KR102037405B1 - Parenteral bioactive substance delivery composition based on low molecular methylcellulose - Google Patents

Abstract

The present invention relates to a composition for delivery of a parenteral bioactive substance using a micelle based on only low molecular weight methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa and a bioactive substance carrier.
The bioactive substance carrier according to the present invention can completely solve the extracorporeal excretion problem which is not solved in the low molecular weight methyl cellulose of the existing weight average molecular weight of 10-20 kDa, thereby improving safety, and micelles are formed so as to solubilize poorly soluble drugs. It is expected to be very useful for sustained release parenteral drug delivery.

Description

Parenteral bioactive substance delivery composition based on low molecular methylcellulose

The present invention relates to a low molecular weight methyl cellulose-based micelle particles, a composition for delivery of parenteral bioactive substances and a bioactive substance carrier.

In drug delivery systems that solubilize and deliver most poorly soluble drugs that are insoluble in water. Drug delivery technology using various kinds of surfactants or alcohol-based additives, drug delivery technology using polymers, drug delivery technology using a copolymer of a hydrophilic block such as PEG-PLGA and two or more polymer blocks having hydrophobic block properties Etc. are the majority [Non-patent Documents 1,2].

The solubilization method of such poorly soluble drugs is reported to cause side effects in the nervous system and digestive system because of the addition of various kinds of surfactants or alcohol-based additives for injection into the blood vessels in the body.

In addition, in the case of drug delivery technology using a copolymer, problems of lack of biocompatibility of a synthetic polymer of a copolymer unit and a complicated process are raised. In addition, the emission efficiency in vivo is reduced due to the high molecular weight.

Therefore, there is an urgent need to develop new formulations that can use poorly soluble drugs as drug carriers having high biocompatibility and biodegradability without the above problems.

Kanjiro Miyata, R. James Christie, Kazunori Kataoka, Polymeric micelles for nano-scale drug delivery, Reactive and Functional Polymers, Volume 71, Issue 3, March 2011, Pages 227-234 Kataoka Kazunori, Kwon Glenn S, Yokoyama Masayuki, Okano Teruo, Sakurai Yasuhisa, Block copolymer micelles as vehicles for drug delivery, Journal of Controlled Release, Volume 24, Issues 1-3, 1 May 1993, Pages 119-132

Accordingly, the present inventors can completely solve the kidney toxicity problem that is not solved in the existing low molecular weight methyl cellulose having a weight average molecular weight of 10-20 kDa, thereby improving safety, and having a weight average molecular weight of 6-9.5 kDa that can be solubilized by forming micelles. The present invention has been completed by developing a composition for delivery of parenteral bioactive substances based on methyl cellulose.

Therefore, an object of the present invention is to provide a micelle particle for delivering a physiologically active substance containing methyl cellulose having a weight average molecular weight of 6 ~ 9.5 kDa.

In addition, the present invention has another object to provide a composition for delivery of sustained-release parenteral bioactive substances that solve the kidney toxicity problem.

Another object of the present invention is to provide a physiologically active substance carrier in which a physiologically active substance is supported in a micelle containing methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa.

Another object of the present invention is to provide an external preparation for skin containing methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa and a bioactive substance.

Definitions of terms used in the present invention are as follows.

“Micelles” formed by polymers are generally formed as self-assembly of amphiphilic polymers in aqueous solution and have their unique core-shell structure, in which poorly soluble drugs are enclosed in the core. Can be. Amphiphilic polymers composed of hydrophilic residues having hydrophilicity and hydrophobic blocks having hydrophobicity and having both hydrophilicity and hydrophobicity are used. When these amphiphilic polymers are dispersed in an aqueous solution, hydrophobic interactions by hydrophobic blocks tend to aggregate themselves (ie, self-assembly) to minimize contact with water and to stabilize free energy. The microcores are formed by the aggregated hydrophobic blocks, and a shell is formed by hydrophilic blocks around the microcores to form polymer micelles having intermolecular physical bonds. The polymer micelle is increased in solubility in aqueous solution by the hydrophilic block. "Micell" in the present invention is a self-aggregated form of methyl cellulose having a weight average molecular weight of 6 ~ 9.5 kDa, as shown in Figure 9, the hydrophilic block of the hydroxyl group (hydroxyl group, methoxide group Hydrophobic blocks of the (methoxide group) are formed in a core-shell structure, the average particle size of which is 50 to 400 nm or 100 to 350 nm.

"Biodegradable" means that the block copolymer can be chemically degraded in the body to form non-toxic compounds. The rate of degradation is equal to or different from the rate of release of the bioactive substance (drug, etc.).

"Biocompatibility" means the ability to interact with the body without undesirable subsequent effects.

"Sustained release" refers to the continued release of a bioactive material (drug, etc.) over a period of time.

"Controlled release" refers to the control of the rate and / or amount of bioactive material delivered in accordance with the bioactive material (drug, etc.) delivery formulation of the present invention. Controlled release can be continuous or discontinuous and / or linear or nonlinear. This may be accomplished by the use of one or more types of polymer compositions, bioactive materials (drugs, etc.) loading, excipients or degradation enhancers, or the inclusion of other modifiers, alone, in combination or in series to provide the desired effect. have.

A “bioactive substance” refers to any compound or composition that, when administered to an organism (human or non-human animal), induces the desired pharmacological, immunogenic and / or physiological effects by local and / or systemic action. To mention. Thus, the term includes compounds or chemicals that are conventionally considered to be biopharmaceuticals that include drugs, vaccines, and molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, and the like. In addition to the drug, it may be included as an active ingredient (active ingredient) of cosmetics, such as whitening, antioxidant, moisturizing, UV protection, conditioning, anti-inflammatory, wrinkle improvement, elasticity enhancement.

"Drug" means an organic or inorganic compound or substance that is bioactive and is used or modified for therapeutic purposes. Poorly soluble drugs, hydrophilic drugs, low molecular weight drugs, polymer drugs, proteins, oligonucleotides, DNA and gene therapeutics are broadly included in the drug definition.

