KR101520539B1 - Novel tacrolimus-loaded liquid crystalline nanoparticles and process for preparing the same - Google Patents

Abstract

The present invention relates to a novel tacrolimus-supported liquid crystal nanoparticle containing monoolein, a release modifier and tacrolimus and capable of controlling the release rate of tacrolimus, a process for producing the novel tacrolimus-supported liquid crystal nanoparticle, and a novel tacrolimus- ≪ / RTI > The tacrolimus-supported liquid crystal nanoparticles of the present invention can control the release rate of tacrolimus by changing the type of the release modifier contained therein, and thus the tacrolimus-containing liquid crystal nanoparticles can be widely used for customized treatment for patients prescribed tacrolimus.

Description

[0001] The present invention relates to novel tacrolimus-supported liquid crystal nanoparticles, and to novel tacrolimus-

The present invention relates to a novel tacrolimus-supported liquid crystal nanoparticle and a process for producing the same. More specifically, the present invention relates to a novel tacrolimus-supported liquid crystal nanoparticle containing monoolein, a release modifier and tacrolimus and capable of controlling the release rate of tacrolimus , A process for producing the novel tacrolimus-supported liquid crystal nanoparticles, and a pharmaceutical composition comprising the novel tacrolimus-supported liquid crystal nanoparticles.

Tacrolimus (FK-506) is a macrolide immunosuppressant found to be more than 100 times more effective than cyclosporine. It can be produced by fermentation of Streptomyces tsukubaensis , a monotypic species of Streptomyces . Such tacrolimus is marketed under the trademark PROGRAF® (available from Fujisawa USA, Inc.) and inhibits humoral immunity and is useful for allograft rejection, delayed type hypersensitivity, collagen Such as arthritis-induced arthritis, allergic encephalomyelitis, and graft versus host disease. Thus, tacrolimus prolongs survival of host and graft fragments in animal models transplanted with liver, kidney, heart, bone marrow, small intestine and pancreas, lung and bronchial, skin, cornea, and limb.

However, since tacrolimus is not well soluble in water, there is a problem that the bioavailability is very low when administered orally. Thus, studies have been actively conducted to develop formulations capable of increasing the solubility of tacrolimus, and some have been commercialized. These commercialized formulations had the advantage of being easily administered to patients, but had the disadvantage that they had to be frequently administered in order to inhibit the immune rejection reaction. Thus, there was a need to develop a sustained release formulation of tacrolimus.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a tacrolimus sustained-release preparation. As a result, they have confirmed that the release rate of tacrolimus can be controlled by using a novel tacrolimus-supported liquid crystal nanoparticle containing a release modifier, Completed.

One object of the present invention is to provide tacrolimus-supported liquid crystal nanoparticles comprising monoolein, a release modifier and tacrolimus.

Another object of the present invention is to provide a method for producing the tacrolimus-supported liquid crystal nanoparticles.

It is still another object of the present invention to provide a pharmaceutical composition comprising the tacrolimus-supported liquid crystal nanoparticles.

In one embodiment, the present invention provides a tacrolimus-supported liquid crystal nanoparticle comprising monoolein, a release modifier, and tacrolimus.

More specifically, the tacrolimus-supported liquid crystal nanoparticles of the present invention have the following characteristics: (a) monoolein, a release modifier and tacrolimus; (b) has a particle structure in liquid crystal form; (c) having a particle size of 180 to 200 nm; (d) a polydispersity index of less than 0.3. The particle structure of the liquid crystal type has a higher durability than that of conventional micelles or liposomes, and is not easily disintegrated even after a lapse of time, so that it can be administered to the body and used as a sustained-release preparation.

The present inventors paid attention to liquid crystal nanoparticles while carrying out various studies to develop tacrolimus sustained-release formulations.

The liquid crystal nanoparticle is a substance used as a carrier of a drug delivery system and exhibits biocompatibility and bio-bonding synthesis. It can collect and hold both hydrophilic and hydrophobic drugs, release the drug in a sustained release form, It has the advantage of not only protecting the drug but also prolonging the active period of the drug.

The present inventors have found that when a release modifier capable of changing the structure of the nanoparticles is added to liquid crystal nanoparticles composed of a monoolein known in the art, the structure of the liquid crystal nanoparticles can be modified by the added release modifier, It is expected that the drug release characteristics of liquid crystal nanoparticles will be changed due to the modification of the structure.

Thus, when various tacrolimus-supported liquid crystal nanoparticles were prepared using various compounds that can be used as a release modifier and their characteristics were analyzed, it was found that when fatty acid, glycerin, glycol compounds and the like were used as the release modifier, It was confirmed that the release rate of tacrolimus can be controlled depending on the type of release modifier used without changing the efficiency.

Specifically, the tacrolimus-supported liquid crystal nanoparticles prepared to contain lauric acid, myristic acid, palmitic acid, arachidic acid, etc. as the release modifier are superior to tacrolimus-supported liquid crystal nanoparticles not containing the release modifier, The tacrolimus-supported liquid crystal nanoparticles prepared to contain lauric acid showed the lowest tacrolimus release rate, and tacrolimus-bearing liquid crystals prepared to contain myristic acid, palmitic acid and arachidic acid The nanoparticles exhibited different release rates of tacrolimus in the slower range than the tacrolimus-supported liquid crystal nanoparticles, which were faster than tacrolimus-supported liquid crystal nanoparticles prepared to include lauric acid but without release modifiers.

