CN108452324B - Method for rapidly evaluating transdermal delivery enhancer - Google Patents
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- CN108452324B CN108452324B CN201810083002.XA CN201810083002A CN108452324B CN 108452324 B CN108452324 B CN 108452324B CN 201810083002 A CN201810083002 A CN 201810083002A CN 108452324 B CN108452324 B CN 108452324B
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- 239000003961 penetration enhancing agent Substances 0.000 claims abstract description 37
- 230000001737 promoting effect Effects 0.000 claims abstract description 26
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- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 5
- 229930003268 Vitamin C Natural products 0.000 claims description 5
- 239000003638 chemical reducing agent Substances 0.000 claims description 5
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 33
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- 241000723346 Cinnamomum camphora Species 0.000 description 1
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- 210000004369 blood Anatomy 0.000 description 1
- CKDOCTFBFTVPSN-UHFFFAOYSA-N borneol Natural products C1CC2(C)C(C)CC1C2(C)C CKDOCTFBFTVPSN-UHFFFAOYSA-N 0.000 description 1
- 229940116229 borneol Drugs 0.000 description 1
- 229930008380 camphor Natural products 0.000 description 1
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- 239000004202 carbamide Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- DTGKSKDOIYIVQL-UHFFFAOYSA-N dl-isoborneol Natural products C1CC2(C)C(O)CC1C2(C)C DTGKSKDOIYIVQL-UHFFFAOYSA-N 0.000 description 1
- 239000013583 drug formulation Substances 0.000 description 1
- 238000005370 electroosmosis Methods 0.000 description 1
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- 229940041616 menthol Drugs 0.000 description 1
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- 239000012466 permeate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229960004063 propylene glycol Drugs 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0006—Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dermatology (AREA)
- Immunology (AREA)
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Abstract
The invention discloses a method for rapidly evaluating transdermal delivery penetration enhancer, which is characterized in that the transdermal effect of the penetration enhancer is evaluated through fluorescence imaging, wherein the used fluorescence imaging agent is a fluorescence probe sensitive to reducing environment, the fluorescence imaging agent and the penetration enhancer are mixed and smeared on the skin of an experimental animal; after a certain time, the penetration promoting effect of the penetration enhancer is evaluated according to the change degree of fluorescence intensity by detecting through a fluorescence imaging system. The invention has the advantages that: (1) the experimental method is simple to operate and very quick; (2) the result obtained by experimental tests is based on animal skin, and is closer to the actual situation of real skin compared with in-vitro skin or model skin; (3) the fluorescence imaging experiment has little damage to the experimental animal and does not need to process or dissect the experimental animal.
Description
(I) technical field
The invention relates to the field of chemical analysis and biological analysis, in particular to a method for rapidly evaluating a transdermal delivery enhancer, which can be used for evaluating the effect of the transdermal delivery enhancer.
(II) background of the invention
Transdermal drug delivery is an important research direction in the design and development of children drug formulations in developed countries abroad. Transdermal administration has many advantages: the percutaneous absorbed medicine does not pass through the first pass effect of the liver and the damage of the gastrointestinal tract; the skin interlayer has certain storage function, so that the drug concentration curve is smooth, and the peak valley phenomenon is avoided, thereby maintaining stable and lasting blood drug concentration; reduce the frequency of medication, is convenient to use and improves the medication compliance of patients. Transdermal drug delivery systems have therefore received constant attention from the medical and industrial communities and industries both at home and abroad.
