KR20200072322A - Method for manufacturing controlled releasable DDS device using thermosensitive hydrogel - Google Patents

Abstract

The present invention relates to a method for preparing a sustained release drug delivery system using thermosensitive hydrogel. Particularly, the method uses a thermosensitive polymer composite to realize a lyophilized form, wherein the thermosensitive polymer composite is present in a low-viscosity aqueous polymer solution state by being combined with a desired drug before being applied to a patient and has a liquid form before drug administration, and forms a reversible lower critical solution temperature (LCST) at a body temperature upon the application to a target site of the human body after being mixed with the desired drug so as to be converted into a hydrogel form to allow sustained release of the drug for 1-3 days.

Description

Method for manufacturing controlled-release drug delivery system using temperature sensitive hydrogels {method for manufacturing controlled releasable DDS device using thermosensitive hydrogel}

The present invention relates to a method for preparing a sustained-release drug delivery system using a temperature-sensitive hydrogel, and in particular, a drug required before application to a patient by having a lyophilized form using a thermosensitive polymer complex. When mixed, it is in the form of a low-viscosity polymer aqueous solution, which takes the form of a liquid prior to drug administration, and forms a reversible lower critical solution temperature (LCST) at the body temperature when it is applied to the site of use after mixing the required drug. It relates to a method for preparing a sustained-release drug delivery system using a temperature-sensitive hydrogel so that it can be changed into a hydrogel and gradually release the drug for 1 to 3 days.

The hydrogel has excellent cell adhesion while maintaining the temperature sensitivity of the polymer, and can be biodegraded after a certain period of time in vivo. Accordingly, various applications have been made in tissue engineering supports and drug delivery systems.

Particularly, in the case of severe organ damage with current medical technology, treatment with drug therapy alone is not possible, and transplantation is the only treatment method.

Accordingly, artificial tissue and organ supply are in progress compared to the increase in the number of patients requiring transplantation, and in some cases, drug treatment or histological regeneration may be performed using an injectable hydrogel without surgical surgery.

However, the existing hydrogel has poor cell affinity, and when used for drug delivery, reactivity according to the temperature of the body is weak, and thus, supplementation is required.

Republic of Korea Registered Patent No. 10-1780391 (September 14, 2017),'Method of manufacturing a hydrogel with improved adhesion, including a support supporting a skin temperature-sensitive pure hydrogel' Republic of Korea Registered Patent No. 10-1424172 (July 22, 2014),'Electric field-sensitive hydrogel, drug delivery system using the same and method for manufacturing the same' Republic of Korea Registered Patent No. 10-1570300 (November 12, 2015),'Drug delivery system comprising a temperature-sensitive micelle for multiple delivery of nucleic acid substances and drugs and a method for manufacturing the same'

The present invention was created to solve this problem in consideration of various problems in the prior art as described above, and a drug required before application to a patient by having a lyophilized form using a thermosensitive polymer complex When mixed with, it is in the form of a low-viscosity polymer aqueous solution, which takes the form of a liquid prior to drug administration, and then mixes the necessary drugs and applies a reversible lower critical solution temperature (LCST) at the biological temperature when applied to the body's use site. The main objective is to provide a method for preparing a sustained-release drug delivery system using a temperature-sensitive hydrogel, which is formed and changed into a hydrogel to slowly release the drug for 1 to 3 days.

The present invention is a means for achieving the above object, the polymer blending step for gel formation; A mixing step of adding a drug delivery substance to the formulation; Filtering the mixture and filling it into a vial; Provided is a method for preparing a sustained release drug delivery system using a temperature-sensitive hydrogel, comprising: freeze-drying the filled vial.

At this time, the gel-forming polymer blending step is a step of adding 3200 g of Phloxamer 407 (Poloxamer 407) and 12 L of water for injection into a blending tank and then mixing while stirring; The mixing step of adding the drug delivery material to the formulation is to add 84.8 g of sodium hyaluronate and then add water for injection to adjust the level to the 16L mark, which is the capacity indication in the mixing tank, and then mix and stir. It also has its features.