"Peptide" "polypeptide" "oligopeptide" and "protein" can be used equally when referring to peptides or protein drugs and are not limited to particular molecular weight, peptide sequence or length, bioactivity or therapeutic field.

"Therapeutic effect" means any improvement in the disease of a subject, human or animal treated according to the method, and means a prophylactic or preventive effect, or a disease that can be detected by physical investigation, laboratory or mechanical methods And obtaining any alleviation in the severity of the signs and symptoms of the disease or illness.

As used herein, unless otherwise defined with respect to a particular disease or condition, the term “treatment” or “treating” means (i) an animal that is susceptible to disease, disease and / or disease but has not yet been diagnosed with disease or To prevent the occurrence of a disease, illness or illness in a human; (ii) inhibit a disease, disorder or condition, ie inhibit its progression; And / or (iii) alleviate a disease, disorder or condition, ie cause disease, disease and / or disease regression.

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are intended to aid the understanding of the invention. Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers.

Hereinafter, the present invention will be described in detail.

The present invention relates to a sustained-release parenteral bioactive substance delivery composition comprising a micelle nanoparticle for delivery of a physiologically active substance comprising methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa and methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa. will be.

In the case of methyl cellulose is a polysaccharide based on the monomer represented by the following formula (1), the degree of saturation (DS) of the methyl group is 1.5-1.9, and has a viscosity of 15 cPs in 80 ℃, 2% aqueous solution of methyl cellulose, It has a property of approximately weight average molecular weight 14,000 Mw (60 kDa).

[Formula 1]

In Formula 1, R is hydrogen or methyl.

Methyl cellulose (MC) having a weight average molecular weight of 6 to 9.5 kDa according to the present invention has a substitution degree of 1.5 to 1.9, and has a property of melting well in a low temperature aqueous solution. The hydrophobic part of the methyl cellulose (methoxide group) forms a nucleus (core) surrounding the poorly soluble material, and the hydrophilic part forms a surface in contact with water to form a micelle [Fig. 9] to be dissolved in water This is called solubilization.

The inventors have significantly reduced the weight average molecular weight of methyl cellulose to 6 ~ 9.5 kDa through enzyme treatment, it was possible to solve the kidney toxicity problem by micelle formation and in vitro discharge.

The low molecular weight methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa of the present invention can be obtained by reducing the molecular weight through, for example, an enzyme treatment such as cellulase.

The methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa of the present invention may be referred to herein as "low molecular weight methyl cellulose" or "LMwMC". In addition, the low molecular weight methyl cellulose of the present invention also includes a methyl cellulose derivative that falls within the range of forming micelles.

In particular, the micelle particles comprising methyl cellulose (LMwMC) having a weight average molecular weight of 6 to 9.5 kDa of the present invention may enclose (or support) a bioactive substance to be delivered into the body in micelles.

At this time, the mixing ratio of the LMwMC and the bioactive material is preferably a weight ratio of 10 ~ 25: 1, if too little bioactive material may not be solubilized because the micelle nanoparticles containing the bioactive material is not sufficiently formed. If there is too much bioactive material, there is a problem in that unnecessary methyl cellulose micelle nanoparticles containing no bioactive material increase to increase the toxicity of the material or decrease the efficiency of treatment.

A bioactive substance (eg, a drug (anticancer agent)) is released by active diffusion from micelles formed using the low molecular weight methylcellulose of the present invention. In addition, after a certain period of time, the remaining drug is released due to degradation due to reduced stability of micelles. It is delivered to cancer cells by the EPR effect (Enhanced permeability and retention effect) in the body to deliver drugs into the cells.

As confirmed in Example 1, micelles made of low molecular weight methyl cellulose are gradually decomposed in the body and are converted into low molecular weight substances which are harmless to the human body and are discharged out of the body. Not required. And this discharge is mainly through the kidneys.

The low molecular weight methylcellulose according to the present invention can be formulated to be bioabsorbable, biodegradable, and biocompatible.

By "bioabsorbable" is meant that the polymer may disappear in the initial application in the body, without degradation or degradation of the dispersed polymer molecules. Biodegradability means that the polymer can be broken down or degraded in the body by hydrolysis or enzymatic degradation.

The term "biocompatible" means that all components are nontoxic in the body.

The bioactive substance delivery composition of the present invention may be delivered by injection or other method suitable for a human or other mammal having a biologically effective disease state or symptom included in the bioactive substance delivery system. Implantation, body cavity or possible space, coating the surface of a tissue of the body or coating the surface of an implantable device), but in particular the composition is preferably delivered parenterally.

The term 'parenteral' includes intramuscular, intraperitoneal, intraperitoneal, subcutaneous, intravenous and intraarterial.

Compositions of the present invention can typically be formulated in injection formulations.

In order to apply the low molecular weight methyl cellulose to the bioactive substance carrier of the injection formulation, it has to have a low molecular weight for easy discharge to the outside of the body. In the present invention, the low molecular weight methyl cellulose is used to maintain the low molecular weight, which is one of the requirements of micelles. It will be possible.

Injectable compositions of the invention can be injected or inserted into the body of a human or other mammal by any suitable method, preferably by injection through a hypodermic needle. For example, by injection or in any other way, Intravenous, genitourinary, subcutaneous, intramuscular, subcutaneous, intracranial, intracardiac, pleural, or other body cavity or space available. Or, via a catheter or syringe, for example, during arthroscopy, intraarticularly, into the genitourinary tract, into the vasculature, into the palate or into the pleura, or into any body cavity or possible space in the body, surgery, surgery, diagnosis or Can be introduced during interventional procedures. In other applications, it may be applied topically to open surgery or trauma wounds, burns, or skin or other tissue surfaces.

In particular, the present invention is characterized by a sustained release composition that allows the drug to be released slowly in the body when injected into a specific site in the body.

