JP2012001461A - S/o formulation comprising gold nanorod - Google Patents

Abstract

PROBLEM TO BE SOLVED: To further improve skin permeability toward utilization of an S/O formulation for percutaneous drug delivery anticipating a systemic action.SOLUTION: The S/O formulation for percutaneous drug delivery comprises: a drug; and a gold nanorod having an absorption band in the region of 650 to 2,000 (preferably, of 680 to 1,200 nm, more preferably, of 700 to 900 nm). The gold nanorod is the one made to be biocompatible, preferably, is the one subjected to surface modification with polyethylene glycol. In the particularly preferable embodiment, the drug is included in a solid phase (S, a dispersion phase) together with the gold nanorod and a surfactant.

Description

  The present invention relates to a preparation containing gold nanorods. The formulations of the present invention are particularly suitable for transdermal drug delivery and are useful in fields such as medicine and pharmaceuticals.

  A transdermal drug delivery system (TDDS) is a system that continuously administers a drug from the skin and performs treatment. There are two types of TDDS, one that expects local effects of drugs and one that expects systemic effects. When systemic action is expected, the drug must reach the deep part of the skin and be taken into the blood, but the skin has a barrier function in the stratum corneum that forms the uppermost part of the epidermis. The penetration of the drug into the stratum corneum is the rate-limiting step. Therefore, transdermal patches (a system that reduces the barrier function by preventing water evaporation from the skin and hydrates the stratum corneum and promotes the penetration of drugs into the skin), chemical enhancers (functions that improve skin permeability) Technology that promotes permeation of the drug into the stratum corneum by coexisting a drug with a drug, and iontophoresis (a technology that attracts a charged drug into the skin by passing a weak electric current through the skin). I came. However, application has been limited to transdermal delivery of relatively low molecular weight hydrophobic drugs.

  Therefore, in order to transdermally deliver a high-molecular substance such as insulin, which is considered to be difficult to apply by these methods, researches focusing on the stratum corneum as a barrier are being actively promoted. For this, electroporation (a technology that penetrates macromolecules into the skin by irreversibly generating small pores in the stratum corneum by applying high voltage electricity in a short time), sonophoresis Method (Technology that administers high-molecular substances using the rise of skin temperature by applying ultrasonic waves to the skin, destabilization of lipids between keratinocytes, generation of cavitation, etc.), microneedle method (micrometer size) There is a technique in which a small hole is made in the stratum corneum without reaching the nerve by using a needle to penetrate a macromolecular drug into the skin. However, each of them has a problem that it requires a large-sized device, lacks in convenience, and damages the skin, and has not been put into practical use.

  Recently, as a transdermal drug delivery system using thermal energy, reports of increasing the transdermal delivery efficiency of human growth hormone, DNA, etc. by applying heat using high frequency to the skin stratum corneum, and laser irradiation There is a report that the skin permeability of molecules such as dextran is improved (Non-patent Document 1). Furthermore, the S / O formulation in which the solid phase containing the drug is dispersed in the oil phase is stable against hydrolysis because the drug exists as a solid phase, and the tape formulation is easy to apply to the skin. Have been studied as an effective means for a drug delivery system (Drug Delivery System, DDS) (Patent Documents 1 to 3).

  On the other hand, gold nanorods are rod-shaped gold nanoparticles and have the property of absorbing light in the vicinity of 800 to 900 nm (near infrared region) derived from the long axis direction and converting the absorbed light into heat. Because near-infrared light has high tissue permeability, gold nanorods are expected to be used as a material to support medical technologies such as bioimaging. On the other hand, gold nanorods must be used during manufacturing and exist as a bilayer on the gold nanorod surface. Since hexadecyltrimethylammonium bromide (CTAB), which contributes to the dispersion stability of gold nanorods in water, has strong cytotoxicity, its application to living bodies has been limited. In order to solve this problem, gold nanorods that have been surface-modified with polyethylene glycol (PEG) chains have been developed instead of CTAB as a biocompatible material while maintaining dispersion stability. 2) Application to biological near-infrared spectroscopy has been studied (Patent Document 4).

International Publication WO2005 / 094789 International Publication WO2006 / 025582 Publication JP 2008-179561 A JP 2009-240586

Jung-Hwan Park et al., International Journal of Pharmaceutics, 359, 94-103, (2008) Niidome, M. Yamagata, Y. Okamoto, Y. Akiyama, H, Takahashi, T. kawano, Y. Katayama, Y. Niidome, J. Controlled Release, 114, 343-347 (2006)

  The inventors of the present invention administered an S / O preparation encapsulating insulin having a molecular weight of about 6,000 daltons fluorescently labeled with fluorescein isothiocyanate (FITC) as a drug, and evaluated skin permeability. As a result, fluorescence was observed up to the dermis 48 hours after administration, and it was confirmed that the penetration of the drug into the stratum corneum was promoted. Also. A similar experiment was conducted with horseradish peroxidase (HRP) having a molecular weight of approximately 39,000 daltons, and the improvement of skin permeability with S / O preparations was confirmed, and the enzymatic activity of HRP penetrating the skin was retained. I also confirmed that. However, the insulin S / O preparation has a skin permeability that is almost as low as that of the negative control insulin solution 12 hours after administration, and a significant decrease in blood glucose level can be confirmed in in vivo experiments using rabbits. There wasn't. Therefore, it is necessary to further improve the skin permeability in order to put the S / O preparation for transdermal delivery expected to have a systemic effect into practical use.