In addition, the polymer for gel formation is N-isopropyl acrylamide polymer, ethylhydroxyethyl cellulose, poly(ethylene oxide-propylene oxide-ethylene oxide), poloxamer, PLURONICS(R) polymer, poly(ethylene Glycol)/poly(D,L-lactic acid-co-glycolic acid) block copolymer, polysaccharide, alginate, polyphosphine, polyacrylate, tetronics(TM) polymer, and polyethylene oxide-polypropylene glycol Among the block copolymers, poly(ethylene oxide-propylene oxide-ethylene oxide) is a block copolymer and is characterized by being any of Poloxamer 407, a poly(ethylene oxide-b-propylene oxide-b-ethylene oxide).

In addition, the drug delivery material is a mixture of poly(ethylene oxide-propylene oxide-ethylene oxide) and sodium hyaluronate, which is a powdery solid, and the gel is also characterized by using a temperature sensitive hydrogel.

According to the present invention, the following effects can be obtained.

First, since it is manufactured in a lyophilized form using a thermosensitive polymer complex, it is easy to manage and use.

Second, it is possible to maintain a low-viscosity polymer aqueous solution state by mixing with a drug required before application to a patient, thereby enabling accurate dosing.

Third, before the drug is administered, it takes the form of a liquid, and after mixing the necessary drugs, it forms a reversible lower critical solution temperature (LCST) at the biological temperature when applied to the body's use site. It has the advantage of being able to slowly release the drug for 1 to 3 days by changing to form.

1 is an exemplary view showing the concept of a sustained-release drug delivery system using a temperature-sensitive hydrogel according to the present invention.
Figure 2 is an exemplary view showing a drug delivery example of a sustained-release drug delivery system using a temperature-sensitive hydrogel according to the present invention.
3 is an exemplary schematic diagram showing an example of dissolution and dissolution in the body of a sustained-release drug delivery system using a temperature-sensitive hydrogel according to the present invention.
4 is a photograph of an exemplary sample of a sustained-release drug delivery system using a temperature-sensitive hydrogel according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

Prior to the description of the present invention, the following specific structures or functional descriptions are merely exemplified for the purpose of illustrating the embodiments according to the concept of the present invention, and the embodiments according to the concept of the present invention may be implemented in various forms, It should not be construed as being limited to the embodiments described herein.

In addition, embodiments according to the concept of the present invention can be applied to various changes and may have various forms, so specific embodiments will be illustrated in the drawings and described in detail herein. However, this is not intended to limit the embodiments according to the concept of the present invention to a specific disclosure form, and it should be understood that it includes all modifications, equivalents, and substitutes included in the spirit and scope of the present invention.

In relation to the present invention, most polymers have a solubility that increases with increasing temperature, but temperature-sensitive polymers that exhibit a lower critical solution temperature (LCST) have a characteristic that the solubility decreases with increasing temperature.

Hydrogels formed of temperature-sensitive polymers shrink at higher temperatures than LCST.

Temperature-sensitive polymers have a hydrophobic group in common, and in response to a change in temperature, water molecules bound to the hydrophobic group are released to cause phase separation, which causes shrinkage.

Therefore, since the drug release behavior can be controlled at a temperature similar to body temperature, it can be usefully applied in a temperature-sensitive drug delivery system.

The method for preparing a sustained release drug delivery system using a temperature-sensitive hydrogel according to the present invention, as shown in FIG. 1, combines a gel-forming aid with a thermosensitive polymer to make a complex that is a sustained release drug delivery chain.

At this time, the complex has a lyophilized form, and when mixed with a drug required before application to a patient, it changes to a low-viscosity aqueous polymer solution.

