JP2014193817A - Liposome carrier for percutaneous absorption agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liposome carrier for a percutaneous absorption agent which is particularly suitable for a percutaneous vaccine.SOLUTION: A liposome with high alcohol resistance enables provision of the liposome suitable for a carrier for a percutaneous absorption agent.

Description

  The present invention relates to a liposome useful for improving absorbability in a transdermal absorption agent. In particular, the present invention relates to a liposome having excellent utility as a carrier for a transdermal vaccine.

  A transdermally absorbable administration agent such as a transdermal vaccine has high utility because it is noninvasive. However, since it is necessary to penetrate from the epidermis, it is necessary to develop a carrier having high skin permeability.

Transdermal vaccine is a technology that is expected to prevent infectious diseases in livestock because it is safe and can induce a high immune response, but it is a technology that penetrates the vaccine antigen from the epidermis in development. Is important.
However, development of a practical technique for penetrating a vaccine antigen from the epidermis and delivering it to Langerhans cells, which are skin immune equivalent cells (dendritic cells), has not yet been made.

  Liposomes function as an excellent delivery system in delivering antigens to immunocompetent cells. The present inventors have so far conducted research on the development of liposome vaccines for livestock and have provided liposomes containing phospholipids such as phosphatidylcholine that are useful as adjuvants (Patent Documents 1 to 3, etc., Non-Patent Documents 1 to 3, etc.) . However, these are for mucosal administration such as oral, nasal, or intraocular administration, and no liposomes suitable for transdermal administration have been researched and developed.

On the other hand, for cosmetic liposomes, a liposome containing phosphatidylcholine and phosphatidic acid is disclosed, and regarding the liposome, there is a conventional technique (Patent Document 4) that describes a flexible membrane structure and high skin absorbability. This has not been studied for application to transdermal vaccines.
In addition, there is a document that describes the use of liposomes for percutaneous administration of influenza vaccines (Patent Document 5). Here, the liposomes to be used are specifically examined and disclosed. Not in.

JP 2006-111540 A WO2007 / 091580 JP 2009-286730 A JP 2009-269871 A JP 2001-151698 A

J. Vet. Med. Sci., 59 (12), 1109-1114 (1997) Development and Comparative Immunology 28, 29-38 (2004) Biomaterials 31, 943-951 (2010)

  An object of the present invention is to provide a liposome that is excellent as a transdermal absorbent carrier and that is capable of inducing a high immune response and is optimal as a transdermal absorbent carrier.

The present inventor has intensively studied to apply the liposomes developed by the present inventors for mucosal administration to transdermal administration.
For transdermal administration, it is essential that the liposomes have excellent epidermal permeability.
That is, for non-invasive transdermal administration, passage through the skin barrier is essential, but since there are intracellular lipids in the epidermis, liposomes that are lipids are difficult to pass through.
Therefore, the transdermal administration agent is dispersed and administered in alcohol for permeation of the epidermis. However, since the alcohol dissolves oil, the present inventors have established that the liposome for transdermal absorption agent has a certain level of alcohol stability. I found that sex is necessary.

  Furthermore, as a result of intensive studies on a liposome composition that is stable in alcohol suitable for a transdermal absorbent and soaked in the epidermis, the present inventor has formulated a combination of phospholipids having a high phase transition temperature, so that the liposome membrane has a certain amount. The present inventors have found that liposomes that can give hardness and constant fluidity and that are excellent in alcohol stability can be provided.

More specifically, DPPC, DSPC, DPPS, DSPE-PEG-SA, HePC, and PEG-PE are selected as such phospholipids having a high phase transition temperature and alcohol stability, and are used in combination. Thus, it was possible to construct an optimal liposome for transdermal administration.
In the present specification, the following abbreviations are used.
DPPC: dipalmitoylphosphatidylcholine
DPPS: dipalmitoylphosphatidylserine
DSPC: distearoylphosphatidylcholine
DSPE-PEG-SA (same as PEG-PE): polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (polyethylen glycol (PEG) and stearic acid (SA) -conjugated distearoylphosphatidylethanolamine)
HePC: Hydrogenated egg yolk lecithin Chol: Cholesterol
DMPC: dimyristylphosphatidylcholine

The phase transition temperatures of these compounds are as follows.
DPPC: 41-42 ° C
DPPS: 65 ° C
DSPC: 54-58 ° C
DSPE-PEG-SA (PEG-PE): 65-70 ° C
HePC: 50-56 ° C
Chol: None DMPC: 23-24 ° C

  Further, as a result of studying transdermal immunity by preparing a skin absorbent using the liposome of the present invention and applying it to the skin, it was found that mucosal immunity can be unexpectedly induced.