  Gold nanorods absorb near infrared light and generate heat. Therefore, when a preparation containing gold nanorods is applied to the skin and irradiated with near-infrared light, the stratum corneum is heated locally in a short time, and improvement in skin permeability is expected. Therefore, an S / O preparation containing a drug and gold nanorods was actually applied to the mouse skin and irradiated with near infrared light using a xenon lamp as a light source. As a result, the penetration of more drugs into the skin was confirmed, and the present invention was completed.

The present invention provides the following:
1) An S / O preparation for transdermal drug delivery comprising a drug and a gold nanorod having an absorption band in a region of 650 to 2000 nm (preferably 680 to 1200 nm, more preferably 700 to 900 nm).
2) The preparation according to 1), wherein the gold nanorod is biocompatible, preferably surface-modified with polyethylene glycol.
3) The preparation according to 1) or 2), wherein the drug forms a solid phase (S, dispersed phase) together with the surfactant.
4) The preparation according to 3), wherein gold nanorods are contained in the solid phase.
5) The preparation according to any one of 1 to 4 for treatment or prevention.
6) The preparation according to 5), wherein the drug is a molecule or polypeptide having a molecular weight of 500 daltons or more.
7) The preparation according to 6), wherein the polypeptide has a molecular weight of 6000 daltons or more, preferably 30,000 daltons or more.
8) The preparation according to any one of 5) to 7), wherein the drug is an antigen.
9) The preparation according to any one of 1) to 8), which is a tape preparation.
10) Use of gold nanorods in therapeutic or prophylactic formulations.

NR-S / O formulation. From the appearance, it was found that PEG-NR was stably dispersed in the oil. Absorption spectrum of NR-S / O formulation. It was found that the prepared NR-S / O preparation showed a peak in the near infrared region. Electron microscope image of NR-S / O preparation. It was confirmed that PEG-NR was encapsulated in the particles. Temperature change of NR-S / O preparation solution by near infrared irradiation. Ten minutes after irradiation, the temperature of the solution rose to around 40 ° C., confirming that the NR-S / O preparation exhibited a photothermal effect. Skin penetration of FITC-insulin by fluorescence microscopy. NR-S / O preparations with near-infrared irradiation showed higher skin penetration than S / O preparations and negative control FITC-insulin solution. It was found from the fluorescence intensity inside the skin. Skin permeability of FITC-insulin by mean fluorescence intensity distribution. It was suggested that the skin thermal permeability of insulin is further improved by the photothermal effect of NR. Skin penetration of FITC-ovalbumin by fluorescence microscopy. It can be seen that the combination of the NR-S / O preparation and near-infrared light irradiation shows higher skin permeability than the S / O preparation containing no NR. Distribution of gold nanorods in tissue by scanning electron microscope. A strong signal (white area) was observed on the skin surface or outside the skin, and it was found that the gold nanorods did not penetrate into the skin. EDX analysis of white area. It was confirmed that gold nanorods exist in the strong signal (white region).

The preparation of the present invention is an S / O preparation containing a drug and gold nanorods, using the solid phase (S) as a dispersed phase and the oil phase (O) as a dispersion medium.
[Gold nanorods]
In the present invention, “gold nanorod” refers to rod-shaped gold fine particles having a major axis length of less than 400 nm and an aspect ratio (major axis length / minor axis length) of greater than 1, unless otherwise specified. Say.

  In the preparation of the present invention, gold nanorods having an absorption band in the near infrared region, more specifically in the region of 650 to 2000 nm are used. In the present invention, when “having an absorption band in the region of λ1 to λ2 nm” for a gold nanorod, the maximum absorption wavelength is determined when the spectral characteristics (absorption spectrum) of the gold nanorod is measured, unless otherwise specified. When it is in the range of ± 100 nm of the range. For example, when a certain gold nanorod “has an absorption band in the region of 650 to 2000 nm”, it means that the peak of the maximum absorption wavelength of the gold nanorod is in the region of 550 to 2100 nm. Methods for measuring spectral characteristics are well known to those skilled in the art. For example, an absorption spectrum can be obtained using a spectrophotometer that can prepare an aqueous dispersion of gold nanorods having an appropriate concentration and can measure absorption in the near infrared region.

  The gold nanorod used in the preparation of the present invention preferably has an absorption band at 680 to 1200 nm. Since 700-900 nm near infrared light has the property of being easily transmitted through living tissue including skin, it is effective to irradiate such near infrared light, and gold having an absorption band in the 700-900 nm region. A preparation using nanorods is a particularly preferred embodiment of the present invention.

  The size of the gold nanorod that can be used in the present invention is not particularly limited as long as the long axis has a length of 400 nm or less and has near-infrared light absorption as described above. It is preferably 200 nm or less, and more preferably 100 nm or less. In general, gold nanorods have an absorption ratio that shifts to the longer wavelength side as the aspect ratio increases. Therefore, when designing gold nanorods having a preferable absorption spectrum, the aspect ratio may be about 4 to about 7. it can.