As described above, after mixing the required drug in the form of a liquid prior to drug administration, as shown in FIG. 2, the reversible lower critical solution temperature (LCST) at the biological temperature is applied at the site of use of the human body. Formed to form a hydrogel (hydrogel), it is possible to manufacture a sustained-release drug delivery system using a temperature-sensitive hydrogel that slowly releases the drug for 1 to 3 days.

In particular, it is very important to control the component content of the composition so that mass-production is possible and temperature-sensitive biodegradable hydrogels can change sol-gel at 35-50°C.

This is a very important factor for establishing a reversible lower critical solution temperature (LCST) at the biological temperature, and the drug delivery agent has a low viscosity at room temperature (for example, a viscosity of 10 Pas at 20 degrees Celsius). Drug injection can be facilitated, and the viscosity in the body is relatively high (e.g., 300 Pas at 37 degrees Celsius), so that the drug should be slowly released.

To confirm this, the hydrogel prepared by the manufacturing method according to the present invention was confirmed by an in-vitro experiment, and about 40-50% by weight of the drug was released on the first day, and on the third day, the hydrogel As this was broken, it was confirmed that not only the drug but also the hydrogel was absorbed into the body and disappeared.

This method may be described in the form of the example of FIG. 3 attached.

More specifically, the method of preparing a sustained release drug delivery system using a temperature-sensitive hydrogel according to the present invention includes a polymer mixing step for gel formation, a mixing step of adding a drug delivery material to the formulation, and a vial after filtering the mixture. ), and lyophilizing the filled vials.

At this time, since the manufacturing method according to the present invention is performed in a sterile room in a sterile state, there is no need to go through a separate sterilization process.

The gel-forming polymer mixing step is a step in which 3200 g of Phloxamer 407 (Poloxamer 407) and 12 L of water for injection are added to the compounding tank, followed by stirring.

In this case, Chilling Temp: 2℃, RPM: 80 / 250, Time: 60/140 min.

In addition, the polymer for gel formation is N-isopropyl acrylamide polymer, ethyl hydroxyethyl cellulose, poly(ethylene oxide-propylene oxide-ethylene oxide), poloxamer, PLURONICS(R) polymer, poly(ethylene Glycol)/poly(D,L-lactic acid-co-glycolic acid) block copolymer, polysaccharide, alginate, polyphosphine, polyacrylate, tetronics(TM) polymer, and polyethylene oxide-polypropylene glycol Among the block copolymers, poly(ethylene oxide-propylene oxide-ethylene oxide) can be used as a block copolymer, Poloxamer 407, which is poly(ethylene oxide-b-propylene oxide-b-ethylene oxide).

However, in the present invention, Phloxamer 407 was exemplarily performed.

In addition, in the mixing step of adding the drug delivery material to the formulation, after adding 84.8 g of sodium hyaluronate, adding water for injection to adjust the level to the 16L mark, which is the capacity indication in the mixing tank, and then mixing and stirring to be.

In this case, it has the conditions of RPM:150 / 250, Time:120 / 600 min, and the drug delivery material is an auxiliary agent for forming a temperature sensitive hydrogel.

At this time, the drug delivery material is a drug delivery material in a solid state, and may be a mixture of poly(ethylene oxide-propylene oxide-ethylene oxide) and sodium hyaluronate in a powder state, and the gel is used as a temperature sensitive hydrogel. This is preferred.

The temperature-sensitive hydrogel refers to a temperature-sensitive hydrogel that is liquid at room temperature and solid or gel at body temperature, for example, a solid temperature-sensitive hydrogel-forming polymer and a solid temperature-sensitive hydrogel forming aid Is dissolved or suspended in a solvent and is in a liquid state at room temperature, and has a mechanism for converting from a body temperature to a solid or gel state.

In addition, the vial filling step is a step of filling with a filling amount per vial of 10g/total 1368 vial after filtering for Filter size 10.0um, Time 300 min.