  Furthermore, the present inventor has also examined the relationship between the particle size of the liposome and the alcohol stability and immunity-inducing property, and found that there is an optimum particle size for alcohol stability and immunity-inducing property.

More specifically, the present invention relates to the following.
[1] A carrier for a transdermal absorbent comprising liposomes that are stable in alcohol.
[2] The carrier for transdermal absorption according to [1] above, comprising liposomes having a release rate of carboxyfluorescein (CF) of 50% or less when reacted in PBS containing 20% ethanol for 2 hours.
[3] The percutaneous absorption carrier according to the above [1] or [2], comprising a liposome formed by blending at least 50% of a phospholipid having a phase transition temperature of 50 ° C. or higher.
[4] Phospholipids are dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylserine (DPPS), hydrogenated egg yolk lecithin (HePC), and polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine ( A carrier for a transdermal absorbent comprising the liposome according to any one of the above [1] to [3], which is one or more selected from DSPE-PEG-SA).
[5] Transcutaneous comprising the liposome according to any one of [1] to [4] above, comprising hydrogenated egg yolk lecithin (HePC), cholesterol (Chol), and dipalmitoylphosphatidylserine (DPPS) Absorbent carrier.
[6] The carrier for transdermal absorbent comprising liposomes according to claim 5, wherein the molar ratio of hydrogenated egg yolk lecithin (HePC): cholesterol (Chol) is 1: 1 to 4: 1.
[7] The liposome is distearoylphosphatidylcholine (DSPC) / dipalmitoylphosphatidylserine (DPPS) / cholesterol (Chol) / polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SA), hydrogenated egg yolk lecithin (HePC) / cholesterol (Chol) / polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SA), hydrogenated egg yolk lecithin (HePC) / dipalmitoylphosphatidylserine (DPPS) / cholesterol (Chol) / Polyethylene glycol-stearic acid-conjugated distearoyl phosphatidylethanolamine (DSPE-PEG-SAE), hydrogenated egg yolk Chin (HePC) / dipalmitoyl phosphatidylserine (DPPS) / cholesterol (Chol) of the percutaneous absorption agent for the carrier is either.
[8] The carrier for a transdermal absorbent according to any one of [1] to [7], wherein the average particle size is 0.6 to 1 μm.
[9] A transdermal vaccine containing the carrier for transdermal absorbent according to any one of [1] to [8].

The liposome carrier for a transdermal absorbent of the present invention is useful for controlling livestock infections, particularly when used for a transdermal vaccine. Since the present invention can be applied to new prevention methods and treatment methods for infectious diseases of livestock, it can be used for safer production of livestock products.
Moreover, the existing injection vaccine can be made into a transdermal vaccine by using the existing vaccine antigen. This indicates the possibility that the transdermal vaccine can be applied in a form that suppresses the development cost of the vaccine.

It is a figure which shows the immunity induction (serum IgG) by transcutaneous immunity. It is a figure which shows the immunity induction (serum IgA) by transcutaneous immunity. It is a figure which shows the induction | guidance | derivation (42nd day) of the intestinal antibody by transdermal immunity. It is a figure which shows the antibody production (serum anti-OVA-IgG) after 20% ethanol stable liposome percutaneous immunization. It is a figure which shows the antibody production (serum anti- OVA-IgA) after 20% ethanol stable liposome transcutaneous immunization. It is a figure which shows the antibody production (intestinal anti-OVA-IgG) after 20% ethanol stable liposome percutaneous immunization. It is a figure which shows the antibody production (intestinal anti-OVA-IgA) after 20% ethanol stable liposome percutaneous immunization. Immunity induction with 20% ethanol-resistant liposome transdermal vaccine (Adjuvant comparison: serum IgG) Immunity induction with 20% ethanol-resistant liposome transdermal vaccine (adjuvant comparison: serum IgA) Immunity induction with 20% ethanol-resistant liposome transdermal vaccine (adjuvant comparison: IgG subclass, Day 42) Immunity induction with 20% ethanol-resistant liposome transdermal vaccine (Adjuvant comparison: Intestinal IgA)

The present invention will be described in detail below, but the present invention is not limited to the following.
As described above, the present invention has been found to be suitable as a carrier for transdermal absorption by making liposomes alcohol-stable, but in the present invention, 20 is used as an index indicating the alcohol stability of liposomes. The degree of release of carboxyfluorescein (CF) when reacted in PBS containing% ethanol for 2 hours was used (hereinafter sometimes simply referred to as “CF degree of release”). For example, even if the degree of CF release is 80%, it can be used as a carrier for transdermal absorption. However, the liposome membrane may be broken and the encapsulant such as an antigen may leak and the encapsulant passing through the skin may be reduced. Is not efficient. Therefore, the CF release rate preferred in the present invention is about 50% or less, more preferably about 30% or less, and still more preferably about 20% or less.