The compositions of the present invention are suitable for use as sustained and controlled release matrices of bioactives. When the matrix is coupled with one or more bioactive substances homogeneously contained therein, a biodegradable sustained release bioactive substance delivery system is provided.

As used herein, the term "sustained release" (ie, sustained release or controlled release) is introduced into a human or other mammal, or on an open wound, burn, or tissue surface or in a body cavity or latent body space. By introduction, it is meant to continuously release one or more bioactive substance streams over a predetermined time at a therapeutic level sufficient to achieve the desired therapeutic effect over a predetermined time.

In one embodiment of the present invention it was confirmed that the majority of the encapsulated bioactive material (drug) can be continuously released drug for about 21-25 days without initial burst.

In addition, as described above, the composition for delivery of the physiologically active substance of the present invention is decomposed into a substance harmless to the human body after a certain period of time and is discharged to the outside through the kidney, the composition according to the present invention using a common syringe or catheter When injected into a specific site of the body, the biologically active substance (drug) is slowly released and the biologically active substance (drug) is maintained at a constant concentration for a long time in the circulating blood, so the efficacy of the bioactive substance is not only excellent, There is an advantage that does not require a separate surgical removal surgery process for removing the active substance (drug) carrier.

Thus, according to the sustained release bioactive substance carrier of the present invention, the bioactive substance may be released in a controlled manner to the target site of the subject.

In one embodiment, micelles are used to provide site-specific release of a bioactive material to a subject. In another embodiment, the micelles comprise one or more bioactive substances that can be administered to the subject such that the bioactive substances are released by diffusion from the micelles and / or degradation of the micelles.

At this time, the dosage value of the composition will vary depending on the type, severity, and condition of the disease, disorder or condition being treated. For any particular subject, it should be further understood that the specific dosage regimen should be adjusted over time in accordance with the needs of the individual and the professional judgment of the person administering or directing the composition. In vivo administration can be based on in vitro release studies in cell culture or on in vivo animal models.

As such, the compositions of the present invention release the bioactive material in a controlled manner, thus providing optimal delivery of the bioactive material. As a result of controlled delivery, the bioactive material is delivered for the desired period of time. Slower and more constant rates of delivery may, in turn, result in a decrease in the frequency at which the bioactive material should be administered to the animal.

As such, the low molecular weight methyl cellulose of the present invention has high biocompatibility and biodegradability, is suitable for long-term sustained release formulations, and is very useful as a carrier for bioactive substances by increasing the stability and effect of the bioactive substances.

The composition according to the present invention may be 0.01 to 90% by weight of the low molecular weight methyl cellulose of the present invention based on 100% by weight of the total composition. Specifically, it may be about 0.1 to 80% by weight, more specifically about 0.1 to 70% by weight, and more specifically about 0.1 to 60% by weight relative to the total composition, and in the case of the dosage, 0.5 per day It may be administered once or several times to ~ 100 mg / kg, but may be appropriately changed depending on the extent of disease symptoms, age, weight, health status, sex, route of administration and duration of treatment.

In addition, the composition according to the present invention may include a carrier that is acceptable to a bioactive material, pharmaceuticals, cosmetics or health food in addition to the low molecular weight methyl cellulose.

On the other hand, the micelle of the present invention can be effectively used in sustained release bioactive substance carrier. In particular, it is more effective for poorly soluble drug delivery.

The poorly soluble drug refers to a drug that is difficult to solubilize due to low water solubility, and may also mean a hydrophobic drug, and for example, anticancer drugs or therapeutic agents for cardiovascular diseases such as arteriosclerosis and hyperlipidemia, but are not limited thereto. It can include all difficultly soluble drugs. Specifically, the poorly soluble drugs include, for example, paclitaxel, docetaxel, tamoxine, anasterazole, carboplatin, topotecan, velotecan, imatinib, irinotecan, phloxuridine, vinorelbine, gemcitabine, and rotrolide. (leuprolide), flutamide, zoleronate, methotrexate, camptothecin, cisplatin, vincristine, hydroxyurea, streptozosin, valubisin, lovastatin, simvastatin, fluvastatin, atorvastatin, pitavastatin, pravastatin, Or rosuvastatin and the like, but is not limited thereto.

In addition to poorly soluble drugs, drug substances that may be included in the micelles of the present invention include, for example, proteins, polypeptides, carbohydrates, inorganic substances, antibiotics, antineoplastic agents, local anesthetics, antiangiogenic agents, vasoactive agents, anticoagulants, immunomodulators. , Cytotoxic agents, antiviral agents, antibodies, neurotransmitters, mental agents, oligonucleotides, lipids, cells, tissues, tissues or cell aggregates, and combinations thereof. In addition, cancer chemotherapeutic agents such as cytokines, chemokines, lymphokines and indeed purified nucleic acids, and vaccines such as attenuated influenza viruses. Actually purified nucleic acids that may be incorporated include genomic nucleic acid sequences, cDNA encoding proteins, expression vectors, antisense molecules and ribozymes that bind to complementary nucleic acid sequences to inhibit translation or transcription. As such, there is no limit to the kind of drugs that can be used basically.

In addition, various techniques are known that can encapsulate (support) bioactive substances in micelles.

The bioactive material is included in the micelle at about 0.01 to about 100% by weight, preferably about 1 to about 95% by weight, and more preferably about 10 to about 70% by weight. The amount or concentration of the bioactive material contained in the micelle will depend on the rate of absorption, deactivation and release of the bioactive material.

In one embodiment, the micelle of the present invention, prepared by the above method, enters the body through intravenous injection, circulates through the bloodstream, and loosens cells that form cancer tissues by an enhanced permeability and retention (EPR) effect. It is delivered to the target site where the tumor is located, and over time, the bioactive substance (drug) contained in the micelles will be actively diffused, or the micelles will be broken down and released by controlled means to the target site.

The present invention also includes a bioactive substance carrier in which a bioactive substance is encapsulated (supported) in a micelle containing methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa.

