of a polymeric micelle preparation comprising a water-miscible drug.

[Equation 1]

(%) = The sedimentation rate of the water-soluble drug (%) = (the water-soluble drug concentration of the initial sample solution - the water-insoluble drug concentration of the sample solution at the time t) / the poorly soluble drug concentration of the initial sample × 100 = A ₀ - A _t ) / A ₀ × 100

A ₀ = Peak solubility of the initial sample solution Peak area / Water solubility of the standard solution Peak area × Water solubility drug concentration of standard solution × Dilution times × Volume of release solution

A _t = Peak solubility of the sample solution at time t Peak area of the drug / Peak solubility of the standard solution Peak area × Water solubility drug concentration of the standard solution × Dilution factor × Volume of the release solution

A second aspect of the present invention provides a method for the preparation of a medicament comprising the steps of: examining the release pattern of a poorly soluble drug according to the method according to the first aspect of the invention; Evaluating the quality or efficacy of the polymeric micelle preparation from the irradiated pattern, wherein the poorly soluble drug is encapsulated in a polymeric micelle formed from an amphiphilic block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B) A method for evaluating a polymeric micelle preparation containing a poorly water soluble drug.

The third aspect of the present invention is characterized in that the water-soluble drug release rate in the test is 90% or more in 3 hours according to the method according to the first aspect of the present invention, wherein the water-insoluble drug is a hydrophilic block (A) Soluble polymer contained in the polymeric micelle formed from the amphiphilic block copolymer containing the hydrophobic block copolymer (B).

The above-described task solution is merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary implementations described above, there may be additional implementations and embodiments described in the drawings and detailed description of the invention.

According to the present invention, it is possible to provide a new release test method for a polymer micelle preparation containing a water-insoluble drug. The method of the present invention can be used as an indicator of the quality and performance of polymeric micelle formulations comprising a water-insoluble drug, and can also be used to reproduce in vivo conditions and to correlate the release mechanism with an in vitro- RTI ID = 0.0 > IVIVC). ≪ / RTI >

FIG. 1 is a graph showing the concentration of paclitaxel according to shaking time in various concentrations of mPEG-PLA polymer solution.
FIG. 2 is a graph showing the concentration of paclitaxel according to shaking time in an mPEG-PLA polymer solution at 25 ° C. and 37 ° C. FIG.
FIG. 3 is a graph showing supersaturation solubility (FIG. 3 a) and intrinsic solubility (FIG. 3 b) of paclitaxel according to the concentration of polymer in distilled water and 4% albumin phosphate buffer.
4 is a graph showing the kinetic stability of polymeric micelles supersaturated with paclitaxel.
Figure 5 is a graph showing paclitaxel precipitation of gelexol pM in 4% albumin phosphate buffer at 37 占 폚.
6 is a graph showing the calibration curve of paclitaxel standard solution.
FIG. 7 is a graph showing paclitaxel release behavior of gelexol pM in a 4% albumin phosphate buffer solution at 37 ° C. using a precipitation method depending on the concentration of paclitaxel.
FIG. 8 is a graph showing the release behavior of paclitaxel in a 4% albumin phosphate buffer solution at 37 ° C. for the reproducibility confirmation test of the Nexesol pM release test method.
Figure 9 is a graph showing paclitaxel release behavior of Jenesol PS at 37 ° C in 4% albumin phosphate buffer according to various product numbers.
10 is a graph showing the release behavior of paclitaxel in a 4% albumin phosphate buffer solution at 37 ° C of a gelexol pem defect product (50: 500) and a normal product (100: 500, GP11511).
11 is a graph showing the release mechanism of paclitaxel in vivo of Genexole pM.

Hereinafter, embodiments and examples of a polymer micelle preparation containing a water-miscible drug of the present invention, a method for evaluating a polymeric micelle formulation containing a water-soluble drug, and a polymeric micelle composition containing a water-soluble drug are described. A detailed description will be made with reference to examples and drawings. However, the present invention is not limited to these embodiments and examples and drawings.