Such alcohol-stable liposome can be achieved by blending a phospholipid having a high phase transition temperature. This is because, by combining phospholipids having a high phase transition temperature in combination, the liposome membrane can be given a certain hardness and a certain fluidity.
Liposomes are preferably formed by blending at least 50 mol% of phospholipids having a phase transition temperature of 50 ° C. or higher. Here, to form liposomes by blending at least 50 mol% or more of phospholipids having a phase transition temperature of 50 ° C. or higher, select phospholipids having a phase transition temperature of 50 ° C. or higher and blending at least 50 mol% or more thereof. It is sufficient to form liposomes, and if it does not exceed 50 mol%, it indicates that it may contain phospholipids and other lipids (such as cholesterol) having a phase transition temperature of 50 ° C. or higher. More preferably, the phospholipid having a phase transition temperature of 50 ° C. or higher is 80 mol% or more, and conversely, if it is about 50 mol% or less, the alcohol stability is undesirably lowered.
Phospholipids having a phase transition temperature of 50 ° C. or higher include distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylserine (DPPS), hydrogenated egg yolk lecithin (HePC), and polyethylene glycol-stearic acid-conjugated distearoyl phosphatidylethanolamine ( DSPE-PEG-SA) and hydrogenated egg yolk lecithin (HePC). Hydrogenated egg yolk lecithin (HePC) is particularly preferable because it is inexpensive and easily available.

  Liposomes can contain cholesterol other than phospholipids (Chol), but when cholesterol (Chol) is added to hydrogenated egg yolk lecithin (HePC), hydrogenated egg yolk lecithin (HePC): cholesterol (Chol) More preferably, the molar ratio is 1: 1 to 4: 1.

As a specific formulation example, distearoylphosphatidylcholine (DSPC) / dipalmitoylphosphatidylserine (DPPS) / cholesterol (Chol) / polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SA); hydrogenated egg yolk Lecithin (HePC) / cholesterol (Chol) / polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SA); hydrogenated egg yolk lecithin (HePC) / dipalmitoylphosphatidylserine (DPPS) / cholesterol (Chol) / Polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SAE); hydrogenated egg yolk Lecithin (HepC) / dipalmitoyl phosphatidylserine (DPPS) /, and the like cholesterol (Chol).
Here, for example, distearoylphosphatidylcholine (DSPC) / dipalmitoylphosphatidylserine (DPPS) / cholesterol (Chol) / polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SA) is DSPC, DPPS, The combination of cholesterol and DSPE-PEG-SA is shown.

  In addition, the average particle size of the liposome used as a transdermal absorption carrier is preferably about 0.6 to 1 μm. This average particle size is a value indicated by the pore size of the extruder through which the liposome passes.

  The liposome of the present invention can be suitably used as a carrier for a transdermal vaccine. In that case, an adjuvant may be further added to the transdermal vaccine in addition to the liposome. As an adjuvant that can be used, poly I: C, MPL, imidazoquinoline derivatives, pathogen-related molecular patterns such as TDM (Toll like receptors, NOD-like receptors, RIG-I-like receptors, C-type lectin receptlrs, etc.) In addition, saponin, α-galactosylceramide (αGalCer), squalene, ADP-ribosylated exotoxin, cytokine, chemokine and the like can be mentioned, saponin, αGalCer and MPL are preferable, and αGalCer is particularly preferable.

  The following examples specifically show that alcohol-resistant liposomes were constructed in the present invention, and the optimum particle size and usefulness as a transdermal absorbent were confirmed. Furthermore, the suitability was tested by changing the type of adjuvant used in the transdermal vaccine.