On the contrary, the tacrolimus-supported liquid crystal nanoparticles prepared to contain stearic acid, glycerin, propylene glycol, polyethylene glycol, etc. as the release modifier increased the release rate of tacrolimus as compared with the tacrolimus-supported liquid crystal nanoparticles not containing the release modifier.

These tacrolimus-supported liquid crystal nanoparticles, which exhibit different tacrolimus release rates, can be used for patient-tailored therapy. Generally, tacrolimus is used as an immunosuppressive agent because the level of immunity rejection varies depending on the patient's constitution and physical condition, so that the administration level of the immunosuppressant to the patient is also different from each other, Since the dosage levels are each different, the release rate of the immunosuppressant in the sustained release formulation containing the immunosuppressant should also be designed differently according to the condition of the patient. The tacrolimus-supported liquid crystal nanoparticles provided in the present invention exhibit different tacholimus release rates depending on the type of the release modifier contained therein, and thus can be suitably used for customized treatment for individual patients in which different levels of immunosuppressive agents should be administered .

The term "monoolein " of the present invention means a compound having a chemical formula of C ₂₁ H ₄₀ O ₄ , a molecular weight of 356.54, a melting point of 35-37 ° C, hydrophobicity, and solubility in chloroform . Generally, triolein is produced by hydrolysis with lipase. Nanoparticles prepared using the monoolein are known to have a cubic structure. For the purpose of the present invention, the monoolein is used as a material forming a basic skeleton constituting the tacrolimus-bearing liquid crystal nanoparticles.

The term "release modifier" of the present invention means a substance capable of controlling the release rate of tacrolimus in association with the particle structure of the nanoparticles as a constituent of tacrolimus-supported liquid crystal nanoparticles provided in the present invention.

In the present invention, fatty acid, glycerin, and glycol compounds may be used alone or in combination as the release modifier.

The term "fatty acids " of the present invention means a compound in which carbon and carbon are covalently bonded to each other in a fatty acid which is a chain-structured hydrocarbon. The fatty acid generally has a very high melting point and remains solid at room temperature.

In the present invention, the fatty acid may be used as a kind of a release modifier which is inserted between monoolefins constituting tacrolimus-supported liquid crystal nanoparticles to change the structure and physical properties of the liquid crystal nanoparticles. Although not limited, saturated or unsaturated fatty acids including a C12 to C20 hydrocarbon chain can be used, and more preferably, lauric acid, myristic acid, palmitic acid, acid, stearic acid, arachidic acid, etc., can be used alone or in combination. Among these fatty acids, lauric acid, myristic acid, palmitic acid or arachidic acid may exhibit an effect of lowering the release rate of tacrolimus from the tacrolimus-supported liquid crystal nanoparticles, and stearic acid may release the release rate of tacrolimus from the tacrolimus- It is possible to exhibit an effect of increasing

The term "glycol-based compound" of the present invention means a divalent alcohol represented by the general formula R (OH) ₂ , also referred to as a diol, which is soluble in water or alcohol but not soluble in ether Lt; / RTI > The glycol can be produced by reacting a halogen compound with an acetic acid or an acetic acid to form a diacetic acid ester, and hydrolyzing the diacetic acid ester.

In the present invention, the glycol compound may be used as a kind of a release modifier that is inserted between monoolefins constituting tacrolimus-supported liquid crystal nanoparticles and changes the structure and physical properties of the liquid crystal nanoparticles. The compound is not particularly limited, but propylene glycol, polyethylene glycol and the like can be used singly or in combination. Among the above glycol compounds, propylene glycol, polyethylene glycol and the like can exhibit an effect of increasing the release rate of tacrolimus from tacrolimus-supported liquid crystal nanoparticles.

The term "tacrolimus" of the present invention refers to 3S- [3R * [E (1S *, 3S *, 4S *)], 4S *, 5R *, 8S *, 9E, 12R *, 14R * , 16R *, 18S *, 19S *, 26aR * -5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19 -dihydroxy-3- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylethenyl] -14,16-dimethoxy-4,10,12,18-tetramethyl-8- -epoxy-3H-pyrido [2,1- c] [1,4] oxaazacyclotricosine-1,7,20,21 (4H, 23H) -tetrone, compounds represented by compound named & monohydrate, C ₄₄ H ₆₉ O ₁₂ , and has a molecular weight of 804.018, which has a very low water solubility. It is usually classified as a type 2 drug of the BSC (Biopharmaceutical Classification System) and can be produced by Streptomyces tsukubaensis . In addition, for pharmaceutical use, it can be used as a therapeutic agent for inflammatory diseases by autoimmune reaction. When used as an immunosuppressive agent, the immunosuppressive action is stronger than that of cyclosporin, and is used for transplantation .

In the present invention, the tacrolimus is used as an active ingredient contained in the tacrolimus-supported liquid crystal nanoparticles provided in the present invention, and the liquid crystal nanoparticles enable the use of tacrolimus having a very low water-solubility as a water-soluble preparation.