In addition, the freeze-drying step is a step for ensuring safety while maintaining good storage conditions for a long time.

At this time, the freeze-drying condition is important because the rate of dissolution must be maintained within a certain range when made into an aqueous solution for use.

In the present invention, the freeze-dried conditions were set by measuring the dissolution rate over time in a state of 37° C. of a freeze-dried drug several times using HPLC, and then averaging.

The freeze-drying conditions according to the present invention allow freezing to reach -15 to -50°C over 60 to 120 minutes, and drying to -45 to 30°C is 1 minute at -45°C and 180-° at -30°C. It is configured to dry by heating for 240 minutes, 840 minutes at -20°C, 600 minutes at 0°C, and 10 minutes at 30°C.

By doing so, it is possible to manufacture a sustained release drug delivery system using a safe temperature-sensitive hydrogel while having the desired physicochemical properties.

To confirm the properties of the drug delivery system according to the present invention, a sample solution was prepared as follows.

First, a 5 mL physiological saline solution was taken using a disposable sterile syringe, and then lightly shaken after being injected into a vial containing lyophilized powder. Then, it was stored and dissolved for about 8 hours in a refrigerated state (2-8° C.), and the sample was maintained as shown in FIG. 4.

Then, the biological safety of the drug delivery system according to the present invention was evaluated.

The safety evaluation was carried out by the Invitro cytotoxicity test method, and the Invitro cytotoxicity test is for evaluating whether the test substance induces cytotoxicity to mouse fibroblasts (NCTC Clone 929) in the cell culture environment, ISO 10993-5: 2009 (Biological Evaluation of Medical Devices, Part 5: Cytotoxicity of mice forming a uniform monolayer according to the items of Tests for invitro) Agar diffusion test method. Microscopically observe the effect of the material that diffuses through the agar from the material on the cells, observe the reactivity of the Neutral live cell staining, and also observe how microscopically the actual cell lysis occurred on the discolored area. Is the way to do it.

At this time, the sample was carried out by placing the temperature of the sample up to 37°C to gel and then placing it on a plate.

In addition, the name of the cell line to be used was NCTC Clone 929 (L-929), Source: American Type Culture Collection (ATCC).

In addition, culture conditions, culture medium and antibiotics were 10% Horse serum [Penicillin (100 Units/mL)/Streptomycin] Minimum Essential Medium (pH 7.4), and the conditions were cultured at (5±1)% CO2, (37±1)℃. Therefore, it was passaged every 3-4 days.

As a test method, trypsin was treated on monolayer cultured cells to adjust the cell concentration to 105 (1 mL of Trypsin/EDTA) and inoculated at about 10 cm 2.

And, the final result reading of the cytotoxicity test was performed according to the following ISO 10993-5, 8.5 Determination of cytotoxicity.

Table 2 shows the state of discoloration and degree of dissolution in the test.

As a result, lysis of some cells was observed in cells to which the test substance was administered, but discoloration on the middle layer was not observed, and cytotoxicity was not observed in each control group.

Through this, it was confirmed that the drug delivery system according to the present invention secures biological safety.

In addition, the results of Tissure distribution after subcutaneous administration of Lidocaine (20 mg/kg) were evaluated to evaluate drug delivery performance.

As a result of comparing the drug exposure (AUC) in the tissue after administration, as shown in Table 3 below, there was a significant difference in the liver, but no significant difference from other tissues.

At this time, the composition of the administration solvent for each group is as follows, and the dosage of Group1 was 0.607 mL/kg, and the dosage of Group2 was 0.506 mL/kg, respectively. During the experiment, the animal (white rat) status was good and specific. There was no matter.

-Group 1: HDC-3D (1 vial) mixed with 20 mL of 2% lidocaine hydrochloride injection solution

-Group 2: 2% Lidocaine Hydrochloride Hydrate Injection

In addition, in order to confirm the change in the physical properties of the drug delivery system according to the present invention, a physical property according to the temperature change of the temperature-sensitive hydrogel was confirmed using a UTM (Universal Testing Machine).

