KR20220082975A - Method for preparing polymeric micelle nanoparticle capable of reducing reconstitution time - Google Patents

Abstract

The present invention relates to a method for preparing polymeric micellar nanoparticles, and more specifically, by preparing drug-containing nanoparticles under specific conditions using an amphiphilic block copolymer having a specific level of low molecular weight, physiological saline, etc. The present invention relates to a method for preparing polymeric micellar nanoparticles capable of providing a formulation that is rapidly reconstructed in an aqueous medium, has excellent stability and uniformity, and has a reduced content of related substances.

Description

Method for preparing polymeric micelle nanoparticles capable of reducing reconstitution time

The present invention relates to a method for preparing polymeric micellar nanoparticles, and more specifically, by preparing drug-containing nanoparticles under specific conditions using an amphiphilic block copolymer having a specific level of low molecular weight, physiological saline, etc. The present invention relates to a method for preparing polymeric micellar nanoparticles capable of providing a formulation that is rapidly reconstructed in an aqueous medium, has excellent stability and uniformity, and has a reduced content of related substances.

For a bioactive agent to achieve the desired therapeutic effect, an appropriate amount of the administered drug must be delivered into the body's target cells. To this end, a submicronic particulate drug delivery system using a biodegradable polymer is being studied, and representatively, a nanoparticle system and a polymeric micelle system using a biodegradable polymer, It has been reported as a technology to reduce side effects and improve efficacy by changing the body distribution of intravenously administered drugs. It is reported that this drug delivery system can control the release of a drug to a target organ, tissue or cell, has excellent biocompatibility, and improves the solubilization ability of a poorly soluble drug and the bioavailability of the drug.

Currently, the polymer mainly used for the production of polymer nanoparticles or polymer micelles is a double block amphiphilic block copolymer (mPEG-PLA) composed of a hydrophilic block such as monomethoxypolyethylene glycol (mPEG) and a hydrophobic block such as polylactic acid (PLA). As a result, since this polymer has a property of being hydrolyzed in aqueous solution, the final product form is provided as a freeze-dried powder or cake. It should be re-dissolved in distilled water immediately before use after storage. However, these polymers have a disadvantage in that it takes a long time to rebuild when the molecular weight is increased.

For example, docetaxel-containing formulations are unstable in aqueous solution, so the drug-polymer cake produced after freeze-drying is reconstructed in physiological saline at the time of use. In this process, the drug-polymer cake dissolves well in water. Reconstruction time of the drug is prolonged as the drug does not dissolve or unravel, which causes inconvenience during dispensing in actual clinical situations, and of course, the reproducibility of the drug effect is lowered because the cake injected into the reconstruction does not melt or unravel.

In addition, if the molecular weight of the polymer is reduced to reduce the reconstruction time, precipitation occurs during the manufacturing process, or the stability of nanoparticles containing the polymer is reduced to precipitate during storage or transportation, or achieve sufficient bioavailability when administered to a patient There is a risk that the quality of the product will be significantly lowered, such as difficult to do.

Therefore, there is a need to develop a nanoparticle drug containing a low molecular weight polymer that can reduce the reconstruction time without affecting the stability of the nanoparticles.

An object of the present invention is to provide a method for preparing polymeric micellar nanoparticles capable of providing a formulation that is rapidly reconstructed in an aqueous medium such as physiological saline, has excellent stability and uniformity, and has a reduced content of related substances will be.

One aspect of the present invention, (1) mixing the active ingredient, an amphiphilic block copolymer having a number average molecular weight of 3,700 or less and a C ₁ to C ₅ alcohol; (2) removing C ₁ to C ₅ alcohol from the resultant of step (1); (3) adding the resultant of step (2) to an aqueous medium, and forming micelles at 20° C. or less; and (4) freeze-drying the resultant of step (3).

According to the present invention, a uniform drug-containing freeze-dried formulation with a shortened reconstruction time can be prepared, and when it is used in actual clinical practice, a uniform drug-containing polymer micelle solution can be quickly obtained, so that it can be easily prepared for use. , the uniform drug-containing polymer micelle solution prepared in this way can achieve reproducible and reduced side effects when administered to a patient.