According to one embodiment of the present invention, tacrolimus-supported liquid crystal nanoparticles containing various amounts of monoolein, various amounts of monoolein and various release modifiers (lauric acid, myristic acid, palmitic acid, stearic acid, Arachidic acid) was prepared (Example 1).

As a result of analyzing the characteristics of the prepared tacrolimus-supported liquid crystal nanoparticles, it was found that the liquid crystal nanoparticles not containing a release modifier had a size of about 150 nm, but the liquid crystal nanoparticles containing a release modifier had a size of 180 to 200 nm (Example 2-1), and the morphology of each liquid crystal nanoparticle was analyzed by an optical microscope and a scanning electron microscope. As a result, liquid crystal nanoparticles not containing a release modifier exhibited a cubic-like structure showing the shape of a cubosome, The liquid crystal nanoparticles containing a release modifier showed a hexagonal structure showing the shape of hexosomes (FIGS. 2 and 3).

In addition, the capturing efficiency of tacrolimus captured by each liquid crystal nanoparticle was analyzed. As a result, it was confirmed that the capturing efficiency was not influenced by the addition of a release modifier (Table 4). In vitro release of tacrolimus from liquid crystal nanoparticles As a result of the analysis, it was found that, in terms of both the in vitro release profile and the released cumulative content of tacrolimus, the liquid crystal nanoparticles prepared from lauric acid, myristic acid, palmitic acid and arachidic acid It was confirmed that the level of the liquid crystal nanoparticles prepared from stearic acid was higher than that of the liquid crystal nanoparticles not containing the release modifier (FIGS. 4 and 5).

Finally, it was confirmed that when glycerin, propylene glycol, ethylene glycol or the like was used instead of fatty acid as a release modifier, the release rate of tacrolimus was increased more than that of liquid crystal nanoparticles not containing a release modifier (FIGS. 6 to 8).

Accordingly, it was found that the tacrolimus-supported liquid crystal nanoparticles of the present invention can control the release rate of tacrolimus by changing the type of the release modifier contained therein, and thus can be used for patient-customized treatment.

As another embodiment for achieving the above object, the present invention provides a method for producing the tacrolimus-supported liquid crystal nanoparticles.

Specifically, a method for producing tacrolimus-supported liquid crystal nanoparticles of the present invention comprises the steps of: (a) dissolving a mixture comprising monoolein, a release modifier and a surfactant in water at 50 to 70 ° C to obtain a first reaction solution; (b) adding tacrolimus to the first reaction solution and stirring to obtain a second reaction solution; And (c) adding distilled water while vortexing the secondary reaction solution, followed by ultrasonic treatment. At this time, the ultrasonic treatment can be carried out at a room temperature for 10 to 1 hour under the condition of 20 to 60 kHz, and the release modifiers used are the same as those described above, and the tacromimus according to the release modifier of the finally prepared tacrolimus- The emission characteristics are also the same as described above.

The term "surfactant" of the present invention means a substance which exhibits both hydrophilic and hydrophilic groups in a molecule and adsorbs to the interface in a dilute solution to reduce the surface tension thereof do. The surfactant is classified into an anionic surfactant, which is ionized in an aqueous solution to become an anion in the active agent, a cationic surfactant in which the active agent becomes a cationic cation, an amphoteric amphoteric surfactant in an ionic form, and a nonionic surfactant in an ionized form . For the purpose of the present invention, the surfactant acts on the hydrophobic monoolein and the water-soluble solvent to assist the monoolein to form the liquid crystal nanoparticles. Here, the surfactant to be used is not particularly limited, but preferably a nonionic surfactant can be used, more preferably a poloxamer-based surfactant can be used, and most preferably, poloxamer 407 407) can be used.

The term " poloxamer 407 "of the present invention means a poloxamer-based copolymer and means a hydrophilic nonionic surfactant. The poloxamer 407 is structurally divided into one hydrophobic region and two hydrophilic regions, wherein the hydrophobic region is a region composed of a centrally located polypropylene glycol, and the hydrophilic region is a polyethyleneglycol located at both sides of the hydrophobic region .

The content of the monoolein used in the preparation of the tacrolimus-supported liquid crystal nanoparticles is not particularly limited, but is preferably 1 to 20% (w / w) based on the weight of the primary reaction liquid, more preferably 1 To 10% (w / w), most preferably from 2 to 8% (w / w); The content of the release modifier is not particularly limited, but is preferably 10 to 40% (w / w) of the monoolein content, more preferably 20 to 30% (w / w) , Most preferably 25% (w / w) of said monoolein content; The content of the surfactant is not particularly limited, but is preferably 10 to 50% (w / w) of the mixed amount of the monoolein and the release modifier, more preferably 10 To 30% (w / w), most preferably 12% (w / w) of the monoolein and release modifier mixed content; The content of tacrolimus is not particularly limited, but is preferably 0.01 to 0.1% (w / w) based on the weight of the primary reaction liquid, more preferably 0.01 to 0.05% (w / w) , And most preferably 0.03% (w / w) based on the weight of the primary reaction liquid.

In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the tacrolimus-supported liquid crystal nanoparticle as an active ingredient.

