CN116670232B

CN116670232B - Preparation method of polymer micelle nano-particles capable of shortening reconstruction time

Info

Publication number: CN116670232B
Application number: CN202180081953.3A
Authority: CN
Inventors: 金东湜; 金峰五; 李在永; 赵重雄
Original assignee: Sanyang Holding Co
Current assignee: Sanyang Holding Co
Priority date: 2020-12-10
Filing date: 2021-12-09
Publication date: 2024-10-18
Anticipated expiration: 2041-12-09
Also published as: KR20220082975A; CN116670232A; WO2022124818A1; KR102688394B1

Abstract

The present invention relates to a method of preparing polymer micelle nanoparticles, and more particularly, to a method of preparing polymer micelle nanoparticles, in which drug-containing nanoparticles, which are rapidly reconstituted in an aqueous medium such as physiological saline, have excellent stability and uniformity, and are capable of providing a formulation with reduced content of related substances, are prepared under specific conditions by using amphiphilic block copolymers having a specific level of low molecular weight.

Description

Preparation method of polymer micelle nano-particles capable of shortening reconstruction time

Technical Field

The present invention relates to a method of preparing polymer micelle nanoparticles, and more particularly, to a method of preparing polymer micelle nanoparticles, in which drug-containing nanoparticles are prepared under specific conditions using amphiphilic block copolymers having a specific level of low molecular weight, so as to be rapidly reconstituted in an aqueous medium of physiological saline or the like, and have excellent stability and uniformity, and it is possible to provide a formulation having reduced content of related substances.

Background

In order for a bioactive agent to achieve the desired therapeutic effect, an appropriate amount of the administered drug must be delivered into the target cells of the body. For this reason, submicron particle drug delivery systems (submicronic particulate drug DELIVERY SYSTEM) using biodegradable polymers are being studied, and typically, nanoparticle systems (nanoparticle system) and polymer micelle systems (polymeric MICELLE SYSTEM) using biodegradable polymers have been reported as techniques for reducing side effects and improving therapeutic effects by changing the distribution of intravenously administered drugs in the body. It has been reported that such a drug delivery system can regulate the release of a drug to a target organ, tissue or cell, has excellent biocompatibility, and improves the solubilizing ability of poorly soluble drugs and the bioavailability (bioavailability) of drugs.

Currently, the polymer mainly used for preparing polymer nanoparticles or polymer micelles is a diblock amphiphilic block copolymer (mPEG-PLA) composed of a hydrophilic block such as monomethoxy polyethylene glycol (mPEG) and a hydrophobic block such as polylactic acid (PLA), which is provided in the form of a freeze-dried powder or cake (cake) as a final product due to its hydrolysis property in an aqueous solution, and thus, in order to use the polymer as a drug carrier, should be freeze-dried and stored in a solid state and re-dissolved in distilled water immediately before use. However, when the molecular weight of such a polymer increases, there is a disadvantage in that it takes a long time for the reconstitution.

For example, since a formulation containing docetaxel is unstable in the state of an aqueous solution, when a drug-polymer cake produced after freeze-drying is used, it is reconstituted in distilled water, but in this process the drug-polymer cake is not well dissolved or dispersed in water, and thus the reconstitution time of the drug becomes long, thereby causing inconvenience in formulation in actual clinical situations, and since the cake for reconstitution is not completely dissolved or dispersed, reproducibility of the drug effect is lowered.

Furthermore, when the molecular weight of the polymer is reduced to reduce the reconstitution time, there may be a risk that the quality of the product is significantly reduced, such as precipitation during the preparation, or that the stability of the nanoparticles comprising the polymer is reduced, resulting in precipitation during storage or transportation, or that it is difficult to achieve adequate bioavailability when administered to a patient, etc.

Therefore, there is a need to develop a nanoparticle drug comprising a low molecular weight polymer that can shorten the reconstitution time without affecting the stability of the nanoparticle.

Disclosure of Invention

Technical problem to be solved

An object of the present invention is to provide a method of preparing polymer micelle nanoparticles that can provide a formulation capable of rapid reconstitution in an aqueous medium such as physiological saline, having excellent stability and uniformity, and reduced content of related substances.