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

The method for preparing the polymer micelle nanoparticles of the present invention comprises the steps of: (1) mixing an active ingredient, an amphiphilic block copolymer having a number average molecular weight of 3,700 or less, and a C ₁ to C ₅ alcohol; (2) removing C ₁ to C ₅ alcohol from the resultant of step (1); (3) adding the resultant of step (2) to an aqueous medium, and forming micelles at 20° C. or less; and (4) freeze-drying the resultant of step (3).

In one embodiment, the “nanoparticles” may have a size (eg, particle size) of sub-microns, ie, less than 1 micrometer (eg, 10-900 nm, or 10-500 nm).

The "amphiphilic block copolymer" is capable of forming micelles in an aqueous solution, and may be used in the same sense as 'micelle polymer'. The amphiphilic block copolymer may be a type A-B double block copolymer or a B-A-B type triple block copolymer composed of a hydrophilic block (A) and a hydrophobic block (B).

In one embodiment, the content of the hydrophilic block of the amphiphilic block copolymer may be 20 to 95 wt%, more specifically 40 to 95 wt%, based on 100 wt% of the total copolymer. In addition, the content of the hydrophobic block of the amphiphilic block copolymer may be 5 to 80% by weight, more specifically 5 to 60% by weight, based on 100% by weight of the total copolymer.

The hydrophilic block is a polymer having biocompatibility, specifically polyethylene glycol or a derivative thereof, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, and a combination thereof. As may include one selected from the group consisting of polyethylene glycol, monomethoxypolyethylene glycol, and combinations thereof.

The hydrophobic block is a biodegradable polymer, and may be a polymer of an alpha (α)-hydroxy acid-derived monomer, specifically polylactide, polyglycolide, poly(lactic-glycolide), polymandelic acid, Polycaprolactone, polydioxan-2-one, polyamino acids, polyorthoesters, polyanhydrides, polycarbonates, and may include those selected from the group consisting of combinations thereof, more specifically polylactide, It may include one selected from the group consisting of polyglycolide, poly(lactic-glycolide), and combinations thereof.

In the present invention, the number average molecular weight (unit: g/mol) of the amphiphilic block copolymer is 3,700 or less. When the number average molecular weight of the amphiphilic block copolymer exceeds 3,700, the reconstruction time of the polymer micelle nanoparticles prepared using the same increases, and the content of related substances increases as the micellization temperature increases.

In one embodiment, the number average molecular weight of the amphiphilic block copolymer may be 3,700 or less, 3,600 or less, 3,550 or less, 3,500 or less, 3,450 or less, or 3,400 or less. The lower limit of the number average molecular weight of the amphiphilic block copolymer is not particularly limited, and may be, for example, 1,500 or more, 2,000 or more, 2,500 or more, 3,000 or more, or 3,300 or more, but is not limited thereto. If it exceeds the number average molecular weight range, the reconstruction time may increase, and if it is less than the molecular weight range, the ability to encapsulate the drug may decrease and precipitation may be accelerated.

In one embodiment, the C ₁ to C ₅ alcohol may be methanol, ethanol, isopropanol, or a mixture thereof. The alcohol may be in the form of an aqueous solution dissolved in water, or may be a mixture with an organic solvent such as acetone, tetrahydrofuran, acetic acid, acetonitrile, dioxane, and combinations thereof.

In one embodiment, the mixing of the active ingredient, the amphiphilic block copolymer and the alcohol in step (1) may be performed at a temperature of 40 ° C. to 70 ° C. for 1 hour to 6 hours.

In one embodiment, the removal of the C ₁ to C ₅ alcohol in step (2) may be performed at a temperature of 30° C. to less than 50° C. for a time of 2 hours or less.

More specifically, the removal of the alcohol may be performed at a temperature of 30°C to 45°C, or 32°C to 42°C, for a time of 30 minutes to 100 minutes, 40 minutes to 80 minutes, or 50 minutes to 70 minutes. . If the alcohol removal temperature is too high, there is a fear that related substances increase, and if it is too low, it may be difficult to sufficiently remove the alcohol.

In one embodiment, the C ₁ to C ₅ alcohol content in the resulting mixture (ie, the mixture before micelleation) from which the alcohol is removed may be 2.5 w/v% or less (ie, 0 to 2.5 w/v%), More specifically, it may be 2.0 w/v% or less, 1.5 w/v% or less, or 1.0 w/v% or less. If too much alcohol remains in the mixture before micelle formation, a phenomenon in which drugs or polymers are precipitated may occur.