As described above, since tacrolimus is used as an immunosuppressive agent or a therapeutic agent for inflammatory diseases, the pharmaceutical composition can also be used as a pharmaceutical composition for immunosuppression or a therapeutic agent for inflammatory diseases.

In addition, as described above, the tacrolimus-supported liquid crystal nanoparticles of the present invention may lower or increase the release rate of tacrolimus depending on the type of the release modifier contained therein. Therefore, the tacrolimus- The pharmaceutical composition may be used as a tacrolimus release rate controlling pharmaceutical composition, i.e., a tacrolimus immediate release pharmaceutical composition or a tacrolimus slow release pharmaceutical composition.

In addition, as described above, the tacrolimus-supported liquid crystal nanoparticles of the present invention can be used as a patient-customized pharmaceutical composition because the release rate of tacrolimus can be controlled according to the type of release modifier contained therein.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable diluent, excipient or carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablet pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

In the present invention, the administration route of the pharmaceutical composition may be administered through any ordinary route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, rectally, but not exclusively, as desired. In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell.

The content of tacrolimus-supported liquid crystal nanoparticles contained in the pharmaceutical composition of the present invention is not particularly limited, but may be in the range of 0.0001 to 50% by weight, preferably 0.001 to 10% by weight, As shown in FIG.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment The effective dose level refers to the type and severity of the disease, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, May be determined according to known factors. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

The dose of the immunosuppressive pharmaceutical composition containing the tacrolimus-bearing liquid crystal nanoparticles of the present invention is determined depending on the purpose of use, the degree of addiction of the disease, the age, body weight, sex, history of the patient, A person skilled in the art can decide. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 100 mg / kg, preferably 1 ng to about 10 mg / kg, per adult, and the frequency of administration of the composition of the present invention is particularly It is not limited, but it can be administered once a day or divided into several doses.

The tacrolimus-supported liquid crystal nanoparticles of the present invention can control the release rate of tacrolimus by changing the type of the release modifier contained therein, and thus can be widely used for customized treatment of patients when tacrolimus is administered.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the particle size of liquid crystal nanoparticles containing different emission control agents, wherein A represents a T1 series, B represents a T2 series, C represents a T3 series, D represents a T4 series, (See Tables 1 and 2 for the composition of the T1, T2, T3 and T4 series).
Fig. 2 is an optical microscope image of the T1 series tacrolimus-supported liquid crystal nanoparticles, in which A represents particles without fatty acid, B represents particles to which lauric acid is added, C represents particles to which myristic acid has been added, E represents particles added with stearic acid, and F represents particles added with arachidic acid.
FIG. 3 is a scanning electron microscope image of tacrolimus-supported liquid crystal nanoparticles, wherein A represents T1-type liquid crystal nanoparticles without fatty acid and B represents T1-series liquid crystal nanoparticles to which a release modifier is added.
FIG. 4 is a graph showing in vitro release profiles of tacrolimus-supported liquid crystal nanoparticles containing different release modifiers, wherein A represents a T1 sequence, B represents a T2 sequence, C represents a T3 sequence, D represents a T4 sequence (See Tables 1 and 2 for the compositions of the T1, T2, T3 and T4 series).
FIG. 5 is a graph showing the cumulative content of tacrolimus released from liquid crystal nanoparticles containing different release modifiers for 14 days, where A represents a T1 series, B represents a T2 series, C represents a T3 series, D (See Tables 1 and 2 for the compositions of the T1, T2, T3 and T4 series).
6 is a graph showing the in vitro release profile of tacrolimus-bearing liquid crystal nanoparticles containing glycerin as a release modifier, wherein (o) represents a control group, (Δ) represents tacrolimus-bearing liquid crystal nanoparticles containing 5% glycerin , And () represents tacrolimus-supported liquid crystal nanoparticles containing 5% glycerin.
7 is a graph showing the in vitro release profile of tacrolimus-supported liquid crystal nanoparticles containing propylene glycol as a release modifier, wherein (o) represents a control group, and (Δ) represents tacronimus-bearing liquid crystal nanoparticles containing 10% propylene glycol () Indicates tacrolimus-supported liquid crystal nanoparticles containing 20% propylene glycol.
8 is a graph showing an in vitro release profile of tacrolimus-supported liquid crystal nanoparticles containing polyethylene glycol (PEG400) as a release modifier, wherein (o) represents a control group and (Δ) represents a tacrolimus- (1) represents tacrolimus-supported liquid crystal nanoparticles containing 15% PEG400.

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

Example  One: Tacrolimus  Preparation of supported liquid crystal nanoparticles

Example  1-1: Various contents of Monoolein  Included Tacrolimus  Preparation of supported liquid crystal nanoparticles

Monoolein and Poloxamer 407 were dissolved in water at 60 ° C, then tacrolimus was added and stirred until completely dissolved. Subsequently, the mixture was vortexed with an appropriate amount of distilled water for 1 minute, and ultrasonicated at 40 kHz for 30 minutes at room temperature to prepare a suspension containing tacrolimus-supported liquid crystal nanoparticles, which was then stored at room temperature . At this time, the content of monoolein was 2 to 8% (w / w), and the content of poloxamer 407 was 12% (w / w) of the content of monoolein (Table 1). At this time, the monoolein was obtained from Danisco (Tokyo, Japan); Poloxamer 407 was purchased from BASF (Ludwigshafen, Germany); Tacrolimus was obtained from Shanghai Qiao Chemical Science (Shanghai, China).