  In the present invention, the gold nanorods are preferably biocompatible. Commonly, gold nanorods are synthesized in water dissolved in hexadecyltrimethylammonium bromide (CTAB), a quaternary ammonium salt, which is a cationic surfactant, and have a bilayer film of CTAB on the surface (Japanese Patent Laid-Open No. 2004-292627) JP, 2005-97718, JP 2006-169544, JP 2006-118036), the preparation of the present invention administered to a living body, except for surface CATB by an appropriate method, The rod surface may be modified with a compatible material.

  As a method for this, for example, surface modification with α-methoxy-ω-mercaptopolyethylene glycol (ME-PEG-SH) having a polymerization average molecular weight of 1000 to 40000, preferably 2000 to 30000, more preferably 2500 to 7500 is possible. Can be mentioned. When the polymerization average molecular weight is small, the dispersion stability in a polar solvent may be inferior. The upper limit value of the average molecular weight may be determined from an economical viewpoint.

  Surface modification with ME-PEG-SH can be performed by adding ME-PEG-SH to an aqueous dispersion of gold nanorods from which excess CTAB has been removed and stirring. ME-PEG-SH is adsorbed on the gold nanorod surface by the terminal thiol group. Unadsorbed mPEG-SH can be removed by dialysis or the like. Gold nanorods surface-treated with ME-PEG-SH can be stably dispersed in a polar solvent such as water or alcohol (eg, ethanol).

  An example of a preferred modifier is ME-PEG-SH with an average molecular weight of about 5000. The amount of addition is preferably in the concentration range of 0.01 to 10 mM, preferably 0.5 to 2 mM, with respect to the gold concentration of 1 mM in the dispersion. If the concentration of ME-PEG-SH is lower than 0.01 mM, the dispersion in the solvent becomes unstable and cannot be stably dispersed. On the other hand, when the concentration of ME-PEG-SH is higher than 10 mM, the amount of excess agent that is not adsorbed on the gold nanorods cannot be ignored.

  The content of gold nanorods in the preparation of the present invention includes the effect of heat on the application site, the effect of promoting skin permeation of the target drug, the dispersion stability of the gold nanorod itself or the solid phase containing it, and the relative to the drug. It can be determined as appropriate in consideration of the amount and economy. In the study by the present inventors, an S / O preparation in which the gold nanorod content was changed in the range of 0.1 mM to 1.0 mM (in terms of gold atom) was applied to the extracted pig skin at ambient temperature, and a near infrared laser beam was applied. As a result of measurement with a radiation thermometer, the skin temperature rose to about 38 ° C to 45 ° C. On the other hand, when the concentration was less than 0.1 mM, no increase in the temperature of the application site could be observed under the experimental conditions. Therefore, it is preferable that the lower limit of gold nanorods in the preparation is 0.1 mM or more. From the viewpoint of further enhancing the thermal effect on the application site, it is more preferably 0.3 mM or more, and further preferably 0.5 mM or more. On the other hand, when the gold nanorod content is 5.0 mM (in terms of gold atoms) or more, the gold nanorods are likely to aggregate and the near-infrared absorption is expected to decrease rather. Therefore, from the viewpoint of dispersion stability, the upper limit value of the gold nanorods is preferably 5.0 mM or less.

  In this specification, when referring to the amount (concentration) of gold nanorods in the preparation, it means the amount (concentration) converted to gold atoms, unless otherwise specified. The number of moles per unit volume. Methods for measuring the concentration of gold nanorods are well known to those skilled in the art. For example, an aqueous dispersion of gold nanorods having an appropriate concentration can be prepared, and the concentration can be obtained from absorbance at an appropriate wavelength (for example, near the maximum absorption wavelength) using a spectrophotometer.

  In the preparation of the present invention, the photothermal (light heat) effect of gold nanorods is utilized. It is understood that the heat generation due to the photothermal effect of the gold nanorods is a local and temporary heat generation limited to a range of several tens of μm on the surface, unlike the case of a normal heat source outside the body. Therefore, when the preparation of the present invention is applied to the skin, fever is limited only to the stratum corneum, and it is considered that the barrier function of the stratum corneum is destabilized without damaging living tissues below the stratum corneum. By reducing the barrier function of the stratum corneum, the preparation of the present invention can further promote the skin permeability of the drug.

  According to the study by the present inventors, it was confirmed that the gold nanorods in the preparation did not permeate into the skin when the preparation of the present invention was applied to the skin under the experimental conditions (see Example 6). ).

[Drug]
Various drugs can be used as the active ingredient of the preparation of the present invention. The drug is preferably present in the solid phase and dispersed in the oil phase in the formulations of the present invention.