Technical proposal

In one aspect, the present invention provides a method of preparing a polymeric micelle nanoparticle comprising the steps of: (1) Mixing the effective component, amphiphilic block copolymer with number average molecular weight below 3700 and C ₁ -C ₅ alcohol; (2) Removing C ₁ to C ₅ alcohol from the product of step (1); (3) Adding the product of step (2) to an aqueous medium and forming micelles at a temperature below 20 ℃; and (4) freeze-drying the product of step (3).

Advantageous effects

According to the present invention, a uniform drug-containing lyophilized preparation having a shortened reconstitution time can be prepared, a uniform drug-containing polymer micelle solution can be rapidly obtained when the lyophilized preparation is used in actual clinical practice, can be easily formulated when used, and a result having reproducibility and reduced side effects can be obtained when the uniform drug-containing polymer micelle solution thus prepared is administered to a patient.

Detailed Description

The present invention will be described in more detail below.

The method for preparing the polymer micelle nano-particles comprises the following steps: (1) Mixing the effective component, amphiphilic block copolymer with number average molecular weight below 3700 and C ₁ -C ₅ alcohol; (2) Removing C ₁ to C ₅ alcohol from the product of step (1); (3) Adding the product of step (2) to an aqueous medium and forming micelles at a temperature below 20 ℃; and (4) freeze-drying the product of step (3).

In a particular embodiment, the "nanoparticle" may have a submicron size (e.g., particle size), i.e., may be less than 1 micron (e.g., 10-900nm or 10-500 nm).

The "amphiphilic block copolymer" may form micelles in an aqueous solution phase, and may be used in the same meaning as "micelle polymer". The amphiphilic block copolymer may be A type A-B diblock copolymer consisting of A hydrophilic block (A) and A hydrophobic block (B) or A type B-A-B triblock copolymer.

In a specific embodiment, the hydrophilic block may be present in the amphiphilic block copolymer in an amount of from 20 to 95% by weight, more specifically from 40 to 95% by weight, based on 100% by weight of the total copolymer. In addition, the content of the hydrophobic block in 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 with biocompatibility, and specifically may include one or more selected from polyethylene glycol or derivatives thereof, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, and combinations thereof, and more specifically may include one or more selected from polyethylene glycol, monomethoxy polyethylene glycol, and combinations thereof.

The hydrophobic block is a biodegradable polymer, which may be a polymer of a monomer derived from an alpha-hydroxy acid, and may specifically comprise one or more selected from the group consisting of polylactide, polyglycolide, poly (lactide-glycolide), polymyalonic acid, polycaprolactone, polydioxanone-2-one, polyamino acid, polyorthoester, polyanhydride, polycarbonate, and combinations thereof, and more specifically may comprise one or more selected from the group consisting of polylactide, polyglycolide, poly (lactide-glycolide), and combinations thereof.

In the present invention, the amphiphilic block copolymer has a number average molecular weight (unit: g/mol) of 3700 or less. When the number average molecular weight of the amphiphilic block copolymer exceeds 3700, the reconstitution time of the polymer micelle nanoparticle prepared using the amphiphilic block copolymer becomes long, and the content of the related substances increases as the temperature of micelle formation increases.

In a specific embodiment, the amphiphilic block copolymer may have a number average molecular weight of 3700 or less, 3600 or less, 3550 or less, 3500 or less, 3450 or less, or 3400 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, 1500 or more, 2000 or more, 2500 or more, 3000 or more, or 3300 or more, but is not limited thereto. When the number average molecular weight of the amphiphilic block copolymer exceeds the above range of number average molecular weights, the reconstitution time may be increased, and when the number average molecular weight of the amphiphilic block copolymer is less than the above range of molecular weights, the ability to capture a drug is reduced, and thus precipitation may be accelerated.

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

In a specific 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 to 70 ℃ for 1 to 6 hours.

In a specific embodiment, the removal of the C ₁ to C ₅ alcohols in step (2) may be performed at a temperature of 30 ℃ to less than 50 ℃ for a period of less than 2 hours.

More specifically, the removal of the alcohol may be performed at a temperature of 30-45 ℃ or 32-42 ℃ for 30-100 minutes, 40-80 minutes, or 50-70 minutes. When the removal temperature of the alcohol is too high, the related substances may increase, and when the removal temperature of the alcohol is too low, it may be difficult to sufficiently remove the alcohol.