Composition of tacrolimus-supported liquid crystal nanoparticles containing various amounts of monoolein Composition Monoolein (g, content ratio) Poloxamer 407 (g) Water (ml) Tacrolimus (mg) T1
T2
T3
T4 1.0 (2%, w / w)
2.0 (4%, w / w)
3.0 (6%, w / w)
4.0 (8%, w / w) 0.12
0.24
0.36
0.48 48.88
47.76
46.64
45.52 15.0
15.0
15.0
15.0

Example  1-2: Various contents of Monoolein and  Preparation of tacrolimus-supported liquid crystal nanoparticles containing different release modifiers

Monoolein, poloxamer 407 and different release modifiers (lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid ) Was dissolved in water at 60 DEG C, tacromimus was added and stirred until completely dissolved. At this time, the lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid was obtained from Sigma-Aldrich (St. Louis, USA). Table 2 shows the melting points of the respective fatty acids and the state at room temperature.

Characteristics of release modifiers Carbon chain length designation The Melting point (℃) State at room temperature C12 Lauric acid CH ₃ (CH _{2) 10} COOH 44.2 Solid phase C14 Myristic acid CH ₃ (CH _{2) 12} COOH 58.8 Solid phase C16 Palmitic acid CH ₃ (CH _{2) 14} COOH 63.1 Solid phase C18 Stearic acid CH ₃ (CH _{2) 16} COOH 69.6 Solid phase C20 Arachidic acid CH ₃ (CH _{2) 18} COOH 76.5 Solid phase

As shown in Table 2, the release modifier is a fatty acid having a carbon chain length of C12 to C20, which has a melting point of 44.2 DEG C to 76.5 DEG C and exhibits a solid phase at room temperature.

Subsequently, the mixture was vortexed with an appropriate amount of distilled water for 1 minute, and ultrasonicated at 40 kHz for 30 minutes at room temperature to prepare a suspension containing tacrolimus-supported liquid crystal nanoparticles, which was then stored at room temperature . Wherein the content of the release agent is 25% (w / w) of the content of the monoolein, and the content of the poloxamer 407 is in the range of 2 to 8% (W / w) of the total fatty acid content (Table 3).

Composition of tacrolimus-supported liquid crystal nanoparticles containing various amounts of monoolein and different release modifiers Composition Monoolein (g, content ratio) Poloxamer 407 (g) X (g) Water (ml) Tacrolimus (mg) T1X
T2X
T3X
T4X 1.0 (2%, w / w)
2.0 (4%, w / w)
3.0 (6%, w / w)
4.0 (8%, w / w) 0.15
0.30
0.45
0.60 0.25
0.50
0.75
1.00 48.88
47.76
46.64
45.52 15.0
15.0
15.0
15.0

X represents a release modifier.

Example  2: Tacrolimus  Characterization of supported liquid crystal nanoparticles

Example  2-1: Particle size analysis

The particle size analysis was performed by appropriately diluting each of the tacrolimus-supported liquid crystal nanoparticle suspensions prepared in Example 1 to a final concentration of 0.02% (w / w) and then applying it to a Zetasizer Nano ZS (Malvern, UK) 1).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the particle size of liquid crystal nanoparticles containing different emission control agents, wherein A represents a T1 series, B represents a T2 series, C represents a T3 series, D represents a T4 series, Represents the polydispersity index. As shown in FIG. 1, the size of the nanoparticles having four different monoolein contents was about 150 nm, and when the release modifier was added, the nanoparticles were slightly increased in the range of 180 to 200 nm. However, the polydispersity index of all compositions was less than 0.3, indicating the monodispersibility of the nanoparticles.

Example  2-2: Optical microscope analysis

Each droplet of the tacrolimus-supported liquid crystal nanoparticle suspension prepared in Example 1 was placed on a slide glass and observed with an optical microscope (Olympus Microscope, Japan) (Fig. 2).

FIG. 2 is an optical microscope image of tacrolimus-supported liquid crystal nanoparticles, wherein A represents particles without fatty acid, B represents particles to which lauric acid has been added, C represents particles to which myristic acid has been added, D represents particles added with palmitic acid E represents particles to which stearic acid has been added, and F represents particles to which arachidic acid has been added. As shown in FIG. 2, the composition containing monoolein alone showed a cubic-like structure showing the shape of the cubosome, but the addition of the release-controlling agent showed a hexagonal structure showing the shape of the hexosomes.

Example  2-3: Scanning Electron Microscopy Analysis

2% phosphotungstic acid solution (pH 6.8) was added to each suspension of the tacrolimus-supported liquid crystal nanoparticles prepared in Example 1 to negatively dye, and a drop of the dyed suspension was placed in a carbon coating grid (200 mesh) and air dried. Then, images were obtained using a scanning electron microscope (Hitachi 7600, Japan) (Fig. 3).