In general, hydrophilic, high molecular weight drugs are difficult to penetrate the stratum corneum of the skin. The formulations of the present invention are particularly useful for drugs that are hydrophilic and / or high molecular weight.
The formulations of the present invention are suitable for delivering molecules or polypeptides having a molecular weight of 500 Daltons or higher. When using a polypeptide, its molecular weight can be 6000 daltons or more, and can be 30000 daltons or more. In the present invention, various polypeptides used as active ingredients of proteinaceous pharmaceuticals can be applied. Examples of such polypeptides include insulin, growth hormone (somatropin), somatomedin, glucagon, urokinase, prourokinase, erythropoietin, G-CSF, IL-2, INF (α, β, γ), tPA, VIII Factors, antibodies, recombinants thereof, and pharmaceutically acceptable analogs thereof.

  According to the study of the present inventors, when an NR-S / O preparation encapsulating ovalbumin was applied to mouse skin and irradiated with near infrared light, antibody production was observed in a single immunization operation. However, after the second immunization, antibody production was 8 times higher than when the S / O preparation containing no gold nanorods was applied. This was considered to be because the ovalbumin permeability was improved by the photothermal effect of the gold nanorods (see Example 7). Therefore, the preparation of the present invention can be an immunological preparation containing an antigen. The antigen is not particularly limited as long as it induces immunogenicity (antibody production), and antigens and immunogenic substances that have been conventionally used for immunization can also be used in the preparation of the present invention. Examples of these include antigenic proteins and peptides (eg, allergenic proteins, antigenic region peptides), haptenizing substances bound with proteins and peptides, bacteria and viruses, partial degradation products thereof, inactivation products thereof, There are nucleic acids. The preparation of the present invention can be used as various vaccines (for infectious diseases, poisoning, cancer, neurological diseases, and autoimmunity). As will be described later, the preparation of the present invention will be useful as a safer and easier-to-use vaccine agent by making it an easy-to-apply dosage form impregnated in an appropriate base material.

[Solid phase (complex production method)]
The preparation of the present invention is typically an S / O type preparation in which the dispersed phase is a solid phase (S) whose solid phase is a solid and the continuous phase is an oil phase (O) based on an oily substance. is there. The solid phase can contain a drug that is an active ingredient and can contain gold nanorods. The drug and the gold nanorod can be contained in the same solid phase, or can be contained in different solid phases.

In the S / O formulation of the present invention, the solid phase is typically formed as a complex of a drug and / or gold nanorod and a surfactant.
There is no restriction | limiting in particular in manufacture of surfactant complex, The various method for preparing S / O formulation can be applied. Typically, gold nanorods are dispersed in an aqueous solution of a drug, emulsified and dispersed in an organic solvent containing a surfactant to prepare a W / O emulsion, and then the obtained W / O emulsion is By lyophilization in a conventional manner. The organic solvent is not particularly limited as long as it can dissolve the lipophilic surfactant and can be removed by lyophilization, but usually a lower alcohol such as ethanol or a hydrocarbon having a low boiling point such as hexane is used. Is done. Emulsification / dispersion can be carried out with a conventional apparatus such as a stirrer, a high-speed rotary shear stirrer (such as a propeller mixer), a colloid mill, a homogenizer, a flow jet mixer, an ultrasonic emulsifier, or a vacuum emulsifier. The particle size of the dispersed droplets can be controlled by adjusting the stirring strength (= power × time). The detailed technology can be referred to Japanese Patent Application Laid-Open No. 2004-43355.

  The surfactant used for preparing the complex of the preparation of the present invention is a surfactant that can be added to a pharmaceutical preparation, preferably a surfactant that can be added to a pharmaceutical preparation for transdermal delivery. Thus, the solid phase can be stably dispersed in the oil phase.

  A suitable example of the surfactant used for complex preparation is a lipophilic nonionic surfactant having an HLB (Hydrophile-Lipophile Balance) value of 10 or less. Examples of lipophilic nonionic surfactants include sucrose fatty acid esters (sucrose stearate esters) that have a high degree of esterification (ie, a high proportion of di, tri, and polyester relative to the monoester). , Sucrose palmitic acid ester, sucrose myristic acid ester, sucrose oleic acid ester, sucrose lauric acid ester, sucrose erucic acid ester, sucrose mixed fatty acid ester), polyglycerin condensed ricinoleic acid ester, decaglycerin ester, glycerin There are fatty acid esters, polyglycerin fatty acid esters, polyoxyethylene glycerin fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbit fatty acid esters, polyoxyethylene castor oil and hydrogenated castor oil. Only one surfactant may be used, or two or more surfactants may be mixed and used.

  As the surfactant to be used, since the preparation of the present invention utilizes the photothermal effect of gold nanorods, it is preferable to have excellent heat stability, and the preparation of the present invention is exclusively for transdermal delivery. From the viewpoint of being present, those having actions such as absorption promotion, stimulation relaxation, and moisture retention are further preferred.

[Oil phase]
The oil phase of the preparation of the present invention may be in a liquid form, or may be in a solid form having fluidity or progressability.