A first aspect of the present invention is a method for preparing a standard solution, comprising the steps of: 1) adding an organic solution of a serially diluted poorly water-soluble drug to an albumin-containing aqueous medium, removing the precipitated drug and albumin precipitated therefrom; 2) adding an aqueous solution of a polymeric micelle preparation containing a water-insoluble drug to an albumin-containing aqueous medium to prepare a release solution, removing the water-soluble drug and albumin precipitated therefrom, and preparing a sample solution; 3) analyzing the standard solution of step 1) and the test solution of step 2) by high performance liquid chromatography (HPLC) respectively; And (4) obtaining a release rate (%) of the water-soluble drug by the following equation (1), wherein the water-miscible drug is an amphipathic drug comprising a hydrophilic block (A) and a hydrophobic block Wherein the polymeric micelle is encapsulated in a polymeric micelle formed from a block copolymer. The present invention also provides a method for in vitro release test of a polymeric micelle preparation comprising a water-miscible drug.

[Equation 1]

(%) = The sedimentation rate of the water-soluble drug (%) = (the water-soluble drug concentration of the initial sample solution - the water-insoluble drug concentration of the sample solution at the time t) / the poorly soluble drug concentration of the initial sample × 100 = A ₀ - A _t ) / A ₀ × 100

A ₀ = Peak solubility of the initial sample solution Peak area / Water solubility of the standard solution Peak area × Water solubility drug concentration of standard solution × Dilution times × Volume of release solution

A _t = Peak solubility of the test solution at time t. Drug peak area / Peak solubility of the standard solution Peak area × Water solubility drug concentration of standard solution × Dilution factor × Volume of release solution.

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

For example, the number average molecular weight of the amphiphilic block copolymer can be from 1,000 to 50,000 daltons, and more specifically from 1,500 to 20,000 daltons, but is not limited thereto. The polymer micelle may be formed by a method commonly used in the technical field of the present invention.

For example, the organic solution may comprise an organic solvent selected from the group consisting of water-miscible organic solvents such as alcohols, acetone, tetrahydrofuran, acetic acid, acetonitrile and dioxane and combinations thereof, . The aqueous solution may be selected from the group consisting of ordinary water, distilled water, distilled water for injection, physiological saline, glucose solution (for example, 5% glucose solution), buffer solution and combinations thereof. .

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

According to one embodiment of the present invention, the albumin-containing aqueous medium of step 1) and step 2) may be, but not limited to, distilled water or buffer containing albumin at a concentration of about 0 to 20%. 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) But may not be limited.

According to one embodiment of the present invention, the water-insoluble drug precipitated in steps 1) and 2) may be removed by filtration, but not always 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 占 퐉 or less, but may not be limited thereto. For example, the filtration may be performed using a filter having a pore size of at least about 0.1 탆 but less than 0.8 탆, at least about 0.2 탆 but less than about 0.8 탆, or at least about 0.2 탆 and less than about 0.5 탆.

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

According to one embodiment of the present invention, the initial poorly soluble drug concentration in the effluent solution of step 2) may be at least about 0.5 mg / ml, for example at least 0.5 mg / ml but not more than 30 mg / ml have.

According to one embodiment of the present invention, the high performance liquid chromatography in step 3) may preferably be carried out using the following conditions (a) and (b): (a) pentafluoro Phosphorous porous particle fixed bed; 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 Laid-Open Publication No. 2017-0014057, in which case Korean Patent Publication No. 2017-0014057 is incorporated herein by reference in its entirety.

According to one embodiment of the present invention, the aqueous solution of the polymeric micelle preparation containing the poorly soluble drug of step 2) may be reconstituted with an aqueous solution, but not limited thereto.

According to one embodiment of the present 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) Polylactide, polyglycolide, polydioxan-2-one, polycaprolactone, polylactic-co-glycolide, polylactic-co-caprolactone, polylactic-co-dioxan- co-caprolactone, and derivatives thereof in which the carboxylic acid terminal thereof is substituted with a fatty acid group, but the present invention is not limited thereto.

According to one embodiment of the present invention, the poorly soluble drug may be, but not limited to, an anti-cancer agent.

According to one embodiment of the present invention, the anticancer agent may be but is not limited to taxane anticancer agent. For example, the taxane anticancer agent may be selected from the group consisting of paclitaxel, docetaxel, 7-epipaclitaxel, t-acetylpaclitaxel, 10-desacetylpaclitaxel, 10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel, 10-desacetyl-7-glutarylpaclitaxel ), 7-N, N-dimethylglycylpaclitaxel, 7-L-alanylpaclitaxel, and carbapentase. , More specifically, but not exclusively, paclitaxel.