FIG. 3 is a scanning electron microscope image of tacrolimus-supported liquid crystal nanoparticles, wherein A represents particles without fatty acid and B represents particles to which a release modifier is added. As shown in FIG. 3, the composition including monoolein alone showed a cuboid-like structure showing the shape of the cubosome, but the addition of the release modifier showed a hexagonal structure showing the shape of the hexosome Could know.

Example  2-4: Tacrolimus Capture  Efficiency analysis

The entrapment efficiency (EE) of tacrolimus was measured using an ultracentrifuge tube (Amikon Ultra-4, MWCO 10,000 g / mole, Billerica, USA).

Among the tacrolimus-containing drugs, tacrolimus-free drugs were screened by centrifugation at 3,500 rpm for 15 min. The amount of tacrolimus that was not captured was measured using Class VP computer software, LC 10 AD VP pump and SPD 10A UV-VIS detector 210 nm). ≪ / RTI > The mobile phase was acetonitrile / distilled water (70/30, v / v) at a flow rate of 1.0 ml / min and Inertsil ODS-3 (4.6 × 150 mm, GL Science, Japan) was used as the column. The total drug in the suspension was measured after destroying the liquid crystal nanoparticles by ethanol dilution (x 10). Then, EE was calculated by the following equation (Table 4). At this time, as the control group, liquid crystal nanoparticles prepared without the release modifier were used.

EE (%) = 100 x (Dt-Df) / Dt,

In the above equation, Dt and Df represent the total drug content in the sample and the content of the drug not captured in the liquid crystal nanoparticles, respectively.

Tacrolimus capture efficiency in tacrolimus-supported liquid crystal nanoparticles Composition T1 series T2 series T3 series T4 series Control group
L
M
P
S
A 99.9 ± 0.73
99.8 + 1.31
99.1 + - 0.38
98.8 ± 1.09
99.2 + 1.35
100.2 ± 0.95 100.2 + -0.44
99.5 ± 0.55
99.6 + - 0.64
99.7 ± 0.48
99.6 + - 0.33
99.8 + - 0.85 100.5 ± 0.78
99.7 ± 0.76
100.1 + - 0.30
100.0 ± 2.05
99.8 + 0.76
100.2 ± 0.56 99.9 ± 0.57
99.8 ± 0.95
100.0 + - 0.72
99.6 + 1.51
100.2 + - 0.46
100.3 ± 0.24

L: Lauric acid

M: Myristic acid

P: Palmitic acid

S: Stearic acid

A: Arachidic acid

As shown in Table 4, it was confirmed that the addition of the release modifier did not change the tacrolimus capturing efficiency of the tacrolimus-supported liquid crystal nanoparticles, but showed a collection efficiency of about 99% or more.

Example  2-5: From liquid crystal nanoparticles Tacrolimus in vitro  Emission analysis

A suspension of liquid crystal nanoparticles containing 500 μg tacrolimus was placed in a dialysis membrane (MWCO 10,000 g / mole, Spectra / Por®) and immersed in USP 30 (0.005% hydroxypropyl cellulose solution, pH 4.5) release solution. Then, the release solution was placed in a shaking constant temperature water bath at 37 DEG C, and tachyronimus was released to the outside of the dialysis membrane while being shaken at a rate of 25 times per minute. At this time, the sample was sampled while being exchanged for a specific period of time (1 hour, 6 hours, 12 hours, 24 hours, 48 hours, and then every 24 hours until 14 days) while maintaining the immersion state. The amount of tacrolimus contained in the sample was determined using the HPLC assay described above (Figures 4 and 5). At this time, as the control group, liquid crystal nanoparticles prepared without the release modifier were used.

4 is a graph showing in vitro release profile of tacrolimus-supported liquid crystal nanoparticles containing different release modifiers, wherein A represents a T1 series, B represents a T2 series, C represents a T3 series, and D represents T4 series.

As shown in FIG. 4, it was confirmed that the tacrolimus release profile was changed according to the content of monoolein (T1 to T4). That is, as the content of monoolein increases, the tacrolimus release level decreases.

In addition, it was confirmed that the release profile of tacrolimus changes depending on the type of release modifier. That is, in the liquid crystal nanoparticles prepared from lauric acid, myristic acid, palmitic acid and arachidic acid, the amount of tacrolimus emission was decreased while the amount of liquid crystal nanoparticles prepared from stearic acid was increased compared with the control group Could know.

Finally, it was found that the changes in the tacrolimus release profiles according to the types of these release modifiers show the same type (T1 to T4) even when they have different amounts of monoolein.

Next, FIG. 5 is a graph showing cumulative contents of tacrolimus released from liquid crystal nanoparticles containing different release modifiers for 14 days, in which A represents T1, B represents T2, and C represents T3 And D represents the T4 series.

As shown in FIG. 5, similarly to FIG. 4, it was confirmed that the accumulated amount of tacrolimus was changed according to the content of monoolein (T1 to T4). That is, as the content of monoolein increases, the cumulative release of tacrolimus decreases.

In addition, it was confirmed that the cumulative release amount of tacrolimus changes depending on the type of release modifier. That is, in the liquid crystal nanoparticles prepared from lauric acid, myristic acid, palmitic acid and arachidic acid, the cumulative amount of tacrolimus emission was decreased, whereas in the case of liquid crystal nanoparticles prepared from stearic acid, the cumulative amount of release increased .