Penetration of drug molecules through the skin requires various penetration-promoting methods, which can be divided into chemical and physical methods. Among them, the chemical method is mainly to use transdermal enhancer to promote the drug molecules to permeate the skin. The penetration enhancer is in many kinds, and can be classified into lipophilic penetration enhancer, surfactant, two-component and multi-component penetration enhancer, etc. The research is commonly carried out by azone, linoleic acid, urea, alpha-pyrrolidone, propylene glycol, dimethyl sulfoxide, borneol, menthol, camphor and the like. There are also newly developed penetration enhancers N, N-dimethylaminoisopropanoate dodecyl ester (DDAIP) and N, N-dimethylglycine dodecyl ester (DDAA). Physical methods include percutaneous iontophoresis, ultrasonic iontophoresis, laser-assisted permeation, electroporation, and electroosmosis. In recent years, studies have also been made to combine penetration enhancers with physical penetration-promoting methods. In vitro transdermal drug experiment is an indispensable research step for the development of transdermal drug delivery system
The method for testing the penetration enhancing effect of the penetration enhancer can adopt an in vitro transdermal experiment. In vitro transdermal assays commonly use animal skin and synthetic membranes. The animal skin and the human skin have many similarities in physical and chemical properties and structural composition, and the biological relevance is good, so that the animal skin and the human skin are the most common test objects. The in vitro transdermal diffusion experiment is to clamp the skin of an animal (or an artificial membrane) in two half tanks (a dosing tank and a receiving tank) of a horizontal diffusion tank, to ensure that the horny layer faces the dosing tank, to place the medicine in the dosing tank, and to sample in the receiving tank to determine the concentration of the medicine. In vitro experimental conditions cannot reflect the real condition of the skin of the living body, and the experimental steps are relatively complicated.
The invention aims to provide an experimental method which can quickly evaluate the penetration promoting effect of a penetration enhancer, can directly smear the penetration enhancer on the skin of an experimental animal, evaluates the penetration promoting effect through fluorescence imaging, does not need to treat or dissect the experimental animal, is simple and convenient to operate, and can be used for quickly screening the penetration enhancer.
Disclosure of the invention
The technical problem to be solved by the invention is to provide a method for rapidly evaluating a transdermal delivery enhancer, which can be used for evaluating the effect of the transdermal delivery enhancer.
The technical scheme adopted by the invention is as follows:
a method for evaluating transdermal penetration enhancer of transdermal administration rapidly, characterized by that through the fluorogenic development evaluation penetration enhancer promotes the transdermal effect of the medicament, wherein the fluorogenic developer used is sensitive to reducing the environmental fluorescent probe, mix fluorogenic developer with penetration enhancer, daub the skin of experimental animal; after a certain time, the penetration promoting effect of the penetration enhancer is evaluated according to the change degree of fluorescence intensity by detecting through a fluorescence imaging system.
In the method for rapidly evaluating a transdermal penetration enhancer as described above, the fluorescent probe used therein is reduced by a reducing component such as vitamin C in skin cells and tissues after penetrating the skin epidermis, resulting in an increase in fluorescence intensity.
As described above, the fluorescent probe molecule used has a structure of a fluorophore and a nitroxide radical (FIG. 1), and the fluorescence of the fluorophore is quenched by the nitroxide radical, and the fluorescence intensity of the probe molecule is increased when the nitroxide radical is reduced by a reducing substance, such as vitamin C.
As described above, the fluorescent probe used has the following structural formula (I) (II):
the method for rapidly evaluating transdermal penetration enhancer as described above comprises the following steps: dissolving the fluorescent probe in a proper solvent, enabling the final concentration to be 0.1-10 mu M, mixing the fluorescent probe with a penetration enhancer with a certain volume, smearing a proper amount (0.5-5 mu L) on the skin of a nude mouse, carrying out fluorescence imaging for 1-10 minutes, and evaluating the penetration enhancing effect of the transdermal penetration enhancer according to the enhancement degree of fluorescence intensity, namely, the penetration enhancer with the more increased fluorescence intensity can preliminarily judge that the penetration enhancing effect is better.
The invention has the advantages that: (1) the experimental method is simple to operate, very quick and suitable for quick screening; (2) the result obtained by experimental tests is based on animal skin, and is closer to the actual situation of real skin compared with in-vitro skin or model skin; (3) the fluorescence imaging experiment has little damage to the experimental animal and does not need to process or dissect the experimental animal.
(IV) description of the drawings
FIG. 1: detection principle of fluorescent probe. After the fluorescent molecules penetrate through the skin, the fluorescent molecules enter cells and are reduced by vitamin C and the like in the cells, and the reduction products have strong fluorescence.
FIG. 2: the principle of the invention is schematically shown.