A second aspect of the present invention provides a method for the preparation of a medicament comprising the steps of: examining the release pattern of a poorly soluble drug according to the method according to the first aspect of the invention; Evaluating the quality or efficacy of the polymeric micelle preparation from the irradiated pattern, wherein the poorly soluble drug is encapsulated in a polymeric micelle formed from an amphiphilic block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B) A method for evaluating a polymeric micelle preparation containing a poorly water soluble drug.

For example, the evaluation method of a polymeric micelle preparation containing the poorly water-soluble drug may include evaluating that the water-soluble drug release rate is 10% or more per hour and 90% or more per 3 hours However, the present invention is not limited thereto. For example, when the water-soluble drug release rate is 10% or more and 30% or less in 1 hour and 90% or more in 3 hours, it can be evaluated that the water-soluble drug polymer micelle preparation has a desirable quality, or the water- But it may be evaluated that the water-miscible drug polymer micelle preparation has a desirable quality in the case of not less than 10% and not more than 30% in the time, more than 30% and not more than 70% in 1.25 hours, and more than 90% .

The third aspect of the present invention is characterized in that the water-soluble drug release rate in the test is 90% or more in 3 hours according to the method according to the first aspect of the present invention, wherein the water-insoluble drug is a hydrophilic block (A) Soluble polymer contained in the polymeric micelle formed from the amphiphilic block copolymer containing the hydrophobic block copolymer (B).

According to an embodiment of the present invention, the release rate of the poorly water-soluble drug may be 10% or more per hour and 90% or more, for example, 90% or more but 99.5% or less in 3 hours .

According to one embodiment of the present invention, the release rate of the poorly soluble drug is not less than 10% but not more than 30% in 1 hour and not less than 90% in 3 hours, for example, not less than 90% and not more than 99.5% .

According to one embodiment of the present invention, the composition is characterized in that the 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, 90% or more in 90 hours, % Or more and 99.5% or less, but may not be limited thereto.

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

[Example]

Drug product

Genexol-PM for injection administration is a formulation using a low molecular weight, amphiphilic two-block copolymer for forming macromolecular micelles for dissolving paclitaxel. Each of the 100 mg Gelnexol PE vials for injection administration contains the ingredients shown in Table 1 below.

In vitro in vitro ) Emission test method

1. Reagents and Materials

Paclitaxel Standard: Lot G479 (Samyang Bio-Palm)

- mPEG-PDLLA: lot PM1403 (Samyang Bio Farm)

- Genexol P: Product number (Lot) GP11511, GP114A1, GP114B1, GP114C1 (Samyang Biofarm)

- 20% human serum albumin 100 mL: Lot 161D14240 (Green Cross)

- 0.9% physiological saline (CJ CheilJedang)

- Acetonitrile (Burdick & Jackson. ACS / HPLC Grade)

2. Equipment and equipment

- Shandong constant water bath: Maxturdy 30, DAIHAN Scientific.

- Shaking incubator: Vision Sci. Co.

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

- Centrifuge: Mini spin plus, Eppendorf.

3. Determination of solubility of paclitaxel in copolymer solution

1) Solubility of paclitaxel in distilled water containing mPEG-PDLLA copolymer

- Apparatus: Shaking 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 and dissolved (100.0 mg / mL). This copolymer aqueous solution was diluted with distilled water in order to obtain concentrations of 2.5, 5, 10, 25, 50 and 100 mg / mL.

Addition of paclitaxel to the copolymer solution

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

- Sampling

1 mL of the suspension was taken at each sampling time (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 24 hours) and filtered with a 0.45 ㎛ 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 equation: a = (peak area of paclitaxel - 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 [mu] g / mL)

[HPLC]

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

Detector: UV (? = 227 nm)

- Flow rate: 0.6 mL / min

- Injection volume: 10 μl

- Column temperature: 25 ° C

- Mobile:

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

25-28 min: water / acetonitrile = 45/55 (v / v)

Water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

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

2) Solubility of paclitaxel in 4% albumin phosphate buffer containing mPEG-PDLLA copolymer

- Apparatus: Shaking incubator: Vision Sci. Co.