In a specific embodiment, the content of C ₁ to C ₅ alcohol in the resulting mixture after removal of the alcohol (i.e., the mixture prior to micellization) may be 2.5 weight/volume (w/v) percent or less (i.e., 0-2.5 w/v%), more specifically may be 2.0w/v% or less, 1.5w/v% or less, or 1.0w/v% or less. When too much alcohol remains in the mixture before micellization, a phenomenon of precipitation of the drug or polymer may occur.

In a specific embodiment, the aqueous medium used in the step (3) may be selected from, for example, general water, distilled water for injection, physiological saline, 5% glucose, buffer, and combinations thereof, but is not limited thereto.

In the step (3), the formation of micelles may be performed at a temperature of 20 ℃ or less. When the micelle formation temperature is higher than 20 ℃, the prepared polymer micelle nano particles are reconstituted, and the drug is rapidly separated out, so that the stability of the preparation can be obviously reduced.

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

Furthermore, in a specific embodiment, the micelle formation 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. When the micelle formation time exceeds the above range, the drug and the polymer may be decomposed in the aqueous phase, and when the micelle formation time is less than the above range, the micelle may not be sufficiently formed due to the condition that the temperature is limited.

In a specific embodiment, the step of forming micelles may comprise a process of filtering the formed micelles.

In a specific embodiment, the filtration may be performed at 0-25 ℃, 0-20 ℃, or 0-15 ℃ using a 0.1-0.8 μm, 0.1-0.6 μm, or 0.2-0.5 μm filter. The filtration may be performed at different temperature conditions by subsequently using filters different from each other, if necessary, or may be further repeated several times according to the respective steps.

In a specific embodiment, the formed micelles (or the micelles after formation of the post-filtration) may be maintained at 0-15 ℃ before the subsequent lyophilization, and may be subjected to the lyophilization within 3 hours. More specifically, the formed micelles may be maintained at 0-12 ℃ or 0-10 ℃ prior to freeze-drying, and may be applied to the freeze-drying step within 10 minutes to 3 hours, within 30 minutes to 3 hours, or within 1-3 hours.

In another embodiment, the formed micelles may be directly applied to the freeze-drying step without the need for a storage process. The term "hold" may be used in the same sense as stored.

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

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

The freeze drying aid is added so that the freeze-dried composition can maintain a cake shape. In addition, the freeze drying aid aids in uniformly dissolving in a short time during reconstitution (reconstitution) after freeze drying of the polymer nanoparticle composition. In one embodiment, the content of the freeze-drying aid 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 of step (4), secondary drying for removing residual moisture in the freeze-dried product may be further performed.

In a specific embodiment, the secondary drying may be performed after the freeze-drying in the freeze-drying apparatus in which the freeze-drying in step (4) is performed, or after the freeze-drying in step (4) is performed, the freeze-dried product may also be transferred to a separate apparatus and performed.

In a preferred embodiment, the secondary drying may be performed at a temperature below 31 ℃. When the secondary drying temperature is too high compared to the above level, the polymer in the prepared cake melts and becomes sticky, and thus the reconstitution time of the polymer micelle nanoparticles may become long. The lower limit of the secondary drying temperature is not particularly limited, and may be, for example, at a temperature of 10 ℃ or higher, 15 ℃ or higher, 18 ℃ or higher, or 21 ℃ or higher, but is not limited thereto. The secondary drying may be performed so that the moisture content of the polymer micelle nanoparticles is 5000ppm or less, 4000ppm or less, 3000ppm or less, 2000ppm or less, or 1000ppm or less, and the lower limit of the moisture content may be 300ppm or more, 400ppm or more, or 500ppm or more.

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

In a specific embodiment, the poorly water soluble drug may be selected from drugs having a solubility in water (25 ℃) of 100mg/mL or less. The pharmaceutical composition may be selected from an antitumor agent (antineoplastic agents), an antifungal agent (antifungal agents), an immunosuppressant (immunosuppressants), an analgesic (analgesics), an anti-inflammatory agent (anti-inflammatory agents), an antiviral agent (ANTIVIRAL AGENTS), an anxiolytic sedative (anxiolytic sedatives), a contrast agent (contrasting agents), a corticosteroid (corticosteroids), a diagnostic agent (diagnostic agents), a diagnostic imaging agent (diagnostic IMAGING AGENTS), a diuretic (diuretics), a prostaglandin (prostaglandins), a radiopharmaceutical (radiopharmaceuticals), a sex hormone (sex hormones) including a steroid (steroid), and combinations thereof, but is not limited thereto.