<Production of alcohol-resistant liposomes>
The present inventor, referring to the composition of liposomes containing phospholipids such as phosphatidylcholine (see Patent Documents 1 to 3) that the present inventors have developed and studied for mucosal administration of vaccines to livestock, has the following composition: Alcohol resistance was investigated.
# 1.DPPC: DPPS: Chol: DSPE-PEG-SA = 1: 1: 2: 0.2 (molar ratio)
# 2.DSPC: Chol: DSPE-PEG-SA = 7: 2: 0.2 (molar ratio)
# 3.DSPC: DPPS: Chol: DSPE-PEG-SA = 7: 3: 2: 0.2 (molar ratio)
# 4.DSPC: DPPS: Chol: DSPE-PEG-SA = 1: 1: 2: 0.2 (molar ratio)
# 5.DMPC: DPPS: Chol: DSPE-PEG-SA = 1: 1: 2: 0.2 (molar ratio)
# 6.DSPC: DPPS: Chol = 1: 1: 2 (molar ratio)

Liposomes encapsulating carboxyfluorescein (CF) were prepared by the thin film method shown below. Phospholipid and cholesterol are dissolved in a solvent (chloroform / methanol mixture) to a concentration of 10 mmol and mixed so that the ratio (molar ratio) of phospholipid and cholesterol is shown in # 1 to # 6, respectively. Into a glass container. The lipid film is attached to the side of the container by distilling off the solvent in the glass container with a rotary evaporator. A 0.05 M concentration of CF aqueous solution was added thereto, and the mixture was stirred with a shaker to remove the lipid membrane from the container. Thus, each liposome encapsulating CF was obtained.
Each liposome encapsulating CF was suspended in PBS (phosphate buffer saline) containing 0%, 2%, 5%, 10% and 20% ethanol, and reacted at room temperature for 30 minutes, 1 hour and 2 hours. The amount of CF released during each reaction time was measured, and the lipid composition and composition ratio of alcohol-resistant liposomes were examined.

The results are as shown in Table 1.
The phase transition temperatures of DPPC, DSPC, DPPS, DPSE-PEG-SA, and DMPC used for the compositions # 1 to # 6 are 41 to 42 ° C., 54 to 58 ° C., 65 ° C., 65 to 70 ° C., and 23 to 23 ° C., respectively. It is 24 ° C. and no phase transition temperature is observed in Chol. In the compositions # 1 to # 6, the proportions of phospholipids having a phase transition temperature of 50 ° C. or higher were 28.6 mol% for # 1, 80.0 mol% for # 2, and 86.3 mol% for # 3, respectively. Alcohol resistance increases as the proportion of phospholipids having a phase transition temperature of 50 ° C. or higher increases with 52.4 mol% for # 4, 28.6 mol% for # 5, and 50.0 mol% for # 6. It became high, and when the ratio of the phospholipid which is 50 degreeC or more was less than 50 mol%, it turned out that alcohol resistance becomes low (CF leak rate (%) becomes high). In Table 1,-indicates that no test was performed.

<Confirmation of antibody production>
From the results shown in Table 1, the ability to induce immunity by transdermal administration was confirmed using # 3 liposomes with the highest alcohol resistance. At that time, monophosphoryl lipid A (MPL) was used as an adjuvant for the # 3 liposome (2 μg MPL / mouse). In this experiment, the effect of liposome particle size (1 μm, 0.6 μm) on antibody production was also confirmed.

For transcutaneous immunization, ovalbumin (OVA) was used as a model antigen at 100 μg / mouse, and the induction of serum IgG, serum IgA, and intestinal antibody on day 42 was measured after 0, 14, and 28 days, respectively. 2 and FIG.
Confirmation of immunity induction ability was performed by the following method.
Liposomes encapsulating OVA were prepared in the same manner as CF encapsulated liposomes. This was dispersed in 20% ethanol-containing PBS and immunized by applying to the back of the mouse so that the amount of OVA was 100 μg / mouse. Immunization was performed 3 times at 14-day intervals. Blood was collected before the start of immunization (day 0) and every 14 days after the start (Day 14, Day 28, Day 42), and the serum IgG and IgA antibody titers specific for OVA at each time point were measured by ELISA. The small intestine collected at Day 42 was suspended in PBS, and the OVA-specific IgG and IgA antibody titers in the supernatant were measured by ELISA using intestinal antibodies.
At that time, OVA was used as a control.

From the results shown in FIGS. 1 to 3, OVA-encapsulated liposomes have significantly superior immunity against OVA in any of serum IgG, serum IgA, intestinal IgG, and intestinal IgA, regardless of particle size of 0.6 μm or 1 μm. It turned out that it showed the characteristic.
From these results, it was found that immunity can be induced transcutaneously using liposomes having a high phase transition temperature and high alcohol resistance. In particular, it was found that high transdermal immunity induction was obtained when # 3 containing 80 mol% or more of phospholipid having a CF release degree of 20% or less and a phase transition temperature of 50 ° C. or more was used.

