Method for producing ceramide liposomes

Abstract

To provide a method for producing a ceramide liposome that is stable and usable as an allergy inhibitor without using organic solvent.SOLUTION: A method for producing a ceramide liposome includes emulsifying ceramide by ultrasound emulsification with supercritical carbon dioxide as a solvent.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing ceramide liposomes.

Background Art

[0002] CD300f has two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one immunoreceptor tyrosine-based switch motif (ITSM) in its intracellular region, and is mainly expressed in myeloid cells including mast cells. The present inventors developed a screening system using a binding assay and a reporter assay, and identified lipid ceramide as a CD300f ligand. On the other hand, mast cells express the high-affinity IgE receptor (FcεRI). When FcεRI is crosslinked and stimulated by IgE and an antigen, mast cells are activated (degranulated) to cause an immediate allergic reaction. The present inventors clarified that the binding of CD300f and ceramide suppresses the FcεRI signal in mast cells and suppresses the immediate allergic reaction (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, conventionally produced ceramide liposomes had problems such as the residual organic solvent used as a solvent and low stability of the liposomes. The residual organic solvent causes an allergic reaction, and the instability of the liposomes has been an obstacle to use as an allergy inhibitor. Therefore, an object of the present invention is to provide a method for producing ceramide liposomes that does not use an organic solvent, is stable, and can be used as an allergy inhibitor. [Means for solving the problem]

[0005] Therefore, the inventors investigated a method for producing ceramide liposomes that does not use organic solvents as a solvent for ceramide. As a result, they found that by emulsifying using ultrasonic emulsification with supercritical carbon dioxide as the solvent, stable ceramide liposomes consisting only of ceramide can be obtained because the carbon dioxide evaporates when the pressure is reduced after emulsification is complete, leaving no solvent residue. Furthermore, they discovered that the obtained ceramide liposomes suppress the symptoms of allergic rhinitis when administered intranasally, thus completing the present invention.

[0006] In other words, the present invention provides the following inventions [1] to [6]. [1] A method for producing ceramide liposomes, characterized by emulsifying ceramide by ultrasonic emulsification using supercritical carbon dioxide as a solvent. [2] A method for producing ceramide liposomes according to [1], comprising dissolving ceramide in supercritical carbon dioxide, sonicating it under supercritical conditions, and then reducing the pressure. [3] A method for producing ceramide liposomes according to [1] or [2], wherein the ceramide is one or more ceramides selected from N-palmitoyl-D-erythro-sphingosine, N-stearoyl-D-erythro-sphingosine, and N-lignoceroyl-D-erythro-sphingosine. [4] A preventive or therapeutic agent for allergic rhinitis containing ceramide liposomes, which consist solely of ceramide and have a particle size of 100 nm to 300 nm. [5] The allergic rhinitis prophylaxis or treatment agent described in [4] for intranasal administration. [6] Ceramide liposomes, composed solely of ceramides and having a particle size of 100 nm to 300 nm, for the prevention or treatment of allergic rhinitis. [Effects of the Invention]

[0007] According to the method of the present invention, stable ceramide liposomes consisting solely of ceramide without any residual solvent can be obtained. Furthermore, the obtained ceramide liposomes can be stably stored in the form of fine particles ranging from 100 nm to 300 nm, and are useful as a therapeutic agent for allergic rhinitis, suppressing the symptoms of allergic rhinitis when administered intranasally. [Brief explanation of the drawing]

[0008] [Figure 1] This document presents a test protocol for a mouse model of allergic rhinitis caused by ragweed pollen. [Figure 2] This shows the number of sneezes 10 minutes after nasal administration in a mouse model of allergic rhinitis caused by ragweed pollen. [Modes for carrying out the invention]

[0009] The present invention relates to a method for producing liposome ceramide, characterized by emulsification by ultrasonic emulsification using supercritical carbon dioxide as a solvent. Specifically, ceramide is dissolved in supercritical carbon dioxide, subjected to ultrasonic treatment under supercritical conditions, and then produced by reducing the pressure.