All the contents described above with respect to the composition for delivering micelle particles and bioactive substances may be applied or applied mutatis mutandis to the bioactive substance carrier.

Here also includes a method of treating a disease, disorder or condition comprising the introduction of the bioactive substance into a subject (patient) in need thereof and a method of manufacturing the delivery system of the present invention.

That is, the present invention may include a pharmaceutical composition for releasing a sustained-release bioactive substance comprising a micelle and a desired bioactive substance containing methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa.

In addition, the present invention may include an external preparation for skin containing methyl cellulose having a weight average molecular weight of 6 to 9.5 kDa and a bioactive substance.

All the contents described above with respect to the composition for delivering micelle particles and bioactive substances may be applied or applied mutatis mutandis to the external preparation for skin.

As used herein, the term “skin external preparation” is a concept encompassing the entire composition applied to the skin, and includes various cosmetics such as basic cosmetics, makeup cosmetics, and hair cosmetics; Ointments, creams, lotions, gels, patches, such as pharmaceutical and pharmaceutical composition that is applied to the external use of the skin, including quasi-drugs.

The content of the low molecular weight methyl cellulose of the present invention used in the external preparation is not particularly limited as long as it can secure a desired physiologically active substance delivery power, and may be appropriately determined by those skilled in the art according to a desired degree. For example, it may be 0.01 to 90% by weight relative to 100% by weight of the total composition. Specifically, it may be about 0.1 to 80% by weight, more specifically about 0.1 to 70% by weight, more specifically about 0.1 to 60% by weight relative to the total composition. The cosmetic may be any formulation known as a cosmetic, for example skin, emulsion, cream, sunscreen, foundation, essence, gel, pack, mask pack, foam cleanser, body cleanser, softening lotion, eyeliner, shampoo, It may be, but is not limited to, a formulation selected from the group consisting of rinse, soap, hair dressing, wool, hair cream, hair styling gel, lubricant, toothpaste, and wet wipes.

The parenteral physiologically active substance delivery system according to the present invention was used to solubilize on the basis of only low molecular weight natural polymer methylcellulose, unlike oils or other additives to solubilize poorly soluble substances and has no toxicity problems. It has excellent biodegradability and can deliver desired bioactive substances in vivo without side effects. Therefore, the low molecular weight methyl cellulose of the present invention can be utilized as a new bioactive substance delivery agent solubilizing poorly soluble substances.

Figure 1 shows the principle of cutting endocellulose from the inner chain of polysaccharides among the cellulase enzyme.
Figure 2 is measured by GPC (Gel Permeation Chromatography) of methyl cellulose having a molecular weight of 60 kDa used in the preparation example before the process of producing a low molecular weight polysaccharide.
FIG. 3 shows a 4% methyl cellulose aqueous solution reacted for 3 days after cellulase enzyme treatment and measured by GPC (Gel Permeation Chromatography), showing that it has a molecular weight of about 9,000 Mw (15 kDa).
4 is a result of lyophilization of an aqueous solution of methyl cellulose filtered through a dialysis membrane (Spectrum Lab., Filtration limit: 6-8 kDa) after cellulase enzyme treatment, and measured by GPC (Gel Permeation Chromatography), and about 5,294 Mw (8.5 kDa) It has a molecular weight.
Figure 5 shows the methyl cellulose having an average molecular weight of 8.5 kDa in the form of a powder (solid) finally obtained in the preparation example.
Figure 6 is a schematic diagram showing the manufacturing process of low molecular weight methyl cellulose in Preparation Example 1.
Figure 7 shows the results of the measurement of the critical micelle concentration (CMC) of the low molecular weight methyl cellulose of Preparation Example 1.
Figure 8 shows the cytotoxicity according to the low molecular weight methyl cellulose concentration of Preparation Example 1.
Figure 9 shows the micelle formed in the present invention.
Figure 10a shows the transmission electron microscope (TEM) results and DLS results when micelles were formed with only the low molecular weight methyl cellulose of Preparation Example 1 without drug encapsulation. `
FIG. 10B shows a transmission electron microscope (TEM) result and a DLS result when 1 mg of docetaxel (DTX) forms a low molecular weight methyl cellulose micelle.
11 shows the results of HPLC analysis to determine the retention time of DTX.
12 is a result of HPLC analysis of low molecular weight methyl cellulose micelles containing DTX at concentrations such as 3.9 ug / ml to 500 ug / ml.
Figure 13 shows the drug release rate of the dosage form (the micelle of Example 1) in which docetaxel is encapsulated in the low molecular weight methyl cellulose of the present invention.
FIG. 14 shows cell viability in the 570 nm wavelength band when only docetaxel was treated and when the docetaxel-encapsulated formulation (the micelle of Example 1) was treated with the low molecular weight methylcellulose of the present invention.
FIG. 15 shows tumor size after control and treatment group intra-regional injection (direct injection into the tumor site) in tumor animal models prepared by allografting B16F10 melanoma tumor cancer cells subcutaneously in the right thigh of C57BL / 6 mice. The graph shown.
FIG. 16 is a mouse photograph for confirming tumor size on day 14 after control and treatment groups (micelles of Example 1) local injection (direct injection to tumor site) in tumor animal models.
Figure 17 is a graph confirming the weight change for 14 days after the control and treatment group (micro mice of Example 1) local injection (direct injection to the tumor site) in the tumor animal model
FIG. 18 is a graph showing survival rate for 22 days after the control and treatment groups (the micelles of Example 1) local injection (direct injection to the tumor site) in the tumor animal model.
19 is a table showing no toxicity as a result of renal toxicity test for 125 mg / kg low molecular weight methyl cellulose administration group of Preparation Example 1.
20 is a graph showing the tumor size for 14 days after the intravenous injection of the control group and the treatment group (the micelle of Example 1) in the tumor animal model.
21 is a graph showing the weight change for 14 days after the intravenous injection of the control group and the treatment group (the micelle of Example 1) in the tumor animal model.
22 is a graph showing the survival rate for 25 days after the intravenous injection of the control group and the treatment group (the micelle of Example 1) in the tumor animal model.