In one embodiment, the aqueous medium used in step (3) may be selected from the group consisting of, for example, ordinary water, distilled water, distilled water for injection, physiological saline, 5% glucose, buffer, and combinations thereof. However, the present invention is not limited thereto.

The formation of micelles in step (3) is performed at a temperature of 20° C. or less. When the micellar formation temperature is higher than 20° C., the drug is rapidly precipitated upon reconstruction of the prepared polymeric micellar nanoparticles, thereby significantly reducing the stability of the formulation.

In one embodiment, the micellar formation temperature is 20 °C or less, 19 °C or less, 18 °C or less, 17 °C or less, 16 °C or less, 15 °C or less, 14 °C or less, 13 °C or less, 12 °C or less, 11 °C or less, or It may be 10° C. or less. The lower limit of the micelle formation temperature is not particularly limited, and may be, for example, 1°C or more, 2°C or more, 3°C or more, 4°C or more, or 5°C or more, but is not limited thereto.

Also, in one embodiment, the formation of micelles in step (3) may be performed for 2 hours or more, 2.5 hours or more, 3 hours or more, or 3.5 hours or more. The upper limit of the micelle formation time is not particularly limited, and may be, for example, 8 hours or less, 7.5 hours or less, 7 hours or less, 6.5 hours or less, 5 hours or less, or 4 hours or less, but is not limited thereto. If the micellar formation time exceeds the above range, there is a possibility that drugs and polymers are decomposed in the aqueous phase, and if the time is less than the above range, the micelles may not be sufficiently formed because the temperature is limited.

In one embodiment, the forming of the micelles may include filtering the formed micelles.

In one embodiment, the filtration may be performed at 0 °C to 25 °C, 0 °C to 20 °C, or 0 °C to 15 °C using a 0.1 to 0.8 µm, 0.1 to 0.6 µm, or 0.2 to 0.5 µm filter. . The filtration may be repeated several times additionally according to each step or successively under different temperature conditions using different filters if necessary.

In one embodiment, the formed micelles (or micelles filtered after formation) may be maintained at 0° C. to 15° C. before subsequent freeze-drying, and may be freeze-dried within 3 hours. More specifically, the formed micelles may be maintained at 0° C. to 12° C., or 0° C. to 10° C. until freeze-drying, and within 10 minutes to 3 hours, within 30 minutes to 3 hours, or within 1 hour to 3 hours. It can be applied to the freeze-drying step.

In another embodiment, the formed micelles can be directly subjected to a freeze-drying step without storage. The above “maintenance” may be used in the same meaning as storage.

In a preferred embodiment, the freeze-drying in step (4) may be performed in the presence of a freeze-drying auxiliary (also referred to as a freeze-drying agent).

In one embodiment, the freeze-drying aid may be selected from the group consisting of sugars, sugar alcohols, and mixtures thereof. The sugar may be at least one selected from the group consisting of lactose, maltose, sucrose and trehalose, and the sugar alcohol may be at least one selected from the group consisting of mannitol, sorbitol, maltitol, xylitol and lactitol, preferably may be mannitol.

The freeze-drying adjuvant is added to allow the freeze-dried composition to retain its cake form. In addition, the freeze-drying adjuvant, after freeze-drying of the polymer nanoparticle composition, serves to help the polymer nanoparticle composition to be uniformly dissolved within a short time in the process of reconstitution. In one embodiment, the content of the freeze-drying adjuvant may be 1 to 20 parts by weight, 2 to 18 parts by weight, 3 to 15 parts by weight, or 5 to 10 parts by weight based on 1 part by weight of the active ingredient.

In a preferred embodiment, after the freeze-drying in step (4), secondary drying for removing residual moisture in the freeze-dried product may be additionally performed.

In one embodiment, the secondary drying may be performed following freeze-drying in a freeze-drying apparatus in which freeze-drying in step (4) is performed, or alternatively, freeze-drying in step (4) is After performing, the freeze-dried product may be transferred to a separate device and performed.