- Medium: 4% albumin phosphate buffer

Preparation of copolymer solution

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

Addition of paclitaxel to the copolymer solution

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

- Sampling

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

- Preparation of standard solution

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

≪ Preparation of paclitaxel standard solution >

- analysis

HPLC was used to analyze the concentration of paclitaxel. The concentration of paclitaxel was calculated using the following equation: a = (peak area of paclitaxel - 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 [mu] g / mL)

4. Paclitaxel release test of Genexol PE in albumin solution

- Apparatus: Shundeo constant water 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 mg of physiological saline was added to the gelnexol pM 100 mg vial and reconstituted to prepare a 5.0 mg / mL solution. 4 mL of the gastric juice was taken and placed in 16 mL of 4% albumin phosphate buffer at 37 ° C. This solution was stirred at 37 ° C and 200 rpm.

- Sampling

1 mL of the gastric juice was filtered through a 0.45 μm PVDF syringe filter at each sampling time (0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, Respectively. Then, 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, and 0.5 mL of the supernatant was taken and mixed with 0.5 mL of 80% acetonitrile aqueous solution. This solution was used as a sample solution for HPLC analysis.

- Preparation of standard solution

3. Prepare the same method as in 2).

- analysis

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

5. Jennsol  Experiment of emission of pM

- Preparation of Emissive Solution

20 mg of physiological saline was added to the gelnexol pM 100 mg vial and reconstituted to prepare a 5.0 mg / mL solution. 4 mL of this solution was taken and placed in 16 mL of 4% albumin phosphate buffer at 37 占 폚. This solution was stirred at 37 ° C and 200 rpm, and this solution was used as a release solution.

- Release

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

- Sampling

1 mL of the gastric juice was filtered through a 0.45 μm PVDF syringe filter at each sampling time (0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, Respectively. Then, 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, and 0.5 mL of the supernatant was taken and mixed with 0.5 mL of 80% acetonitrile aqueous solution. This solution was used as a sample solution for HPLC analysis.

- HPLC

1) Preparation of standard solution

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

2) Preparation of initial test liquid

Jenesolex PE 100 mg vials were reconstituted with 20 mL of physiological saline. 4 mL was taken and placed in 16 mL of 4% albumin phosphate buffer at 37 占 폚. 1 mL of the solution was taken and filtered through a 0.45 탆 PVDF filter. 0.2 mL of the filtrate was taken and 0.8 mL of acetonitrile was added to precipitate albumin. This solution was centrifuged at 14,000 rpm for 5 minutes and 0.5 mL of the supernatant was mixed with 0.5 mL of 80% acetonitrile aqueous solution. This solution was used as the initial solution.

The standard solution and the test solution were injected into the HPLC under the following conditions. The release of paclitaxel was calculated as the peak area of the paclitaxel of the test solution and the standard solution.

[HPLC]

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

Detector: UV (? = 227 nm)

- Flow rate: 0.6 mL / min

- Injection volume: 10 μl

- Column temperature: 25 ° C

- mobile phase

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

25-28 min: water / acetonitrile = 45/55 (v / v)

Water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

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

- Calculation

Paclitaxel concentration (%) = precipitated paclitaxel (%) = (paclitaxel concentration of the initial test solution - paclitaxel concentration of the test sample at each sampling time (t)) x 100 = (A0-At) / A0 x 100

- A0 = peak area of paclitaxel in initial test solution / peak area of paclitaxel in standard solution x concentration of paclitaxel in standard solution x 10 x 20

- At = peak area of paclitaxel in test solution at time t / paclitaxel peak area in standard solution x concentration of paclitaxel in standard solution x 10 x 20

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

Results and Discussion

1. Solubility of paclitaxel in polymer solution

1) The concentration of paclitaxel over time in polymer solutions of various concentrations

The solubility of paclitaxel was tested in distilled water and 4% albumin phosphate buffer. When an excess of amorphous paclitaxel is added to various concentrations of polymer solution, the amorphous paclitaxel spontaneously dissolves in the polymer micelles and is supersaturated in the polymer solution for a certain period of time. Figures 1 and 3 show the concentration of paclitaxel over time in various concentrations of mPEG-PLA polymer solution. As shown in Figs. 1 and 3, the concentration of paclitaxel decreased in the supersaturation solubility after the supersaturation state was maintained, and the solubility was high. The reason why the concentration of paclitaxel falls from the supersaturation solubility to the intrinsic solubility concentration is that the amorphous paclitaxel temporarily trapped in the polymer micelle is a form of the less soluble dihydrate.