  The base material of the oil phase of the preparation of the present invention is not particularly limited as long as it is an oily base material that can be used in a pharmaceutical preparation, preferably a pharmaceutical preparation for transdermal delivery. Either oil that is liquid at room temperature (25 ° C.) or fat that is solid at room temperature can be used. There are no restrictions on the origin of the raw material, and any of natural products and synthetic products can be used. For example, vegetable (for example, soybean oil, cottonseed oil, rapeseed oil, sesame oil, corn oil, peanut oil, safflower oil, sunflower Flower oil, olive oil, rapeseed oil, perilla oil, fennel oil, cacao oil, cinnamon oil, mint oil, bergamot oil) or animal (beef tallow, pork oil, fish oil). In addition, neutral lipids (eg, glycerides, triolein, trilinolein, tripalmitin, tristearin, trimyristin, triarachidonin), synthetic lipids, sterol derivatives (eg, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, Cholesteryl palmitate, cholesrelyl arachidate), long chain fatty acid esters (eg, isopropyl myristate, octyldodecyl myristate, cetyl myristate, ethyl oleate, ethyl linoleate, isopropyl linoleate, isopropyl palmitate, butyl stearate) ), Carboxylic acid esters (ethyl lactate, cetyl lactate, triethyl citrate, diisopropyl adipate, diethyl sebacate, diisopropyl sebacate, 2-ethylhexanoic acid Chill), hydrocarbons (for example, petroleum jelly, paraffin squalane, vegetable squalane), and silicones. Only one kind of oily base material may be used, or plural kinds of oily base materials may be mixed and used.

The oily base material preferably has high compatibility with additives and is excellent in stability, antibacterial properties, moisture retention and the like.
A person skilled in the art can appropriately determine the amount of the oil phase in the preparation of the present invention in consideration of the concentration and amount of the active ingredient in the preparation, the area of the application site, and the like.

  Dispersion of the solid phase in the oil phase is performed using the above-described emulsification / dispersion, such as a stirrer, high-speed rotary shear stirrer (propeller mixer, etc.), colloid mill, homogenizer, flow jet mixer, ultrasonic emulsifier, vacuum. It can be performed with a conventional apparatus such as an emulsifier.

[Near-infrared light irradiation]
The formulation of the present invention is applied to the skin for transdermal delivery and subsequently used by irradiation with near infrared light. The means for irradiating near-infrared light to be used, the irradiation intensity, and the irradiation time can make the gold nanorods exhibit a photothermal effect effective for promoting the skin permeation of the drug, and the drug is an active ingredient There is no limit as long as it does not impair the effectiveness of the device and does not cause permanent damage to the application site. A person skilled in the art can determine appropriately with reference to the conditions of the embodiments of the present invention.

  A person skilled in the art can appropriately evaluate whether or not the photothermal effect is exhibited by irradiation with near infrared light and whether or not skin penetration is promoted. Whether or not skin penetration is promoted can also be evaluated based on whether or not a desired effect (for example, a clinical effect due to the systemic action of the drug) is exerted, and the drug concentration.

[Others]
The formulations of the present invention are used for the treatment or prevention of various diseases or conditions for transdermal delivery of drugs. “Prevention” includes reducing the risk of onset, and mildly, even if it occurs. “Treatment” includes coping treatment that suppresses symptoms and fundamental treatment such as eradication of pathogens or improvement of constitution, and also includes maintenance of symptoms, prevention of deterioration, and prevention of related symptoms.

  The preparation of the present invention may further contain a hydrophilic protein and / or a hydrophilic polysaccharide as a stabilizer (Patent Document 1). The formulations of the present invention also contain various pharmaceutically acceptable additives such as isotonic agents, stabilizers (eg, methylparaben, propylparaben, chlorobutanol, benzyl alcohol, phenylethyl alcohol, benzalkonium chloride, phenol , Cresol, thimerosal, acetic anhydride, sorbic acid, preservatives, wetting agents, preservatives, pigments, vitamins, chelating agents, refreshing agents, hormones, antioxidants, pigments, fragrances, etc. Various additives that can be included as long as they are acceptable as a formulation, and used for promoting transdermal delivery, such as hydrophilic organic solvents (alcohols such as ethanol and isopropyl alcohol, glycerin, propylene glycol, polyethylene) Polyhydric alcohols such as glycol), moisturizing and softening agents for the stratum corneum (eg pyrrolidone) , Hyaluronic acid, urea, urea derivatives, salicylic acids), aprotic solvents (DMF, DMSO, DMAC), unsaturated fatty acids (eg oleic acid), surfactants (eg lecithin), accelerators (eg Azone, Decylmethyl sulfoxide, urea on the ring).

  The S / O formulation of the present invention is applied to the skin for transdermal delivery. Application to the skin is, for example, by applying the liquid S / O preparation as it is to a place where the skin is thin and easy to apply, such as the inside of the arm.

  A person skilled in the art can appropriately design the dosage, administration interval, and administration period of the transdermal immunizing agent of the present invention in consideration of the disease or condition, the age, physique, sex, etc. of the subject. When the preparation of the present invention is a transdermal immunizing agent containing only an antigen without an adjuvant, for example, generally, the amount of the active ingredient is generally in the range of 100 to 2000 μg per administration per adult. More preferably, it is preferably administered at least twice or more in the range of 100 to 500 μg over a period of several weeks to several months. When both an antigen and an adjuvant are included, it is usually in the range of 50 to 500 μg, more preferably in the range of 50 to 200 μg, usually from several weeks to several times per administration per adult. It is preferred to administer at least twice over a month.