In a specific embodiment, the poorly water soluble drug may be selected from anticancer agents, and may specifically be taxane anticancer agents. The taxane anticancer agent may be, for example, one or more selected from paclitaxel (paclitaxel), docetaxel (docetaxel), 7-epipaclitaxel (7-epipaclitaxel), t-acetylpaclitaxel (t-acetyl paclitaxel), 10-deacetylpaclitaxel (10-desacetylpaclitaxel), 10-deacetyl-7-epipaclitaxel (10-desacetyl-7-epipaclitaxel), 7-xylosylpaclitaxel (7-xylosylpaclitaxel), 10-deacetyl-7-glutaryl paclitaxel (10-desacetyl-7-glutarylpaclitaxel), 7-N, N-dimethylglycyl paclitaxel (7-N, N-dimethylglycylpaclitaxel), 7-L-alanyl paclitaxel (7-L-alanylpaclitaxel), and cabazitaxel, more specifically, paclitaxel, docetaxel, or a combination thereof.

In a specific embodiment, the reconstitution time of the nanoparticles prepared according to the present invention in an aqueous solution may be within 5 minutes, within 4 minutes, or within 3 minutes. The reconstitution time can be measured by a generally known method, for example, the time in which nanoparticles as a freeze-dried product are stirred at 400 to 600rpm in an aqueous solution at normal temperature until the solution becomes transparent can be measured.

In a specific embodiment, the precipitation time of the drug after the nanoparticles prepared according to the present invention are formulated into a formulation may be 4 hours or more, 6 hours or more, 8 hours or more, or 10 hours or more. The precipitation time may be measured by a generally known method, for example, when nanoparticles are dissolved in an aqueous solution to make a transparent solution, and then left at normal temperature, the time until the solution becomes cloudy or a precipitate is observed may be measured.

The present invention is illustrated in more detail by the following examples, which are merely illustrative of the present invention, and the scope of the present invention is not limited in any way by these examples.

Examples (example)

Example 1, comparative example 1 and comparative example 2: preparation of Polymer micelle containing docetaxel 1

In a round bottom flask, 32g of monomethoxy polyethylene glycol- (poly-D, L-lactic acid) copolymer (mPEG-PDLLA) was added to 10g of ethanol (EtOH) and then stirred at 60 ℃ until completely dissolved and turned into a clear solution with viscosity. While stirring the polymer solution, 1.7g of docetaxel was weighed in a Falcon tube (Falcon tube), and after adding 12g of ethanol (EtOH), the solution was stirred with a roller mixer until it was completely dissolved, and the solution became transparent. After that, docetaxel solution was added to a round bottom flask containing a polymer solution, and then distilled under reduced pressure at 35 ℃ for 1 hour using a rotary vacuum distiller equipped with a flask to remove ethanol. Thereafter, the mixture was reacted into a blue transparent solution under the following micelle formation conditions to form polymer micelles.

8.5G of mannitol as a freeze-drying agent was completely dissolved in physiological saline and stored under refrigeration, and then mixed with the polymer micelle formed as described above. Thereafter, the product was filtered using a filter having a pore size of 0.22 μm, and then freeze-dried to prepare a polymer micelle composition containing docetaxel in powder form. In the prepared micelle composition, the content of docetaxel per 1 bottle (via) was 80mg, the content of mpeg-PDLLA was 1529mg, and the content of mannitol was 400mg.

TABLE 1

Example 2, example 3 and comparative example 3: preparation of docetaxel-containing Polymer micelle 2

The freeze-dried product prepared by the same method as that of example 1 was subjected to secondary drying under the conditions of the following table 2, thereby preparing a polymer micelle composition containing docetaxel in powder form.