The concentration of paclitaxel over time varied with temperature. Figures 2 and 4 show the concentration of paclitaxel in the mPEG-PLA polymer over time at 25 ° C and 37 ° C. As shown in FIG. 2 and Table 4, the supersaturation solubility of paclitaxel at 37 DEG C was higher than that of paclitaxel supersaturation at 25 DEG C, and the intrinsic solubility at 37 DEG C was lower than that at 25 DEG C.

The supersaturation duration was also shorter at 37 ° C than at 25 ° C. This is because, at higher temperatures, the amorphous paclitaxel dissolves rapidly in the polymeric micelles, but can crystallize faster in the form of dihydrates.

2) Solubility of paclitaxel by polymer concentration

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

2. Paclitaxel precipitation of Jenesol PE aqueous solution

Because mPEG-PLA polymer micelles are more dynamically more stable than low molecular weight surfactant micelles, they can contain large amounts of hydrophobic drugs. In the case of low molecular weight surfactant micelles, excess drug is easily deposited upon contact with water and is therefore unsuitable for injections that must contain an excess of drug over a long period of time.

4 is a graph showing the kinetic stability of polymeric micelles supersaturated with paclitaxel. Jennexol PE contains an excess of paclitaxel in polymeric micelles, and the micellar solution in the water phase is stable at room temperature for a long time and can be conveniently used in clinical practice. However, at high temperatures such as body temperature, it becomes mechanically unstable, which means that paclitaxel can easily be released from polymer micelles at high temperatures. Paclitaxel from Genexol pM is precipitated as a 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 gelexol pM in 4% albumin phosphate buffer at 37 占 폚. Using this characteristic of Genexol PE, we were able to develop precipitation method of in vitro emission test method. The inherent 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 the nexxole pM. Actually, 0.9 mg / mL of gelexolepM in 4% albumin phosphate buffer drops to a concentration of 0.08 mg / mL after 2 hours. In fact, after 2 hours and 6 hours 90 and 97% of the paclitaxel in micelles was released and precipitated.

3. Setting of emission test method of Gnexol P

1) a bio-mimetic medium

The biological medium used is gelatin, plasma, and serum, which are not readily available in sufficient quantities for in vitro release testing, and these media have difficulty in drug analysis. Thus, a phosphate buffer solution (PBS, pH 7.4) has been used as a biomaterial-like medium, and phosphate buffer solution containing albumin was used in the in vitro release test of Genexol PE in this example.

2) Separation of paclitaxel precipitate in a bio-mimetic medium

There is a method of separating the paclitaxel precipitate from the biomaterial-like medium. One is centrifugation and the other is filtration.

In centrifugation, it is not easy to select the appropriate centrifugal force and time. Table 7 shows the amount of paclitaxel released from Jenesolepime after 24 hours in 4% albumin phosphate buffer at 37 ° C according to centrifugation time. According to Table 7, the amount of paclitaxel released increased with increasing centrifugation time. During centrifugation (over 5 minutes), the amount of paclitaxel released can be overestimated as paclitaxel continues to precipitate.

However, when using the filtration method, the filtration time is less than 1 minute, so the filtration time will not affect the experimental results. In the filtration method, the release medium containing gelnexol pM is taken at each sampling time and filtered with a PVDF syringe filter. The filter removes the precipitated paclitaxel (crystallized paclitaxel), but the paclitaxel in micelles and the albumin-bound paclitaxel remain in the filtrate. The filtrate may contain paclitaxel dissolved in pure water, but negligible. Therefore, the filtration method is more reliable than the centrifugation method.

Since various pore size filters can be used for filtration, the amount of paclitaxel released from Jenesol PS at 24 ° C in 4% albumin phosphate buffer at 37 ° C, depending on the size of the filter pores, was evaluated (Table 8). The amount of paclitaxel in the filtrate (paclitaxel with no release and negligible albumin with paclitaxel) when using a 0.22 μm filter was slightly less than when the 0.45 μm filter was used. However, the difference was negligible as can be seen in Table 8.