Hereinafter, the embodiment of the present invention will be described in detail. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. These embodiments are provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art, and the invention is defined by the scope of the claims. It is only.

[ EXAMPLE ]

Production Example Weight average molecular weight 8.5 kDa Low molecular weight  Preparation of Methyl Cellulose

8 g of methyl cellulose (molecular weight: 60 kDa) [Sigma aldrich, Inc.] was added to 200 ml of tertiary distilled water to make a total solution of 4%. The aqueous solution of methyl cellulose was stirred at 80 ° C. and 210 RPM for 30 minutes to increase the degree of dispersion. By stirring the aqueous methyl cellulose solution in a low temperature room (4 degreeC) for 1 hour. The solubility of methylcellulose in water again increased. Then, the aqueous methyl cellulose solution was stirred at room temperature (24-25 ° C.), 210 RPM, and 30 minutes.

The target unit of methyl cellulose was 30 Units / ml, and 51.75 mg of endocellulase (Worthington, Inc.) having an enzyme effect of 105 Units / mg was added to the aqueous methyl cellulose solution. Methylcellulose aqueous solution was stored for 3 days in a stirrer at room temperature (24-25 ° C), 210RPM. Since the role of the cellulase enzyme is an endcellulase that cuts from the inside of the polysaccharide chain, the activity of the cellulase after 3 days is not maintained. Therefore, in order to inactivate the cellulase in the aqueous solution of methyl cellulose on the third day, the temperature of the stirrer was raised to 80 ° C. and stirred for 10 to 15 minutes. When the temperature was raised, the solubility of methyl cellulose in water was lowered, so it was stirred for 1 hour in a low temperature chamber (4 ° C.) to increase the solubility of methyl cellulose. Subsequently, the aqueous solution of methylcellulose was first subjected to large polymers with a 0.22 μm Polyvinylidene fluoride (PVDF) filter. The aqueous solution of methyl cellulose passed through the filter was put in a dialysis membrane (Spectrum Lab., Filtration limit: 6-9.5 kDa) and dialyzed in a low temperature chamber (4 ° C.) for 2 days in tertiary distilled water. After 2 days, the dialysis membrane was removed and the third distilled water outside the dialysis membrane was collected and stored in a freezer (-70 ° C.) for one day (the solution outside the dialysis membrane was collected because low molecular weight methylcellulose was required). The frozen solution was vaporized with water only at -76 ° C and 20-24 mTorr lyophilizer for 5-7 days. Weight average molecular weight of 8.5 kDa obtained after lyophilization Only low molecular weight methylcellulose powder was collected.

EXAMPLE  1: weight average molecular weight 8.5 kDa Low molecular weight  Methylcellulose Micelles  Manufacturing and Characterization

The micelles were prepared using only the obtained low molecular weight methyl cellulose and analyzed for their properties.

Micelles were prepared using a thin-film hydration method.

First, dissolve in 1 ml of HFP (hexafluoro-2-propanol) in a vial containing 1 mg of docetaxel (DTX) and 20 mg of a weight average molecular weight of 8.5 kDa low molecular weight methyl cellulose (LMwMC), followed by a rotary evaporator. The two solutions were mixed. Rotate slowly at 36 ° C. until HFP evaporated to form a thin film. In order to make the formed thin-film an aqueous solution, 3 ml of tertiary distilled water was added, and then slowly rotated at 50 ° C. for 10 minutes to increase dispersion. Finally, by slowly rotating for 30 minutes under the condition of minus 4 ℃ to increase the solubility, an aqueous solution containing a low molecular weight methyl cellulose micelle enclosed with an anticancer agent was completed.

(1) CMC (critical micelle concentration) measurement

Dissolve 5 ug of DPH (1,6-Diphenyl-1,3,5-hexatriene, Mw = 232.32) in HFP (Hexafluoro-2-propanol) to make 0.86 mM stock solution, and 2.5 mg of low molecular weight methyl cellulose in 1 ml of HFP. Dissolved in, serial dilution up to 2.4 ug to make a sample of 2.5 mg / ml ~ 2.4 ug / ml. Each low molecular weight methylcellulose sample was placed in a glass beaker, 5uM of stock solution was added thereto, and then conjugated by thin film hydration. First, two mixed solutions were evaporated with an evaprator, and 3 ml of distilled water was added thereto, followed by rotating at 47 ° C. for 10 minutes to increase dispersion. After 30 minutes in ice to increase the solubility, each sample was collected and placed on a 96 well DC plate, excitation 355nm, emission 428nm at the UV / Vis fluorescence spectrophotometer (Infinite M200Pro, TECAN, Switzerland) to measure the absorbance micelles At this concentration, we found out how well it occurs.

As shown in FIG. 7, micelle formation of the low molecular weight methyl cellulose of an average of 8.5 kDa was confirmed from the concentration of 0.0526 mg / ml or more.

(2) cytotoxicity assay

Each well of the 24-well plate was counted to contain as much as 2 X 1010 B16F10 mouse skin melanoma cell lines [available from the Korean cell line bank] and a total of 500 ul of complete media was laid (Seeding). Cells were grown for 24 hours in an environment of 5% CO ₂ , 37 ° C. For the statistical significance, the number of wells in each group was 4, and the control group without any treatment and the treatment groups with different concentrations of low molecular weight methyl cellulose (62.5, 125, 250, 500, 1000 ug / PBS 1ml) were added to each well. After processing 50ul each and incubated for 24 hours in an environment of 5% CO ₂ , 37 ℃ (Treating). After 24 hours, remove all the solution in each well and complete the complete media containing MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide): Media in 1ml: 9ml ratio. After 500 ul per well was incubated for 2 hours in an environment of 5% CO ₂ , 37 ℃. After 2 hours, remove the complete media in each well, add 500 ul of DMSO (dimethyl sulfoxide) to each well, incubate at 37 ℃ for 15 minutes, and shake with a UV spectrophotometer for 5 seconds. Was measured.