TABLE 2

	Number average molecular weight of mPEG-PDLLA	Secondary drying conditions	Moisture content (ppm)
				Example 2	3360	25	967.9
Example 3	3360	29	568.3
				Comparative example 3	3360	32	255.9

Example 4: preparation of docetaxel-containing Polymer micelle 3

Prepared by the same method as in example 1, except that the addition amount of mannitol per 1 bottle was set to 2 times (800 mg), thereby preparing a polymer micelle composition containing docetaxel in powder form.

Test example 1: comparative experiment of reconstitution time and precipitation time

2G of each of the docetaxel-containing polymer micelles of examples 1 to 4 and comparative examples 1 to 2 was added to 38mL of physiological saline injection and stirred with a IKA VIBRAX shaker at a stirring speed of 500.+ -. 50rpm, while measuring the time (reconstitution time) for completely dissolving the freeze-dried cake. The results are shown in table 3 below.

Further, 2g of each of the polymer micelles of examples 1 to 4 and comparative examples 1 to 2 containing docetaxel were added to 38mL of physiological saline injection, dissolved by a IKA VIBRAX shaker, and left to stand, and whether or not docetaxel was precipitated was observed at intervals of 30 minutes. The results are shown in table 3 below.

TABLE 3

	Average reconstitution time (minutes)	Average precipitation time (hours)
			Example 1	2.5	6
Example 2	2.4	8
			Example 3	2.4	8
Example 4	2.1	8
			Comparative example 1	9.3	10
Comparative example 2	2.7	1

Test example 2: comparative experiment of content of related substances

The formulations of docetaxel-containing polymer micelles prepared in examples 1 to 4 and comparative examples 1 to 3 were filtered with 0.45 μm membrane filter paper to obtain 1mL of filtrate, which was mixed with 4mL of HPLC mobile phase solution to prepare sample solutions. The obtained samples were subjected to HPLC analysis under the following conditions, the contents of the related substances were compared, and the contents (%) of the related substances are shown in table 4 below.

HPLC conditions:

-column: at a particle diameter of 5 μm and a pore diameter of Is coated with a C18 stainless steel column with a length of 250mm and an inner diameter of 4.6mm

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

-Flow rate: 1.5 ml/min

-A detector: ultraviolet absorption photometer (measurement wavelength 232 nm)

TABLE 4

ImpC: impurity (Impurity) C in the formulation solution

From the test results, it is apparent that the drug-containing nanoparticles according to the examples produced according to the present invention are rapidly reconstituted in an aqueous medium such as physiological saline, while having excellent stability and uniformity, and provide a formulation with reduced contents of related substances, as compared with the nanoparticles of the comparative examples.

Claims

1. A method of preparing a polymeric micelle nanoparticle comprising the steps of:

(1) Mixing an active ingredient, an amphiphilic block copolymer having a number average molecular weight of 1500 to 3700, and a C ₁ to C ₅ alcohol;

(2) Removing C ₁ to C ₅ alcohol from the product of step (1);

(3) Adding the product of step (2) to an aqueous medium and performing at a temperature of 1 ℃ to 20 ℃ for 2 to 8 hours to form micelles; and

(4) Freeze-drying the product of the step (3),

The effective component is a poorly water-soluble drug.

2. The method for preparing polymer micelle nanoparticles according to claim 1, wherein the freeze-drying in step (4) is performed in the presence of a freeze-drying aid.

3. The method of preparing polymeric micelle nanoparticles according to claim 2, wherein the freeze drying aid is selected from the group consisting of sugars, sugar alcohols and mixtures thereof.

4. The method for preparing polymer micelle nanoparticles according to claim 1, wherein after the freeze-drying of the step (4), further secondary drying for removing residual moisture in the freeze-dried product is performed at a temperature of 31 ℃ or less.

5. The method of preparing polymer micelle nanoparticles according to any one of claims 1 to 4, wherein the amphiphilic block copolymer comprises a hydrophilic block (a) selected from the group consisting of polyethylene glycol or derivatives thereof, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, and combinations thereof, and a hydrophobic block (B) selected from the group consisting of polylactide, polyglycolide, poly (lactide-glycolide), polymyaldonic acid, polycaprolactone, polydioxan-2-one, polyamino acid, polyorthoester, polyanhydride, polycarbonate, and combinations thereof.