  The S / O preparation of the present invention can be a lotion, ointment, plaster, poultice, gel, plaster, reservoir-type patch, matrix-type patch, or tape. The tape for each single application (adhesive: bandage holding the preparation) is prepared for storage and transportation, and it is particularly suitable for application to the skin and removal / recovery after use. One of the preferred dosage forms.

  In the preparation of the present invention, the S / O preparation can be further dispersed in an aqueous phase to form an S / O / W type preparation. As the aqueous phase, those acceptable as a preparation for transdermal delivery, for example, purified water, physiological saline, isotonic solution containing a buffer, and the like can be used.

  While the present invention has been described as an S / O formulation, the present invention also provides the use of gold nanorods in therapeutic or prophylactic formulations. Based on the present invention, application of gold nanorods to various forms of preparation can be expected. For example, W / O emulsion preparations in which gold nanorods and drugs are dissolved in the aqueous layer, gold nanorod surfaces modified with hydrophobic groups such as dodecanethiol, and dispersed in the oil phase, gold nanorods and polymer particles or liposomes A formulation combined with particles is conceivable. The method for producing an S / O preparation containing gold nanorods of the present invention is applied to a preparation containing iron oxide nanoparticles that generate heat in an alternating magnetic field, silver nanoparticles that can be expected to have a bactericidal action, titanium oxide particles, fluorescent semiconductor nanoparticles, and the like. Applications can also be expected.

Hereinafter, the present invention will be specifically described by way of examples. Polytron-2100 manufactured by KINEMATICA was used for high-speed stirring of the solution with a homogenizer. Spectral characteristics were measured with JASCO Corporation V-670. The particle size was measured by dynamic light scattering using Zeta-sizer Nano-ZS manufactured by Malverne. Asahi Spectroscopy Co., Ltd. product MAX-302 was used for irradiation of near infrared light using xenon as a light source. BZ-8000 manufactured by Keyence Corporation was used to evaluate the skin permeability of the fluorescently labeled protein with a fluorescence microscope.
As the gold nanorods, those having a short axis length of about 10 nanometers, a long axis length of about 50 nanometers, and an aspect ratio of 4 to 7 (manufactured by Dainippon Paint Co., Ltd.) were used.

[Example 1]
The following procedure was used to prepare an NR-S / O formulation in which fluorescein isothiocyanate (FITC) -labeled insulin and PEG-coated gold nanorods (PEG-NR) were encapsulated in particles.

[PEG modification of gold nanorods]
A dispersion of gold nanorods modified with PEG was prepared by the following procedure. 1 mL of CTAB-modified gold nanorod (NR) dispersion was placed in multiple centrifuge tubes and centrifuged at 14000 (× g) relative centrifugal acceleration for 10 minutes to allow NR to settle at the bottom of the centrifuge tube. The supernatant was removed and the precipitated NR was redispersed in 0.3 mL of water each and their aqueous NR solution was collected together. Measure the absorption spectrum of the resulting NR dispersion, determine the gold atom concentration from the absorbance, add water so that the gold atom concentration is 1 mM, and add the NR dispersion (CTAB-NRs, gold atom concentration 1 mM). Obtained.

  Next, ME-PEG-SH so that the molar ratio of gold atom to ME-PEG-SH (α-methoxy-ω-mercaptopolyethylene glycol, molecular weight 5000, NOF Corporation) is 20: 1 or 1: 1. Was added to the NR dispersion and stirred for 24 hours to react gold atoms with ME-PEG-SH. Subsequently, the reaction solution was dialyzed for 3 days using a dialysis membrane having a molecular weight cut off of 10,000 to remove CTAB. 1 mL of the obtained PEG-NR dispersion was put into a plurality of centrifuge tubes, and centrifuged at a relative centrifugal acceleration of 14000 (× g) for 10 minutes to precipitate NR at the bottom of the centrifuge tube. Unreacted ME-PEG-SH was removed by removing the supernatant. The precipitated NR was redispersed in 0.3 mL of water, and the PEG-NR dispersions were collected together and the absorption spectrum was measured. The gold atom concentration was determined from the absorbance, and an appropriate amount of water was added to prepare a PEG-NR dispersion having a gold atom concentration of 1 mM.

(Preparation of NR-S / O formulation)
A 1 mL solution (insulin concentration of 1 mg / mL) in which insulin fluorescently labeled with fluorescein isothiocyanate (FITC) was dissolved in water was prepared. A solution of sucrose laurate L-195 (Mitsubishi Chemical Foods, Ltd., HLB approx. 1, bound fatty acid approx. 99%, ester composition: monoester 1%, di / tri / polyester 99%) dissolved in cyclohexane 4 mL (L-195 concentration 50 mg / mL) was prepared. Next, prepared PEG-NR dispersion 1 mL (gold atom concentration 1 mM), FITC-insulin aqueous solution 1 mL (insulin concentration 1 mg / mL), L-195 containing cyclohexane solution 4 mL (L-195 concentration 50 mg / mL) mL) was mixed in a glass vial. This mixed solution was stirred at high speed by a homogenizer (26,000 rpm, 2 minutes). A water-in-oil (W / O) emulsion in which an aqueous phase containing PEG-NR and FITC-insulin was dispersed in a cyclohexane oil phase by L-195 was obtained. Next, the liquid in the W / O state was immediately immersed in liquid nitrogen and frozen (20 minutes). Subsequently, the frozen product was freeze-dried (about 30 Pa, 24 hours) to remove water in the internal phase of the emulsion. Finally, the resulting composite particles of L-195, NR, and FITC-insulin were dispersed in 1 mL of oil phase isopropyl myristate (IPM), and this solution was mixed with NR-S / O preparation (gold atom concentration 1 mM, insulin concentration 1 mg / mL). Similarly, an S / O preparation containing no conventional NR and containing only FITC-insulin in particles was also prepared.