In a preferred embodiment, the secondary drying may be performed at a temperature of 31° C. or less. If the secondary drying temperature is excessively higher than the above level, the polymer melts and becomes sticky in the prepared cake, thereby prolonging the rebuilding time of the polymer micelle nanoparticles. There is no particular limitation on the lower limit of the secondary drying temperature, and for example, it may be carried out at a temperature of 10 °C or higher, 15 °C or higher, 18 °C or higher, or 21 °C or higher, but is not limited thereto. In the secondary drying, the moisture content of the polymer micelle nanoparticles may be 5,000 ppm or less, 4,000 ppm or less, 3,000 ppm or less, 2,000 ppm or less, or 1,000 ppm or less, and the lower limit is 300 ppm or more, 400 ppm or more, or 500 ppm or more. It may be performed as much as possible.

In one embodiment, the active ingredient may be a poorly water-soluble drug.

In one embodiment, the poorly water-soluble drug may be selected from drugs having a solubility in water (25° C. of 100 mg/mL or less). In addition, antineoplastic agents, antifungal agents, immunosuppressants, anesthetics (analgesics), anti-inflammatory agents, antiviral agents, anxiolytic sedatives, contrasting agents, corticosteroids, diagnostic agents, diagnostic imaging agents agents), diuretics, prostaglandins, radiopharmaceuticals, sex hormones including steroids, and combinations thereof, but is not limited thereto.

In one embodiment, the poorly water-soluble drug may be selected from anticancer drugs, specifically, may be a taxane anticancer drug. The taxane anticancer agent is, for example, paclitaxel (paclitaxel), docetaxel (docetaxel), 7-epipaclitaxel (7-epipaclitaxel), t- acetyl paclitaxel (tacetylpaclitaxel), 10-desacetyl paclitaxel (10-desacetylpaclitaxel), 10- Desacetyl-7-epipaclitaxel (10-desacetyl-7-epipaclitaxel), 7-xylosylpaclitaxel (7-xylosylpaclitaxel), 10-desacetyl-7-glutarylpaclitaxel (10-desacetyl-7-glutarylpaclitaxel), It may be at least one selected from the group consisting of 7-N,N-dimethylglycylpaclitaxel, 7-L-alanylpaclitaxel, and cabazitaxel, and more Specifically, it may be paclitaxel, docetaxel, or a combination thereof.

In one embodiment, the nanoparticles prepared according to the present invention may have a rebuild time in an aqueous solution within 5 minutes, within 4 minutes, or within 3 minutes. The reconstruction time may be measured by a commonly known method, for example, by stirring the nanoparticles as a freeze-dried product in an aqueous solution at 400 to 600 rpm at room temperature to measure the time until a transparent solution is obtained.

In one embodiment, the nanoparticles prepared according to the present invention may have a drug precipitation time of 4 hours or more, 6 hours or more, 8 hours or more, or 10 hours or more after making the preparation solution. The precipitation time can be measured by a commonly known method, for example, the time until the solution becomes turbid or a precipitate is observed when the nanoparticles are dissolved in an aqueous solution to make a transparent solution and then left at room temperature. have.

Hereinafter, the present invention will be described in more detail based on the following examples, but these are only for illustrating the present invention, and the scope of the present invention is not limited in any way by these.

[Example]

Example 1 and Comparative Examples 1 and 2: Preparation of polymer micelles containing docetaxel 1

32 g of monomethoxypolyethylene glycol-(poly-D,L-lactic acid) copolymer (mPEG-PDLLA) was added to 10 g of ethanol (EtOH) in a round-bottom flask, and stirred until completely dissolved at 60° C. to become a transparent, viscous solution. did While stirring the polymer solution, 1.7 g of docetaxel was quantified in a Falcon tube, and 12 g of ethanol (EtOH) was added thereto, followed by stirring with a roller mixer until completely dissolved into a transparent solution. Then, the docetaxel solution was added to the round bottom flask containing the polymer solution, and then the ethanol was distilled off under reduced pressure at 35° C. for 1 hour using a mounted rotary vacuum distiller. Thereafter, under the following micellarization conditions, it was reacted until a blue-colored transparent solution was formed to form polymer micelles.

As a freeze-drying agent, 8.5 g of mannitol was completely dissolved in physiological saline and refrigerated, and then mixed with the resulting polymer micelles. Thereafter, the resultant was filtered using a filter having a pore size of 0.22 μm, and then lyophilized to prepare a polymer micelle composition containing docetaxel in powder form. In the prepared micellar composition, the docetaxel content per vial was 80 mg, the mPEG-PDLLA content was 1529 mg, and the mannitol content was 400 mg.