Finally, it can be seen that the change in the cumulative release of tacrolimus depending on the type of the release modifier shows the same type (T1 to T4) even with different amounts of monoolein. In addition, lauric acid showed the lowest tacrolimus release while stearic acid showed the highest tachyronimus release.

The result of Example 2 shows that the addition of the release modifier during the preparation of tacrolimus-supported liquid crystal nanoparticles increases the size of the nanoparticles to a small extent, changes the shape of the nanoparticles to a hexagonal shape, However, it was found that the rate of release of the liquid crystal nanoparticles can be increased or decreased depending on the kind of the release modifier.

Example  3: Containing various release modifiers Tacrolimus  From the supported liquid crystal nanoparticles Tacrolimus in vitro  Emission analysis

Tacrolimus-supported liquid crystal nanoparticles were prepared by using glycerin, propylene glycol, polyethylene glycol (PEG400) or the like as a release modifier other than the fatty acid, and the release rate of tacrolimus in vitro was analyzed from the tacrolimus-supported liquid crystal nanoparticles prepared above.

Example  3-1: Containing glycerin Tacrolimus  From the supported liquid crystal nanoparticles, tacrolimus in vitro  Emission analysis

Monoolein, Poloxamer 407 and glycerin were dissolved in water at 60 占 폚, tacrolimus was added and stirred until completely dissolved. Subsequently, the mixture was vortexed, and an appropriate amount of distilled water was gradually added for 1 minute, followed by ultrasonic treatment for 1 hour to prepare a suspension containing tacrolimus-supported liquid crystal nanoparticles, which was then stored at room temperature. At this time, the contents of monoolein, poloxamer 407, glycerin and water are shown in Table 5.

Composition of tacrolimus-supported liquid crystal nanoparticles containing glycerin as a release modifier Composition Monoolein
(g, content ratio) Poloxamer 407
(g) glycerin
(g) water
(Ml) Tacrolimus
(mg) T3
T3-G 5%
T3-G 10% 3.0 (6%, w / w)
3.0 (6%, w / w)
3.0 (6%, w / w) 0.45
0.45
0.45 0
0.15
0.30 48.88
48.73
48.58 15.0
15.0
15.0

Subsequently, a liquid crystal nanoparticle suspension containing tacrolimus-supported liquid crystal nanoparticles (T3, T3-G 5% or T3-G 10%) containing glycerin was prepared as a release modulating agent on the dialysis membrane (MWCO 10,000 g / mole, Spectra / Por®) and immersed in USP 30 (0.005% hydroxypropyl cellulose solution, pH 4.5) release solution. Then, the release solution was placed in a shaking constant temperature water bath at 37 DEG C, and tachyronimus was released to the outside of the dialysis membrane while being shaken at a rate of 25 times per minute. At this time, while maintaining the immersed state, the discharged solution was sampled while being replaced for a specific period of time (1 hour, 12 hours, 24 hours, 48 hours, and 24 hours afterward until 14 days) The amount of tacrolimus was determined using the HPLC assay described above (Fig. 6). At this time, as a control group, liquid crystal nanoparticles (T3) prepared without a release modifier were used.

6 is a graph showing the in vitro release profile of tacrolimus-bearing liquid crystal nanoparticles containing glycerin as a release modifier, wherein (o) represents a control group, (Δ) represents tacrolimus-bearing liquid crystal nanoparticles containing 5% glycerin , And () represents tacrolimus-supported liquid crystal nanoparticles containing 5% glycerin. As shown in FIG. 6, tacronimus-containing liquid crystal nanoparticles containing glycerin as a release modifier showed increased release of tacrolimus as compared to the control.

Example  3-2: Propylene glycol  Included Tacrolimus  From the supported liquid crystal nanoparticles Tacrolimus in vitro  Emission analysis

Each tacrolimus-supported liquid crystal nanoparticle was prepared in the same manner as in Example 3-1, except that propylene glycol at various concentrations was used as a release modifier. The contents of monoolein, poloxamer 407, propylene glycol and water are shown in Table 6.

Composition of tacrolimus-supported liquid crystal nanoparticles containing propylene glycol as a release modifier Composition Monoolein
(g, content ratio) Poloxamer 407
(g) Propylene glycol
(g) water
(Ml) Tacrolimus
(mg) T3
T3-PG 10%
T3-PG 20% 3.0 (6%, w / w)
3.0 (6%, w / w)
3.0 (6%, w / w) 0.45
0.45
0.45 0
0.30
0.60 48.88
48.58
48.28 15.0
15.0
15.0

The procedure of Example 3-1 was repeated except that the liquid crystal nanoparticles containing the tacrolimus-supported liquid crystal nanoparticles (T3, T3-PG 10% or T3-PG 20%) prepared above were used, The release rate of tacrolimus from tacrolimus-supported liquid crystal nanoparticles was measured (Fig. 7).