  From the appearance of the obtained NR-S / O formulation, it was found that PEG-NR was stably dispersed in the oil (Fig. 1). From the results of the absorption spectrum, it was found that the prepared NR-S / O preparation showed a peak in the near infrared region (FIG. 2). As a result of observation with an electron microscope, it was confirmed that PEG-NR was included in the particles (FIG. 3). Moreover, as a result of measuring the particle size of the particles in the NR-S / O formulation by dynamic light scattering, it was shown that the particle size was not constant and had a distribution of about 100 to 500 nm.

(Example 2)
Using FITC-ovalbumin instead of FITC-insulin, an NR-S / O preparation in which PEG-NR and FITC-ovalbumin were encapsulated in particles was prepared in the same manner as in Example 1. Observation with an electron microscope confirmed that PEG-NR was encapsulated in the particles as in the case of using insulin.

Example 3
150 μL of NR-S / O preparation containing insulin prepared in Example 2 was irradiated with near-infrared light (wavelength: about 700 nm to 1,000 nm) using a xenon lamp (irradiation intensity: 500 mW, irradiation time: 10 Min). The temperature change of the solution before irradiation and immediately after irradiation was measured with a thermometer. The prepared NR-S / O preparation absorbed near-infrared light, and the temperature of the solution rose to about 40 ° C. after 10 minutes. From this result, it was confirmed that the NR-S / O preparation showed a photothermal effect (FIG. 4).

(Example 4)
The near-infrared light was irradiated to the NR-S / O preparation, and the skin permeation enhancement of FITC-insulin by local fever limited to the vicinity of NR in the stratum corneum was examined. The NR-S / O preparation was administered to the mouse skin, and the preparation was irradiated with near infrared light. Subsequently, the skin permeability of FITC-insulin was evaluated by observing the skin cross section with a fluorescence microscope. In evaluating skin permeability, a xenon lamp was used as a light source for near infrared light, and mouse skin was used as a model skin.

<Collecting mouse skin>
Nembutal 48 μL (pentobarbital sodium concentration 50 mg / mL) was injected intraperitoneally into a 6-week-old male ddY mouse (weight of about 32 g), and the mouse was anesthetized. Subsequently, the hair on the back of the mouse was removed using a commercially available hair removal cream, and the hair was washed away with warm water. Thereafter, the hair removal part was gently wiped with Kimwipe. The mouse under anesthesia was euthanized by dislocation of the cervical spine, and the skin on the back of the hair that had been removed was cut with scissors for animal experiments to obtain mouse skin. The obtained skin was immediately immersed in liquid nitrogen and frozen, and after freezing, it was stored in a deep freezer (about −80 ° C.).

<Evaluation of FITC-insulin skin permeability>
Mouse skin that had been stored frozen was thawed on ice at room temperature. Subsequently, a Franz diffusion cell was prepared, and a stirring bar was placed in the receiver phase. Further, vacuum grease was applied to the skin adhesive portion of the receiver phase, and the thawed mouse skin was placed and fixed with a clip. Then add 5.0 mL of phosphate buffered saline (PBS, pH 7.4) to prevent bubbles from entering the receiver phase of the Franz diffusion cell and start stirring with a stirrer (60 rpm). Constant at about 32 ° C. After completing these preparatory work, each sample preparation (table below) was administered to the donor phase of the Franz diffusion cell.

  For those irradiated with near-infrared light, 150 μL of the preparation was administered to the donor phase and irradiated with near-infrared light (wavelength 700 to 1,000 nm, irradiation intensity 500 mW, irradiation time 20 minutes). After irradiation, 350 μL of S / O preparation containing no NR was further added and incubated for 12 hours. For those not irradiated with near-infrared light, 500 μL of the preparation was administered to the donor phase and incubated for 12 hours in the same manner.

  The formulation remaining in the donor phase after incubation was removed, and the skin surface was washed with the solvent of each formulation (water or IPM). Subsequently, the mouse skin was removed from the Franz diffusion cell, and the surface of the mouse skin was gently wiped with a Kimwipe. The mouse skin was immersed in 5.0 mL of 4% paraformaldehyde for 7 hours to immobilize the skin. Thereafter, the skin of the part to which the preparation was administered was cut out, and the skin was put into a plastic section preparation case. Further, an appropriate amount of paraffin glue (HistoPrep ™) was added, a round chuck was installed, and the mixture was frozen in a deep freezer (about −80 ° C.). Thereafter, skin sections having a thickness of 14 μm were prepared with a cryostat microtome (about −20 ° C.), and several sections were pasted on a slide glass. The prepared preparation was immersed in ethanol at −20 ° C. to return to room temperature, and the cellulose contained in HistoPrep ™ was removed with PBS (pH 7.4). Finally, the skin permeability of FITC-insulin was evaluated by observing the skin section with a fluorescence microscope.