The cytotoxicity test results are shown in FIG. 8. It was confirmed that the low molecular weight methyl cellulose micelle of the present invention does not affect the viability of the cells.

(3) Checking micelle size

Methyl cellulose micelles were prepared by dissolving 10 mg of the prepared low molecular weight methyl cellulose in 1 ml of distilled water.

The low molecular weight methyl cellulose micelle containing 1 mg docetaxel was prepared in the same manner as the micelle manufacturing process using the thin film hydration method described above, and was prepared in the same manner only by varying the amounts of docetaxel and methyl cellulose added. That is, 1 mg of docetaxel and 20 mg of low molecular weight methylcellulose were used for 1 mg of micelles.

The micelles thus prepared were identified using a transmission electron microscope.

In order to measure the DLS, each micelle made by the above method was frozen for one day, and then, 1 ml of tertiary distilled water was added to the powder prepared by freeze-drying for about 3-5 days, and the size was measured by a machine. As a result, it can be seen that micelles were formed more compactly than in the case of micelles containing docetaxel as shown in FIG. 10. Although the amount of docetaxel contained in the low molecular weight methyl cellulose is different, it can be observed that micelles having a similar size are formed in an aqueous solution at a PDI (polydispersity) of 0.2 or less and near 200 nm.

DTX and low molecular weight methyl cellulose were spherical micelles using a transmission electron microscopy (TEM), and as shown in FIG. 10, the size of the micelles containing low molecular weight methyl cellulose without DTX and 1 mg DTX was measured. 324.9 nm and 209.98 nm. In addition, when measured by the DLS (Dynamic Light scatter) method, the results similar to the transmission electron microscope results were confirmed.

EXAMPLE  2: Low molecular weight  Methylcellulose Micelles  drug Inclusion rate  Measure

ACN (acetonitrile) based DTX standard graphs were made using the Breeze program (Version 3.30, Waters, Milford, MA) (using C18 HPLC column) to determine drug loading and drug content by HPLC.

Eight samples were prepared by serial dilution of 500 ug DTX / 1ml ACN to create a linear equation of the first-order equation (the R-squared value of the standard curve represents reliability and is more than 0.99; therefore, this is a reliable reference graph).

The aqueous solution of low molecular weight methylcellulose micelles containing 1 mg docetaxel prepared by the method of preparing micelles was frozen for one day and then made into powder form by lyophilization for about 3-5 days. After adding 1ml ACN to the powder, centrifugation at 13000 rpm for 3 minutes, the supernatant (DTX + ACN) separated from the low molecular weight methyl cellulose was extracted and filtered through a column for HPLC. With this sample, the encapsulation rate and the content of the drug were measured by HPLC and Breeze program (Version 3.30, Waters, Milford, MA). Drug encapsulation rate and content rate were calculated by substituting the amount of drug in the solubilized aqueous solution measured by HPLC in the following equations (1) and (2).

Equation 1 and 2 for the micelle of Example 1 Drug loading rates and drug content rates were measured, respectively, and the results are shown in Table 1 below.

[Equation 1]

Drug inclusion rate (%) = total amount of drug in solubilized aqueous solution / amount of initially injected drug × 100

[Equation 2]

Drug content (%) = total amount of drug in solubilized aqueous solution / weight of solubilized aqueous solution × 100

(The first injection was strictly the low molecular weight methyl cellulose containing docetaxel, and since DTX: LMwMC forms micelles in a weight ratio of 1:20, the drug content was quantified by HPLC. Detection of DTX by 4-5% is a desirable result).

division
(n = 3) Amount of Low Molecular Methyl Cellulose Containing First Injected Docetaxel (ug) Drug inclusion rate
(Loading efficiency) Drug content
(Loading contents) value 1000 71.6 ± 15.0% 4.47 ± 0.67%

FIG. 11 is an experiment conducted to determine how many seconds of DTX is detected by HPLC analysis, that is, the retension time of DTX with three samples of low molecular weight methylcellulose micelles containing 1 mg of DTX. It can be seen that the peak that appears is the peak when DTX is detected. In the HPLC analysis result graph of FIG. 11, it can be seen that the peak shown at 3.73 seconds is the absorbance value of the detected DTX.

FIG. 12 was analyzed using an HPLC machine as shown in FIG. 11 with low molecular weight methylcellulose micelles containing DTX at concentrations of 3.9 ug / ml to 500 ug / ml. At this time, the results are obtained as shown in the graph of FIG. 11, but the vertical axis (absorbance) will vary according to the concentration (the higher the amount of DTX, the higher the absorbance is measured). 11 shows that the peak detected at 3.73 seconds is a peak indicating the amount of DTX, and thus the absorbance value detected at 3.73 seconds is converted into a first-order equation graph to set the absorbance at each DTX concentration. . That is, FIG. 12 is used as a reference graph to know how much the amount of DTX is when measuring the release rate of docetaxel in FIG. 13.

EXAMPLE  3: Low molecular weight  Methylcellulose Micelles  Drug release efficacy (In vitro release test)

For micelles of Example 1 Drug release tests were conducted to confirm drug release capability.

The solution obtained by dissolving the docetaxel-containing low molecular weight methyl cellulose powder, which was lyophilized in the above-mentioned micelle, was placed in a dialysis membrane (MWCO: 3,500 Da) and sealed with a string tightly tied up and down. This was put into a release media (saline buffer containing 5% ACN (pH 7.4)) and shaken at 100 RPM and 37 degrees. Since DTX is highly soluble in ACN, it will escape from the dialysis membrane and melt in the release media. Therefore, the new release media was replaced and the harvested release media were quantified by the HPLC machine.

As a result, as shown in FIG. 13, it was confirmed that about 60% of the drug was slowly released from the micelles for 25 days.