The 0.8 ㎛ filter, however, showed that the relatively large size of precipitated paclitaxel penetrated the filter pores and was significantly higher than the 0.45 and 0.22 ㎛ filters. Therefore, a 0.45 ㎛ filter was used for the emission test of Genexol PS.

3) Analysis of paclitaxel in a biomaterial-like medium

The validated HPLC assay was used to quantify the release of paclitaxel in the bioassembly medium. Prior to HPLC analysis, pretreatment was required to remove albumin from the release medium of Genexol PE. If albumin is not removed, HPLC analysis will damage the HPLC and column, so the albumin removal step is a necessary pretreatment for HPLC analysis. It has been reported that proteins can be denatured (precipitated) by adding an organic solvent such as acetonitrile and methanol. The precipitated albumin can be removed by centrifugation or filtration. In this embodiment, centrifugation is used. Since 80% acetonitrile dissolves both paclitaxel and mPEG-PLA polymers, polymeric micelles do not exist, while albumin-free 80% acetonitrile solution contains only paclitaxel and polymer.

Validated HPLC method

[HPLC]

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

Detector: UV (? = 227 nm)

- Flow rate: 0.6 mL / min

- Injection volume: 10 μl

- Column temperature: 25 ° C

- Mobile:

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

25-28 min: water / acetonitrile = 45/55 (v / v)

Water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

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

Calibration curves and results of the paclitaxel standard solution are shown in FIG. 6 and Table 9.

4) Initial concentration of paclitaxel in the release medium

The effect of the concentration of paclitaxel in the release medium on the release behavior of Jenesole pM to determine the concentration of the initial paclitaxel in the release medium was determined by the precipitation method. FIG. 7 is a graph showing the release behavior of Paclitaxel of Genexol pM in 4% albumin phosphate buffer at 37 占 폚. As can be seen from Fig. 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 suggests that gelnexol pem is easily dissociated from blood and that paclitaxel can be easily released from the blood. Therefore, Genexole® P appears to be released immediately after injection into the body.

And the initial low concentration of paclitaxel indicates the situation of slow-rate intravenous injection. However, the paclitaxel released from this precipitation method contains a precipitate that does not bind to 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 Jenesole P (1.0 mg / mL -> 0.1 mg / mL) results in a negligible proportion of albumin-bound paclitaxel. Therefore, the initial paclitaxel concentration of the release medium was selected to be 1.0 g / mL.

3.4. Paclitaxel release of Genexole pM in a bioequivalent medium

1) Reproducibility of emission test

Three repetitive tests were carried out to evaluate the reproducibility of the precipitation method using Jennexol PE (Lot GP11511) with the same product number. The reproducibility test results are shown in FIG. 8 and Table 10, and this method is highly reproducible.

<Genexol pM release test method Percentage of released paclitaxel over time in the reproducibility test>

<Genexol pM release test method> The concentration of paclitaxel released over time in the reproducibility test>

2) Release of Genexol PE according to various product numbers

The results of evaluating the release of Jenesole p. 4 of the product number using the precipitation method are shown in FIG. 9 and Table 11.

&Lt; Percentage of paclitaxel released by gelnexol pM in 37%, 4% albumin phosphate buffer according to various product numbers >

<Paclitaxel Emission Concentration of Jenesol PS in 37, 4% Albumin Phosphate Buffer according to Various Product Numbers>

3) Evaluation of bad products

Since the in-vitro emission method should be able to distinguish the defective product, two defective products were produced to evaluate the behavior of the defective batch by the precipitation method. The composition of the defective batch is shown in Table 12. The defective product with a ratio of 100: 300 of paclitaxel to mPEG-PLA was in an opaque form when reconstituted with physiological saline, so the release test could not be performed. The release behavior of 50: 500 ratio and 100: 500 (GP11511) is shown in FIG. 10 and Table 13.