<result>
The result of fluorescence microscope observation is shown (FIG. 5). Based on the results obtained, the NR-S / O formulation irradiated with near-infrared light has higher skin permeability than the S / O formulation and the negative control solution FITC-insulin. It was found from the fluorescence intensity inside the skin.

  In addition, an average of the fluorescence intensity from the skin surface to the inside of the skin is shown as a graph from the images obtained by fluorescence microscope observation (FIG. 6). These results suggest that the skin permeability of insulin is further improved by the photothermal effect of NR.

Example 5
For the NR-S / O preparation containing ovalbumin instead of insulin, the skin permeability of FITC-ovalbumin was evaluated in the same manner as in Example 4. The result of fluorescence microscope observation is shown (FIG. 7). From this result, it can be seen that the combination of the NR-S / O preparation and near-infrared light irradiation exhibits higher skin permeability than the S / O preparation containing no NR. Including the results of Example 4, it was suggested that ovalbumin having a molecular weight higher than that of insulin also enhances skin permeability due to the photothermal effect of NR.

Example 6
Samples tested for skin permeability of FITC-ovalbumin were observed with a scanning electron microscope to evaluate the distribution of nanorods in the tissue (FIG. 8). As a result, a strong signal (white region) was recognized on the skin surface or outside the skin, and it was found that the gold nanorods did not penetrate into the skin. Furthermore, when the white region was measured by EDX (Energy Dispersive X-ray Spectroscopy), a gold signal was confirmed (Fig. 9), and it was confirmed that gold nanorods existed in this region. .

Example 7
The vaccine effect of the NR-S / O preparation encapsulating ovalbumin was evaluated. A polystyrene cup having a diameter of 1 cm was adhered to the depilated mouse skin, and 180 μL of an NR-S / O preparation encapsulating ovalbumin (0.15 mg ovalbumin, gold atom concentration 2.5 mM) was placed therein. After 30 minutes, near-infrared light using a xenon lamp as a light source was irradiated from the upper part for 10 minutes. After leaving for 1 hour, remove the cup, and add a patch (10 tissue paper stack (0.8 x 1.5 cm)) soaked with 180 μL (ovalbumin 0.15 mg, gold atom concentration 2.5 mM) at the same site. Affixed with film and left for 23.5 hours. Thereafter, the patch was removed and left for 1 week (primary immunization).

  Again, the cylindrical cup was adhered, and 180 μL of an NR-S / O preparation encapsulating ovalbumin (0.15 mg of ovalbumin, gold atom concentration 2.5 mM) was placed not in the same site but in an adjacent site. After 30 minutes, near-infrared light using a xenon lamp as a light source was irradiated from the top for 10 minutes. After leaving for 1 hour, remove the cup, and add a patch (with 10 tissue sheets (0.8 x 1.5 cm)) soaked with 180 μL (ovalbumin 0.15 mg, gold atom concentration 2.5 mM) on the same site. And left for 23.5 hours. Thereafter, the patch was removed and left for 2 weeks (second immunization). Thereafter, serum was collected, and antigen albumin antibody was evaluated by ELISA.

  As a result, when the NR-S / O formulation encapsulating ovalbumin and near infrared light were combined, antibody production was not observed at the time of the first immunization, but high antibody production was observed after the second immunization. It was. Antibody production was also observed in S / O preparations without gold nanorods, but about one-eighth compared to those with gold nanorods (Table 2). Xenon lamp irradiation may have some effects of directly warming the skin. It is thought that the ovalbumin permeability was improved by the photothermal effect of gold nanorods.

Claims (10)

  An S / O preparation for transdermal drug delivery comprising a drug and a gold nanorod having an absorption band in a region of 650 to 2000 nm (preferably 680 to 1200 nm, more preferably 700 to 900 nm).   2. A formulation according to claim 1, wherein the gold nanorods are biocompatible, preferably surface modified with polyethylene glycol.   The preparation according to claim 1 or 2, wherein the drug forms a solid phase (S, dispersed phase) together with the surfactant.   4. The preparation according to claim 3, wherein the gold nanorod is contained in a solid phase.   The preparation according to claims 1 to 4 for treatment or prevention.   6. The preparation according to claim 5, wherein the drug is a molecule or polypeptide having a molecular weight of 500 daltons.   7. The preparation according to claim 6, wherein the molecular weight of the polypeptide is 6000 daltons or more, preferably 30,000 daltons or more.   The preparation according to any one of claims 5 to 7, wherein the drug is an antigen.   The preparation according to any one of claims 1 to 8, which is a tape preparation.   Use of gold nanorods in therapeutic or prophylactic formulations.