EXAMPLE  4: Low molecular weight  Methylcellulose Micelles in vitro Drug Validation

A total of 500 ul of complete media was counted on a 24 well plate by counting 2 × 10 mm B16F10 melanoma cells. Cells were grown for 24 hours in an environment of 5% CO ₂ , 37 ° C. 5,10,25,50,100,250 ug of micelles containing 1 mg of anticancer drug (DTX) made of low molecular weight methyl cellulose were dissolved in 50ul of PBS, respectively. 50 ul was added to the well and incubated for 24 hours in an environment of 5% CO ₂ , 37 ° C (Treating). For the statistical significance, the number of wells in each group was four. After 24 hours, the medium containing the drug contained in each well was removed, followed by MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide): Media in a 1 ml: 9 ml ratio. After 500ul of each well was incubated for 2 hours in an environment of 5% CO ₂ , 37 ℃. After 2 hours, remove 500ul of complete medium in each well, add 500ul of DMSO, incubate for 15 minutes at 37 ℃, and measure absorbance at 570nm wavelength after shaking for 5 seconds using UV spectrophotometer. The survival rate was confirmed.

FIG. 14 is a graph showing cell viability measured by MTT assay, which shows how much B16F10 cells died according to the amount of drug applied based on the number of living cells in the control group at 100%. Compared to Free DTX, DTX-loaded LMwMC has a better anti-cancer effect by killing cancer cells more effectively. (In particular, 5ug of drug is used to show a big difference, so even if a small amount of drug is injected, the difference in treatment is large.)

The MTT solution forms a purple formazan crystal only in the mitochondria of living cells.These crystals are insoluble, so they are dissolved in DMSO to form a liquid, and the light of 570nm wavelength is added to this purple. The more purple) the higher the absorbance is measured.

As shown in Figure 14, the anti-cancer agent encapsulated in a low molecular weight methyl cellulose micelle showed a similar or superior effect than the treatment with only the anticancer agent.

EXAMPLE  5: Low molecular weight  Methylcellulose Micelles in vivo Drug Validation Intratumor  injection)

This experiment is a preliminary experiment prior to intravenous injection, in order to see how much the anticancer effect is obtained by the drug in the tumor when delivered directly to the actual cancer site.

(1) Production of tumor animal model

B16F10 melanoma cells grown in an environment of 5% CO ₂ , 37 ° C were injected into 6-week-old male C57BL / 6 mice.

First, anesthetized with 50ul of anesthetic mixed with ketamine (ketamine (100 mg / kg) and xylazine (10 mg / kg), and then subcutaneously injecting B16F10 melanoma cells into the left posterior flank to produce a tumor animal. It was. When the tumor volume reached ~ 100 mm ³ , it was divided into 6 groups.

(2) tumor size confirmation

After the tumor animal model was produced, the day divided into 6 groups was determined as day0 when the tumor volume became ~ 100mm ³ . Intratumoral injection of 50ul PBS with 50ul PBS and 2mg of low molecular weight methylcellulose without drug was injected into the control group, and the other drug treated group was treated with 1mg DTX-encapsulated low molecular weight methylcellulose powder obtained by lyophilization. Dissolved in PBS according to the concentration of the group, each tumor was injected with intratumoral injection at day 0 (50ul per day) (Control group injected 50ul of PBS on day 0. LMwMC group was dissolved in 50ul PBS LMwMC containing 2mg of drug Intratumoral injection The Taxotere (5 mg / kg) group was prepared in an intravenous formulation at a rate of v / v / v 3.125% Tween80 / 3.125% ethanol / 96.75% PBS to inject 50ul with 0.1 mg of docetaxel intratumorally. The DTX loaded LMwMC (5 mg / kg) group dissolved the prepared 1 mg DTX loaded LMwMC powder in 500 ul of PBS and injected 50 ul per tumor intratumor DTX loaded LMwMC (10 mg / kg) group used the prepared 1 mg DTX loaded LMwMC powder in PB. After dissolving in 250ul S, 50ul per head was injected intratumor DTX loaded LMwMC (20mg / kg) The group prepared 250ul of samples by dissolving two prepared 1mgDTX loaded LMwMC samples in 125ul of PBS, and 50ul per head. After that, the rats were fixed by holding the tail of the mice for a total of 14 days at intervals of 2 days, and the size of the tumor in the left posterior flank was measured using a vernier caliper. Tumor size of the rats was measured using the vernier caliper's main axis, one scale spaced 1 mm apart. After measuring the long axis and short axis of the tumor, the volume of the tumor was measured by the following equation (3).

[Equation 3]

(a: 장축, b: 단축) Tumor volume (mm ³ ) =

(a: long axis, b: short axis)

The data in FIG. 15 shows the mean value of the five tumor sizes corresponding to each group.

As a result, as shown in Figure 15 and 16, compared to the control group, the anti-cancer formulation in the low-molecular weight methyl cellulose micelles confirmed the excellent anti-cancer effect, even when compared to the conventional drug taxotere of DTX to low molecular weight methyl cellulose micelles Formulations enclosed with anticancer agents confirmed excellent anticancer effects.

(3) check the weight change

After intratumoral injection on day0, the rats were placed on an electronic balance for a total of 14 days at intervals of two days, and the measured values were observed.

As a result, as shown in Figure 17, the control group was reduced in weight due to stress caused by tumors and in vivo damage. On the other hand, the treatment group (the formulation in which the anticancer agent was encapsulated in the low molecular weight methylcellulose micelle) was maintained at normal weight without any particular toxicity.

(4) Check survival rate

After intratumoral injection at day0, survival rates were observed at regular intervals (3 pm) at day intervals for 22 days.

As a result, as shown in Figure 18, compared with the control group (the formulation of the anticancer drug in the low molecular weight methyl cellulose micelle, 5, 10, 20 mg / kg) showed a higher survival rate depending on the concentration.