7 is a graph showing the in vitro release profile of tacrolimus-supported liquid crystal nanoparticles containing propylene glycol as a release modifier, wherein (o) represents a control group, and (Δ) represents tacronimus-bearing liquid crystal nanoparticles containing 10% propylene glycol () Indicates tacrolimus-supported liquid crystal nanoparticles containing 20% propylene glycol. As shown in FIG. 7, tacronimus-containing liquid crystal nanoparticles containing propylene glycol as a release modifier showed an increase in the release of tacrolimus as compared with the control group.

Example  3-3: Polyethylene glycol  Included Tacrolimus  From the supported liquid crystal nanoparticles Tacrolimus in vitro  Emission analysis

Each tacrolimus-supported liquid crystal nanoparticle was prepared in the same manner as in Example 3-1, except that polyethylene glycol (PEG400) at various concentrations was used as a release modifier. The contents of monoolein, poloxamer 407, PEG 400 and water are shown in Table 7.

Composition of tacrolimus-supported liquid crystal nanoparticles containing PEF400 as a release modifier Composition Monoolein
(g, content ratio) Poloxamer 407
(g) PEG400
(g) water
(Ml) Tacrolimus
(mg) T3
T3-PEG 7.5%
T3-PEG 15% 3.0 (6%, w / w)
3.0 (6%, w / w)
3.0 (6%, w / w) 0.45
0.45
0.45 0
0.225
0.45 48.880
48.655
48.430 15.0
15.0
15.0

The procedure of Example 3-1 was repeated except that the liquid crystal nanoparticles containing the prepared tacrolimus-supported liquid crystal nanoparticles (T3, T3-PEG 7.5% or T3-PEG 15%) were used. The release rate of tacrolimus from tacrolimus-supported liquid crystal nanoparticles was measured (Fig. 8).

8 is a graph showing the in vitro release profile of tacrolimus-supported liquid crystal nanoparticles containing polyethylene glycol (PEG400) as a release modifier, wherein (o) represents a control group and (Δ) represents a tacrolimus- (1) represents tacrolimus-supported liquid crystal nanoparticles containing 15% PEG400. As shown in FIG. 8, it was found that the release of tacrolimus was increased in the tacrolimus-supported liquid crystal nanoparticles containing polyethylene glycol as the release modifier, as compared with the control group.

The results of Example 3 above show that the addition of glycerin, propylene glycol, polyethylene glycol or the like as a release modifier in the preparation of the taglolite-supported liquid crystal nanoparticles can increase the rate of release from the liquid crystal nanoparticles Respectively.

Accordingly, the tacrolimus-supported liquid crystal nanoparticles of the present invention can be used as a release modifier in addition to the fatty acid, and the tacrolimus-supported liquid crystal nanoparticles provided by the present invention can change the release rate of tacrolimus It can be used for patient - customized treatment.

Claims (22)

delete delete delete delete delete delete delete delete (a) 1 to 20% (w / w) monoolein based on the weight of the primary reaction solution; (W / w) of a monoolein content selected from the group consisting of lauric acid, myristic acid, palmitic acid, arachidic acid, stearic acid, glycerin, propylene glycol and polyethylene glycol ; And 10 to 50% (w / w) of a mixture of monoolein and a release modifier is dissolved in water at 50 to 70 占 폚 to obtain a first reaction solution;
(b) adding tacrolimus in an amount of 0.01 to 0.1% (w / w) based on the weight of the first reaction solution to the first reaction solution and stirring to obtain a second reaction solution; And
(c) adding distilled water while vortexing the secondary reaction solution, and subjecting the reaction liquid to ultrasonic treatment.
10. The method of claim 9,
Wherein the content of the release modifier is 25% (w / w) of the monoolein content.
10. The method of claim 9,
Wherein the content of the surfactant is 12% (w / w) of the monoolein and the release modifier mixed content.
10. The method of claim 9,
Wherein the tacrolimus content is 0.03% (w / w) relative to the weight of the primary reaction liquid.
10. A process for preparing a monoolefin, which is produced by the process of claim 9; Tacrolimus; And a release modifier selected from the group consisting of lauric acid, myristic acid, palmitic acid, arachidic acid, stearic acid, glycerin, propylene glycol, and polyethylene glycol.
14. The method of claim 13,
Wherein the release rate controlling agent is selected from the group consisting of lauric acid, myristic acid, palmitic acid and arachidic acid, wherein the rate of tacrolimus release is reduced as compared to the tacrolimus-supported liquid crystal nanoparticles not containing the release modifier Tacrolimus supported liquid crystal nanoparticles.
14. The method of claim 13,
Wherein the release rate controlling agent is selected from the group consisting of stearic acid, glycerin, propylene glycol, and polyethylene glycol, wherein the release rate of tacrolimus is increased as compared to the tacrolimus-supported liquid crystal nanoparticles not containing the release modifier .
14. The method of claim 13,
Tacrolimus-supported liquid crystal nanoparticles having a particle structure of liquid crystal type, having a particle size of 180 to 200 nm, and exhibiting a polydispersity index of less than 0.3.
delete delete delete delete A tacrolimus-supported liquid crystal nanoparticle prepared by the method of any one of claims 9 to 12 or a tacrolimus-supported liquid crystal nanoparticle of any one of claims 13 to 16, or a pharmaceutical composition for the treatment of an immunosuppressive or inflammatory disease .
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