As another example, alcohol resistance and immunity induction were confirmed for liposomes using HePC (hydrogenated egg yolk lecithin), which is inexpensive and easily available, as the liposome material.
Experiments were performed according to Example 1 except for the liposome composition. In addition, the phase transition temperature of HePC (hydrogenated egg yolk lecithin) is 50 to 56 ° C., and the phase transition temperature of DPPS is 65 ° C. Further, as in Example 1, monophosphoryl lipid A (MPL) was used as an adjuvant (2 μg MPL / mouse).

<Alcohol resistance>
The composition of liposomes in Example 2 and the alcohol resistance thereof are as shown in Table 2 below.

<Confirmation of immune induction>
From Table 2, the compositions of # 1, 3 and 5 having particularly high alcohol resistance were selected, and transdermal immunity induction was performed. The results are shown in FIGS. 4 to 7, # 1, 3 and 5 are shown as groups 3, 2 and 1, respectively. In particular, high alcohol-resistant liposomes having a CF release rate of 20% or less have shown high induction of transcutaneous immunity.

  From the results of Examples 1 and 2 described above, it was found that liposomes useful for transcutaneous immunity can be obtained by using liposomes with high alcohol resistance and high phase transition temperature.

<Adjuvant types>
For the transdermal liposome of the present invention, a test in which the adjuvant was changed was performed in order to select an optimal substance as an adjuvant.
A transcutaneous immunity test was also performed according to Example 1 except that # 3 liposome of Example 1 was used and saponin, MPL, and αGalCer were used as adjuvants.
The results are shown in FIGS.
As can be seen from the results of FIGS. 8 to 11, saponin, MPL, αGalCer, immunity with serum IgG, serum IgA, and intestinal IgA are significantly different from control (no adjuvant). It has been found that αGalCer is most preferable, since the characteristics are obtained.

The carrier for transdermal absorbent of the present invention is useful for controlling livestock infectious diseases, particularly when used as a transdermal vaccine. Since the present invention can be applied to new prevention methods and treatment methods for infectious diseases of livestock, it can be used for safer production of livestock products.
Moreover, the existing injection vaccine can be made into a transdermal vaccine by using the existing vaccine antigen. This indicates the possibility that the transdermal vaccine can be applied in a form that suppresses the development cost of the vaccine.

Claims (9)

A carrier for a transdermal absorbent comprising liposomes that are stable in alcohol. The carrier for a transdermal absorption agent according to claim 1, comprising a liposome having a carboxyfluorescein (CF) release rate of 50% or less when reacted in PBS containing 20% ethanol for 2 hours. The carrier for a percutaneous absorption according to claim 1 or 2, comprising a liposome formed by blending at least 50 mol% of a phospholipid having a phase transition temperature of 50 ° C or higher. Phospholipids are dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylserine (DPPS), hydrogenated egg yolk lecithin (HePC), and polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG) The carrier for transdermal absorbents comprising the liposome according to any one of claims 1 to 3, wherein the carrier is one or more selected from -SA). The carrier for transdermal absorbents comprising liposomes according to any one of claims 1 to 4, comprising hydrogenated egg yolk lecithin (HePC), cholesterol (Chol), and dipalmitoyl phosphatidylserine (DPPS). The carrier for transdermal absorbents comprising liposomes according to claim 5, wherein the molar ratio of hydrogenated egg yolk lecithin (HePC): cholesterol (Chol) is 1: 1 to 4: 1. Liposomes are distearoylphosphatidylcholine (DSPC) / dipalmitoylphosphatidylserine (DPPS) / cholesterol (Chol) / polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SA), hydrogenated egg yolk lecithin (HePC) / Cholesterol (Chol) / polyethylene glycol-stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SA), hydrogenated egg yolk lecithin (HePC) / dipalmitoylphosphatidylserine (DPPS) / cholesterol (Chol) / polyethylene glycol- Stearic acid-conjugated distearoylphosphatidylethanolamine (DSPE-PEG-SAE), hydrogenated egg yolk lecithin HepC) / dipalmitoyl phosphatidylserine (DPPS) / Cholesterol (Chol) transdermal absorbent carrier is either. The carrier for transdermal absorbent according to any one of claims 1 to 7, wherein the average particle diameter is 0.6 to 1 µm. A transdermal vaccine comprising the carrier for a transdermal absorbent according to any one of claims 1 to 8.