[0010] The ceramide used can be any sphingosine lipid, i.e., a compound in which sphingosine and a fatty acid are linked by an amide bond, and many compounds are included depending on the type of sphingosine and fatty acid. Of these, from the viewpoint of acting as a ligand for CD300f and suppressing allergies, it is preferable to use one or more ceramides selected from N-palmitoyl-D-erythro-sphingosine, N-stearoyl-D-erythro-sphingosine, and N-lignoceroyl-D-erythro-sphingosine.

[0011] Ceramide is dissolved or dispersed in supercritical carbon dioxide. Since carbon dioxide has a critical pressure of 7.38 MPa and a critical temperature of 31.1°C, it is sufficient to create conditions above these. Therefore, typically, ceramide should be dissolved or dispersed in carbon dioxide in an environment with a pressure of 10 MPa or higher and a temperature of 50°C or higher. Water is also used at this time to emulsify the mixture. The ceramide concentration in the water is preferably 0.01 g / L or higher, more preferably 0.1 g / L or higher, and even more preferably 1 g / L or higher. The upper limit of the ceramide concentration is preferably 10 g / L or lower, and even more preferably 5 g / L or lower. Furthermore, the amount of water is preferably about 1 to 10 times the mass ratio of the supercritical carbon dioxide fluid.

[0012] Next, the mixture of water and a supercritical fluid solution of ceramide is subjected to sonication under supercritical conditions. The sonication is preferably performed at a frequency of 28 Hz to 100 Hz and an output of 300 W to 600 W. While one sonication is sufficient, it is preferable to perform it two or more times, preferably two to three times, to obtain stable ceramide liposomes. The sonication time is preferably 10 to 60 minutes per session. Furthermore, this supercritical melting and ultrasonic treatment are performed inside a pressure vessel.

[0013] Next, by reducing the pressure, carbon dioxide evaporates, so no organic solvent remains in the resulting ceramide liposomes, making them liposomes composed solely of ceramide.

[0014] The ceramide liposomes obtained by the present invention had a particle size in the range of 100 nm to 300 nm, and this particle size was maintained even after storage at 4°C for 3 months. Here, the particle size of the liposomes was measured by dynamic light scattering, specifically using Malvern's Zetasizer Nano ZS90.

[0015] When the ceramide liposomes obtained according to the present invention were administered intranasally to mice under isoflurane anesthesia using a p20 pipette, it was confirmed that they acted on CD300f in mast cells in the nasal mucosa to suppress degranulation and allergic rhinitis symptoms. Therefore, the ceramide liposomes obtained by the present invention are useful as a preventive and therapeutic agent for allergic rhinitis, particularly as a preventive and therapeutic agent for allergic rhinitis administered intranasally.

[0016] As a nasal administration preparation, a form in which the ceramide liposome of the present invention can be directly sprayed into the nasal cavity in the state of an emulsion is preferable.

Example

[0017] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples at all.

[0018] Example 1 A high-pressure pump for CO2 (PU-2086, Jasco, Japan), an ultrasonic device (ultrasonic multicleaner W-118, Honda Electronics, Japan), a water bath with a thermostat equipped with an ultrasonic device at the bottom, a chiller (TBG020AA, Advantec, Japan), a pressure-resistant steel reaction vessel (SUS-316; inner diameter 20.0 mm; outer diameter 25.0 mm; length 250 mm, GL Sciences, Japan), and a back pressure regulator (BPR; AKICO, Japan) were used. A pressure gauge (GLT-21-25 MPa, Mikishita Seiki Manufacturing Co., Ltd., Japan) was connected between the pressure-resistant container and the BPR to measure the pressure. A pressure-resistant container containing distilled water and ceramide was connected to the system with a 1 / 16-inch tube and placed horizontally in a water bath controlled at 73 °C with a distance of 13 cm from the pressure-resistant container to the vibrator. Carbon dioxide was sent from a siphon-type cylinder through a chiller tube by a pump to maintain the contents in the liquid phase. Next, the contents were heated to the supercritical state in a water bath and then introduced into the pressure-resistant container. The pressure was controlled by a BPR at 20 MPa. When the temperature and pressure in the pressure-resistant container reached the target state, ultrasonic treatment was started at 45 kHz and 600 W. After 60 minutes of treatment, the pressure-resistant container was turned vertically for 15 minutes, and the BPR was adjusted to reduce the pressure from the top. The prepared liposomes were recovered by opening the container and stored in a refrigerator. Cerami Do It was used as a liposome material at a concentration of 0.01 g / L.