EXAMPLE  6: Kidney Toxicity Test

In this study, single intravenous toxicity studies were performed on male mice with medium and low doses of 500 mg / kg, 250 mg / kg and 125 mg / kg dose groups to obtain single-dose toxicity data for mice with an average molecular weight of 8.5 kDa MC. It was. The observation period was 2 weeks, and clinical symptoms, weight change, autopsy findings, kidney weight, and histopathological changes were observed.

Single mouse intravenous toxicity of male mice with an average molecular weight of 8.5 kDa MC was assessed and was limited to the 8.5 kDa MC 500 mg / kg group, with one (1/5; 20%) death occurring 10 days after administration and 250 mg The deaths in the / kg, 125mg / kg administration group did not appear during the 14-day experimental period [FIG. 19].

In addition, Table 2 shows the results of autopsy of the organs after 2 weeks of intravenous injection, and each value means 'the number of animals in which abnormality was observed / the total number of animals in the group' (5/5 of the normal group). Is the normal number of animals / total number of animals in a group). In particular, in the 125 mg / kg administration group, necropsy findings such as renal discoloration and atrophy, and histopathological changes such as tubular local necrosis and fibrosis were not confirmed.

group Vehicle control 8.5kDa MC Treatment:
125mg / kg kidney Normal 5/5 5/5 DC-Atrophy 0/5 0/5   1+  0/5  0/5   3+  0/5  0/5

* Degree = 1 +: weak, 2+: medium, 3+: severe

Example 7: of low molecular weight methylcellulose micelles in vivo Drug efficacy check (intravenous injection)

(1) Production of tumor animal model

B16F10 melanoma cells grown in an environment of 5% CO ₂ , 37 ° C were injected into 6-week-old male C57BL / 6 mice.

First, anesthetized with 50ul of anesthetic mixed with ketamine (ketamine (100 mg / kg) and xylazine (10 mg / kg), and then subcutaneously injecting B16F10 melanoma cells into the left posterior flank to produce a tumor animal. It was. When the tumor volume reached ~ 100 mm ³ , it was divided into 4 groups.

(2) intravenous injection and tumor size identification

After the tumor animal model was produced, the day divided into four groups was determined as day0 when the tumor volume became ~ 100mm ³ . Unlike intratumoral injections, intravenous injections were injected 100ul at a time, four mice per group at a given time (3 pm) a total of five times for two weeks at three-day intervals from day 0. The control group received 100 ul of PBS and the LMwMC group injected 100 ul PBS containing 4 mg of low molecular weight methylcellulose, intravenously 5 times, every other day for 2 weeks, and the Taxotere group contained 0.1 mg of docetaxel. 100 ul (3.125 ul Tween80, 3.125 ul ethanol, 93.75 ul PBS) solution was injected intravenously five times over two weeks at three day intervals. v / v / v 3.125% Tween80 / 3.125% ethanol / 96.75% PBS was prepared at a rate of 100 ul solution containing 0.1 mg of docetaxel and injected intravenously five times for two weeks at three-day intervals. The DTX loaded LMwMC (5 mg / kg) group dissolved the prepared 1 mg DTX loaded LMwMC powder in 1000 ul of PBS, and injected 100 ul per mouse intravenously five times for two weeks at three-day intervals. Tumor size of the rat was fixed by holding the tail of the rat for a total of 14 days at intervals of 3 days, the same as the day of intravenous injection, and then measuring the size of the tumor in the left rear flank using vernier calipers. Tumor size of the rats was measured using the vernier caliper's main axis, one scale spaced 1 mm apart. After measuring the long axis and short axis of the tumor, the volume of the tumor was measured by the following equation (3).

[Equation 3]

(a: 장축, b: 단축) Tumor volume (mm ³ ) =

(a: long axis, b: short axis)

The data in FIG. 20 shows the mean value of four tumor sizes corresponding to each group.

As a result, as shown in Figure 20, compared with the control group was confirmed that the anti-cancer effect of the low-molecular weight methyl cellulose micelle-encapsulated formulation was superior, compared to the conventional drug Taxotere of DTX, the anti-cancer agent was encapsulated. Formulations confirmed good anticancer effects. In addition, as shown in Figure 22 Taxotere group was observed to have a low self-toxicity, even if the anti-cancer effect, even if the survival rate.

(3) check the weight change

As for the change in the weight of the rats, each rat was placed on the electronic balance for 14 days at 3 days intervals, and the value measured after the intravenous injection was observed.

As a result, as shown in FIG. 21, the Taxotere group lost about 15% in weight due to its toxicity. On the other hand, the treatment group (the formulation in which the anticancer agent was encapsulated in the low molecular weight methylcellulose micelle) maintained normal weight without particular toxicity.

(4) Check survival rate

Survival after intravenous injection at day 0 was observed at regular intervals (3 pm) at day intervals.

As a result, as shown in Figure 22, compared to the high mortality caused by the toxicity of the Taxotere group, all the mice survived during the observation period in the treatment group.

Therefore, it was confirmed that the DTX-loaded LMwMC group showed higher anticancer effect with lower toxicity than the Taxotere group.

Claims (13)

delete delete delete A composition for sustained-release parenteral poorly soluble drug delivery comprising methylcellulose having a weight average molecular weight of 8.5 kDa in the form of micelles and a poorly soluble drug in a weight ratio of 20: 1.
delete The method of claim 4, wherein
Wherein said parenteral is intramuscular, intraperitoneal, intraperitoneal, subcutaneous, vein or artery.
delete A poorly soluble drug carrier in which a poorly soluble drug is supported in a micelle made of methyl cellulose having a weight average molecular weight of 8.5 kDa, and containing a methyl cellulose having a weight average molecular weight of 8.5 kDa and a poorly soluble drug in a weight ratio of 20: 1.
delete delete delete The method of claim 8,
A bioactive substance carrier having an encapsulation rate of the drug (encapsulated drug relative to total drug amount) of 60% to 90%.
A skin external preparation comprising methylcellulose having a weight average molecular weight of 8.5 kDa in the form of micelles and a poorly soluble drug in a weight ratio of 20: 1.

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