Percentage of paclitaxel released in 4% albumin phosphate buffer at 37 ° C for the defective products (50: 500, 100: 300) and normal products (100: 500, GP11511)

<Paclitaxel Emission Concentration in 4% Albumin Phosphate Buffer at 37 ° C> of Gennexol ™ Poor Products (50: 500, 100: 300) and Normal Products (100: 500, GP11511)

4. Emission test methods and standards

4.1. Emission test method

1) Preparation of standard solution

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

2) Preparation of sample solution

20 mg of physiological saline was added to the gelnexol pM 100 mg vial and completely dissolved (5.0 mg / mL). 4 mL of this solution was added to 16 mL of 4% albumin phosphate buffer at 37 ° C, and this solution was used as a release solution. 1 mL of this effluent 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 period of 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 into 0.8 mL of acetonitrile and albumin was precipitated. 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 made into a release solution at time t.

3) Release and analysis

The aqueous solution was stirred at 37 ° C and 200 rpm.

[Release condition]

- Emission method: Precipitation method

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

- Capacity: 20 mL

- stirring speed: 200 rpm

- Emission temperature: 37.0 ± 0.5 ℃

The standard and sample solutions were injected into HPLC and the paclitaxel released was calculated using the peak area of paclitaxel according to the following conditions.

[HPLC]

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

Detector: UV (? = 227 nm)

- Flow rate: 0.6 mL / min

- Injection volume: 10 μl

- Column temperature: 25 ° C

- mobile phase

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

25-28 min: water / acetonitrile = 45/55 (v / v)

Water / acetonitrile = 45/55 (v / v)) 65/35 (v / v)

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

- Calculation

Paclitaxel concentration (%) = precipitated paclitaxel (%) = (paclitaxel concentration of the initial test solution - paclitaxel concentration of the test sample at each sampling time (t)) x 100 = (A0-At) / A0 x 100

- A0 = peak area of paclitaxel in initial test solution / peak area of paclitaxel in standard solution x concentration of paclitaxel in standard solution x 10 x 20

- At = peak area of paclitaxel in test solution at time t / paclitaxel peak area in standard solution x concentration of paclitaxel in standard solution x 10 x 20

5. Possible mechanism of paclitaxel release in vivo

Genexol PE is diluted very quickly when injected into the body and is therefore not likely to crystallize and precipitate. Instead, it binds rapidly with other proteins and is distributed throughout the body as well as in the blood. However, it is not easy to reproduce in vivo conditions diluted infinitely by the precipitation method. Therefore, the precipitation of paclitaxel in vitro was regarded as protein binding in vivo. That is, the crystallization of paclitaxel in polymeric micelles in in vitro can be regarded as the combination of paclitaxel and protein in vivo due to the presence of many proteins and blood components and rapid dilution. From the results of the in vitro release test using the precipitation method, the possible release mechanism of the release of paclitaxel from Jenesol PS is shown in FIG.

Step 1:

The supersaturated paclitaxel in the polymeric micelles is released from the polymeric micelles and binds to plasma proteins immediately. This step accounts for more than 95% of the total release.

Step 2

The paclitaxel remaining in the polymeric micelles is dissociated below the CMC of the polymeric micelles or is slowly released by decomposition by the enzymes in the blood. This step accounts for less than 5% of the total release.

6. Release test for polymeric micelle formulations containing conventional and purified polymers

150 g of monomethoxy polyethylene glycol (mPEG, number average molecular weight = 2,000) was added to a 500 ml round-bottomed flask equipped with a stirrer, and water was removed by stirring at 120 ° C and vacuum for 2 hours. 0.15 g of Stannus octoate (Sn (Oct) ₂ ) dissolved in 200 of toluene was added into the reaction flask, and toluene was distilled off while stirring under vacuum for 1 hour. Then, 150 g of D, L-lactide was added and dissolved in a nitrogen atmosphere while stirring. After the D, L-lactide was completely dissolved, the reactor was sealed and polymerization was carried out at 120 ° C for 10 hours.

30 g of the obtained mPEG-PDLLA was put into a one-necked flask and melted at 12 ° C. Lactide was removed by sublimation at less than 1 torr for 7 hours (conventional polymer).

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

The paclitaxel polymer micelle preparation was prepared using the conventional polymer and purified polymer obtained as described above, and a release test was conducted according to the present invention. The results are shown in Table 14.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description and all changes or modifications derived from the meaning and scope of the claims and their equivalents are to be construed as being included within the scope of the present invention do.