[0019] The particle size of the obtained liposomes was measured with a Zetasizer Nano ZS90 from Malvern. As a result, the particle size of the liposomes was in the range of 100 nm to 300 nm. Furthermore, the particle size of liposomes measured after storage at 4°C for 3 months was similar.

[0020] Example 2 Wild-type Balb / c mice were administered ragweed pollen twice at two-week intervals (immunization with ragweed pollen), and two weeks later, ragweed pollen was administered nasally for four consecutive days (challenge), and the number of sneezes was measured. The effect of ceramide liposomes was investigated in this allergic rhinitis model. The protocol is shown in Figure 1.

[0021] <Immunity> Day 0: (First immunization) 100 μg of ragweed pollen and 100 μL of arum adjuvant were mixed at room temperature overnight for each mouse. 50 μL of this mixture was subcutaneously injected into the soles of both feet of the mice. Day 14: (Second immunization) 100 μg of ragweed pollen and 200 μL of PBS were mixed at room temperature for about 1 hour per mouse. 200 μL of this mixture was injected into the peritoneal cavity of the mice.

[0022] <Challenge> Days 28, 29, 30, and 31 (4 consecutive days): 1 mg of ragweed pollen and 20 μL of PBS were mixed at room temperature for approximately 1 hour per mouse. This 20 μL solution was administered nasally to mice under isoflurane anesthesia using a p20 pipette. The number of sneezes was measured for 10 minutes immediately after the mice recovered from anesthesia.

[0023] <Treatment> Days 27, 28, 29, 30, and 31 (5 consecutive days): Mice were divided into two groups: a control group and a ceramide liposome administration group. For 5 consecutive days starting the day before the ragweed pollen challenge (3-5 hours before ragweed pollen nasal administration if applicable), 20 μL of distilled water or ceramide liposomes (0.01 g / L) was administered nasally to mice under isoflurane anesthesia using a p20 pipette. As a result, as shown in Figure 2, we confirmed that the number of sneezes was significantly reduced in the ceramide liposome administration group compared to the control group.

Claims

1. A method for producing ceramide liposomes composed solely of ceramide, characterized by emulsifying ceramide using an ultrasonic emulsification method with supercritical carbon dioxide as the solvent.

2. A method for producing ceramide liposomes consisting solely of ceramide, as described in claim 1, comprising dissolving ceramide in supercritical carbon dioxide, sonicating it under supercritical conditions, and then reducing the pressure.

3. A method for producing ceramide liposomes composed solely of ceramide according to claim 1 or 2, wherein the ceramide is one or more ceramides selected from N-palmitoyl-D-erythrose-sphingosine, N-stearoyl-D-erythrose-sphingosine, and N-lignoceroyl-D-erythrose-sphingosine.

4. An allergic rhinitis preventive or therapeutic agent containing ceramide liposomes, which are composed solely of ceramide and have a particle size of 100 nm to 300 nm.

5. The allergic rhinitis preventive or therapeutic agent according to claim 4, for intranasal administration.

6. Ceramide liposomes, composed solely of ceramides and with a particle size of 100 nm to 300 nm, for the prevention or treatment of allergic rhinitis.

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