[Table 1]

Examples 2 and 3 and Comparative Example 3: Preparation of polymer micelles containing docetaxel 2

The freeze-dried product prepared in the same manner as in Example 1 was subjected to secondary drying under the conditions shown in Table 2 below to prepare a polymer micelle composition containing docetaxel in powder form.

[Table 2]

Example 4: Preparation of polymer micelles containing docetaxel 3

It was prepared in the same manner as in Example 1, except that the amount of mannitol per vial was doubled (800 mg) to prepare a polymeric micellar composition containing docetaxel in powder form.

Test Example 1: Reconstruction time and precipitation time comparison experiment

38 mL of physiological saline injection was added to 2 g of each of the polymer micelles containing docetaxel of Examples 1 to 4 and Comparative Examples 1 to 2, and while stirring at a stirring speed of 500 ± 50 rpm using an IKA VIBRAX stirrer, the freeze-dried cake was completely The dissolution time (reconstruction time) was measured. The results are shown in Table 3 below.

In addition, 38 mL of physiological saline injection was added to 2 g of each of the polymer micelles containing docetaxel of Examples 1 to 4 and Comparative Examples 1 and 2, dissolved using an IKA VIBRAX stirrer, and left to stand, and the precipitation of docetaxel was checked at 30 minute intervals. observed. The results are shown in Table 3 below.

[Table 3]

Test Example 2: Comparative experiment on the content of related substances

A sample solution was prepared by mixing 1 mL of a filtrate obtained by filtering the docetaxel-containing polymeric micelles of Examples 1 to 4 and Comparative Examples 1 to 3 with a 0.45 μm membrane filter paper with 4 mL of a HPLC mobile phase solution. The prepared sample solution was analyzed by HPLC under the following conditions to compare the content of related substances, and the content (%) of the related substances is shown in Table 4 below.

HPLC conditions:

- Column: A stainless column with a length of 250 mm and an inner diameter of 4.6 mm coated with C18 on silica gel particles with a particle diameter of 5 μm and a pore diameter of 100 Å.

- Mobile phase: acetonitrile/methanol/water mixture (26/32/42, v/v/v)

- Flow rate: 1.5 milliliters/min

- Detector: UV absorbance spectrometer (measurement wavelength 232nm)

[Table 4]

As can be seen from the test results, the drug-containing nanoparticles of Examples prepared according to the present invention are rapidly rebuilt in an aqueous medium such as physiological saline, compared to the nanoparticles of Comparative Example, and at the same time have excellent stability and uniformity, , a formulation with a reduced content of related substances was provided.

Claims (7)

(1) mixing an active ingredient, an amphiphilic block copolymer having a number average molecular weight of 3,700 or less, and a C ₁ to C ₅ alcohol;
(2) removing C ₁ to C ₅ alcohol from the resultant of step (1);
(3) adding the resultant of step (2) to an aqueous medium, and forming micelles at 20° C. or less; and
(4) freeze-drying the resultant of step (3); including,
A method for preparing polymeric micellar nanoparticles. The method of claim 1, wherein the freeze-drying in step (4) is performed in the presence of a freeze-drying aid. The method of claim 2, wherein the freeze-drying aid is selected from the group consisting of sugars, sugar alcohols, and mixtures thereof. According to claim 1, After the freeze-drying in step (4), secondary drying for removing residual moisture in the freeze-dried material is additionally performed, the method for producing polymeric micellar nanoparticles. The method of claim 4, wherein the secondary drying is performed at a temperature of 31° C. or less. The method according to any one of claims 1 to 5, wherein the amphiphilic block copolymer is selected from the group consisting of polyethylene glycol or a derivative thereof, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, and combinations thereof. hydrophilic block (A); and polylactide, polyglycolide, poly(lactic-glycolide), polymandelic acid, polycaprolactone, polydioxan-2-one, polyamino acid, polyorthoester, polyanhydride, polycarbonate and A method for producing polymeric micellar nanoparticles comprising a hydrophobic block (B) selected from the group consisting of combinations thereof. The method of any one of claims 1 to 5, wherein the active ingredient is a poorly water-soluble drug.