Claims (16)

1) adding an organic solution of a water-soluble drug sequentially diluted to an albumin-containing aqueous medium, removing the precipitated drug and albumin precipitated therefrom to prepare a standard solution;
2) adding an aqueous solution of a polymeric micelle preparation containing a water-insoluble drug to an albumin-containing aqueous medium to prepare a release solution, removing the water-soluble drug and albumin precipitated therefrom, and preparing a sample solution;
3) analyzing the standard solution of step 1) and the test solution of step 2) by high performance liquid chromatography (HPLC) respectively; And
4) obtaining the release rate (%) of the water-soluble drug by the following equation (1)
Lt; / RTI &gt;
Wherein the water-miscible drug is encapsulated in a polymeric micelle formed from an amphiphilic block copolymer containing a hydrophilic block (A) and a hydrophobic block (B), and the water-insoluble drug is paclitaxel or docetaxel,
In vitro release test of polymeric micelle formulations containing water-soluble drugs:
[Equation 1]
(%) = The sedimentation rate of the water-soluble drug (%) = (the water-soluble drug concentration of the initial sample solution - the water-insoluble drug concentration of the sample solution at the time t) / the poorly soluble drug concentration of the initial sample × 100 = A ₀ - A _t ) / A ₀ × 100
A ₀ = Peak solubility of the initial sample solution Peak area / Water solubility of the standard solution Peak area × Water solubility drug concentration of standard solution × Dilution times × Volume of release solution
A _t = Peak solubility of the test solution at time t. Drug peak area / Peak solubility of the standard solution Peak area × Water solubility drug concentration of standard solution × Dilution factor × Volume of release solution. The method of claim 1 wherein the albumin-containing aqueous medium of step 1) and step 2) is distilled water or buffer containing albumin at a concentration of greater than 0 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 according to claim 3, wherein the filtration is performed using a filter having a pore size of 0.8 μm or less. The method according to claim 1, wherein albumin is precipitated by adding an organic solvent in steps 1) and 2) and removed by centrifugation. The method of claim 1 wherein the initial poorly soluble drug concentration in the effluent of step 2) is at least 0.5 mg / ml. The method according to claim 1, wherein in step 3) the high performance liquid chromatography utilizes the following conditions (a) and (b):
(a) a pentafluorophenyl porous particle fixed bed having a particle size of 4 탆 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 according to claim 1, wherein the aqueous solution of the polymeric micelle formulation comprising the poorly water soluble drug of step 2) is reconstituted with an aqueous solution of the lyophilized preparation. The composition according to claim 1, wherein the hydrophilic block (A) is selected from the group consisting of polyethylene glycol, monomethoxy polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol and polyacrylamide, and the hydrophobic block (B) , Polyglycolide, polydioxan-2-one, polycaprolactone, polylactic-co-glycolide, polylactic-co-caprolactone, polylactic-co-dioxan- Lactones, and derivatives thereof in which the carboxylic acid terminal thereof is substituted with a fatty acid group. delete delete 10. A method for the preparation of a medicament for the preparation of a medicament for the preparation of a medicament for the preparation of a medicament according to claim 1,
Evaluating the quality or efficacy of the polymeric micelle formulation from the irradiated emission pattern:
Lt; / RTI &gt;
Wherein the water-insoluble drug is encapsulated in a polymeric micelle formed from an amphiphilic block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B), wherein the poorly soluble drug is a polymeric micelle comprising a poorly water soluble drug which is paclitaxel or docetaxel Evaluation method of preparation. The method according to any one of claims 1 to 9, wherein the water-soluble drug release rate is 90% or more at 3 hours, wherein the water-soluble drug is a hydrophilic block (A) and a hydrophobic block (B) Wherein the water insoluble drug is paclitaxel or docetaxel, and the water miscible drug is encapsulated in a polymeric micelle formed from an amphiphilic block copolymer containing an amphiphilic block copolymer. 14. The composition of claim 13, wherein the poorly soluble drug release rate is greater than or equal to 10% per hour and greater than or equal to 90% per three hours. The composition according to claim 14, wherein the water-soluble drug release rate is 10% or more and 30% or less at 1 hour and 90% or more at 3 hours. The composition according to claim 15, wherein the water-miscible drug release rate is 10% or more and 30% or less in 1 hour, more than 30% in 70% or less in 90 minutes, or 90% or more in 3 hours.

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