As shown in Table 4, it was confirmed that at room temperature, there was no change in stress due to compression or the change was insignificant, so that it had liquid or incomplete viscosity. On the other hand, the formation of a solid-phase gel and the strengthening of physical properties were confirmed by checking the stress due to compression at a temperature similar to body temperature.

Through this, when the temperature-sensitive hydrogel was injected into the body, the phase change into a solid phase gel through the sol-gel phase transition phenomenon in the body and the physical properties can be predicted through the phase change.

Finally, in order to confirm the formation of a temperature-sensitive hydrogel according to an injection in the body at the level of a small animal, the experiment was conducted as follows.

In order to confirm the formation and duration of the solid phase gel according to the injection of the temperature-sensitive hydrogel into the body, a mixture of a temperature-sensitive hydrogel and a fluorescent agent, rhodamine B, was injected subcutaneously into small animals.

At this time, both the rhodamine B alone and the temperature-sensitive hydrogel and rhodamine B mixture were able to visually confirm the change according to the dosage immediately after dosing, but the rhodamine B alone had a difference compared to the temperature-sensitive hydrogel and rhodamine B mixture. Was incomplete.

In addition, in the case of single rhodamine B, it was possible to confirm the form according to dosing up to 1 hour of dosing, but it was difficult to confirm the form due to the absorption of the drug over time.

On the other hand, in the case of a mixture of temperature-sensitive hydrogel and rhodamine B, the confirmation of the form according to the dosing from the beginning of dosing was more pronounced than that of rhodamine B alone, and the form was confirmed up to 9 hours.

Through this, the formation and duration of the solid-phase gel according to the injection of the temperature-sensitive hydrogel will be predicted, and at the same time, it will show a sustained-release drug release form through mixing with the drug through a longer duration than rhodamine B alone. Prediction was possible, and also, the stability due to the injection in the body could be predicted as no change was observed due to the inflammatory reaction at the injection site due to subcutaneous injection.

Claims (4)

A gel-forming polymer compounding step;
A mixing step of adding a drug delivery substance to the formulation;
Filtering the mixture and filling it into a vial;
A method of preparing a sustained release drug delivery system using a temperature-sensitive hydrogel, comprising: freeze-drying the filled vial.
The method according to claim 1,
The gel-forming polymer blending step is a step of adding 3200 g of Phloxamer 407 (Poloxamer 407) and 12 L of water for injection into a blending tank and then mixing while stirring;
The mixing step of adding the drug delivery material to the formulation is to add 84.8 g of sodium hyaluronate and then add water for injection to adjust the level to the 16L mark, which is the capacity indication in the mixing tank, and then mix and stir. A method for preparing a sustained release drug delivery system using a temperature-sensitive hydrogel.
The method according to claim 1,
The gel-forming polymer is N-isopropyl acrylamide polymer, ethyl hydroxyethyl cellulose, poly(ethylene oxide-propylene oxide-ethylene oxide), poloxamer, PLURONICS(R) polymer, poly(ethylene glycol) /Poly(D,L-lactic acid-co-glycolic acid) block copolymer, polysaccharide, alginate, polyphosphine, polyacrylate, Tetronics(TM) polymer, and polyethylene oxide-polypropylene glycol block copolymer Poly(ethylene oxide-propylene oxide-ethylene oxide) is a block copolymer of poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) Poloxamer 407, which is a temperature sensitive hydrogel. Method for preparing sustained-release drug delivery system.
The method according to claim 1,
The drug delivery material is a mixture of poly(ethylene oxide-propylene oxide-ethylene oxide) and sodium hyaluronate, which is a powdery solid, and the gel uses a temperature-sensitive hydrogel, characterized in that a temperature-sensitive hydrogel is used. Sustained-release drug delivery method.

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