FIG. 3: results of animal fluorography, the penetration-promoting effect of azone (0%, 2%, 5%, 10%) at different ratios was rapidly determined using the method of the present invention. The faster the fluorescent probe (0.1. mu.M) penetrates the skin of the animal, the faster it is reduced by the reducing substance in the living cell, and the stronger the fluorescence intensity. The point of high fluorescence intensity indicates that the ratio is effective in promoting penetration. The results show that 5% and 10% of azone have a better penetration promoting effect than 2%. The ordinate is fluorescence intensity, and the abscissa is different proportions of the penetration enhancer.
FIG. 4: results of animal fluorescence imaging, the penetration-promoting effect of dimethyl sulfoxide DMSO (0%, 2%, 5%, 10%) at different ratios was rapidly determined using the method of the present invention. The faster the fluorescent probe (0.1. mu.M) penetrates the skin of the animal, the faster it is reduced by the reducing substance in the living cell, and the stronger the fluorescence intensity. The results show that the penetration promoting effect of 10% DMSO is better than that of 2% and that of 5% DMSO. The ordinate is fluorescence intensity, and the abscissa is different proportions of the penetration enhancer.
FIG. 5: results of animal fluorography, the penetration-promoting effect of glycerol (0%, 2%, 5%, 10%) at different ratios was rapidly determined using the method of the present invention. The faster the fluorescent probe (0.1. mu.M) penetrates the skin of the animal, the faster it is reduced by the reducing substance in the living cell, and the stronger the fluorescence intensity. The results show that the penetration promoting effect of 2%, 5% and 10% glycerol is not significant. The ordinate is fluorescence intensity, and the abscissa is different proportions of the penetration enhancer.
(V) detailed description of the preferred embodiments
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
It is known that azone and DMSO have certain permeation promoting effect, but glycerol has poor permeation promoting effect, azone and DMSO with different concentrations are selected in the following examples, and compared with glycerol with corresponding concentrations, experimental results also show that azone and DMSO have better permeation promoting effect than glycerol.
Example 1
Dissolving the fluorescent probe (I) in 1ml of physiological saline, wherein the final concentration is 0.1 mu M, respectively adding azone with a certain volume, mixing, and preparing test solutions with different concentrations, wherein the azone concentration in each solution is 0%, 2%, 5% and 10% respectively. Respectively applying 0.5 mu L of the transdermal enhancer to the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 1 minute, and evaluating the penetration enhancing effect of the transdermal enhancer according to the degree of fluorescence intensity enhancement, namely, the better the penetration enhancing effect of the transdermal enhancer can be preliminarily judged according to the permeant with the more increased fluorescence intensity. The results show that the penetration promoting effect of 5% and 10% azone are both comparable and better than that of 2% azone.
Example 2
Dissolving the fluorescent probe (I) in 0.9ml of physiological saline, adding 100 mu L of ethanol for assisting dissolution to enable the final concentration of the probe to be 10 mu M, respectively adding azone with a certain volume, mixing, and preparing test solutions with different concentrations, wherein the azone concentrations in the solutions are respectively 0%, 2%, 5% and 10%. Respectively smearing 5 mu L of the transdermal enhancer on the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 10 minutes, and evaluating the penetration promoting effect of the transdermal enhancer according to the enhancement degree of fluorescence intensity, namely, the penetration enhancer with the more increased fluorescence intensity can be preliminarily judged to have the better penetration promoting effect. The results show that the penetration promoting effect of 5% and 10% azone are both comparable and better than that of 2% azone.
Example 3
1, dissolving the fluorescent probe (I) in 1ml of physiological saline, wherein the final concentration is 0.1 mu M, adding a certain volume of DMSO respectively, mixing, and preparing test solutions with different concentrations, wherein the concentrations of DMSO in the solutions are 0%, 2%, 5% and 10% respectively. Respectively applying 0.5 mu L of the transdermal enhancer to the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 1 minute, and evaluating the penetration enhancing effect of the transdermal enhancer according to the degree of fluorescence intensity enhancement, namely, the better the penetration enhancing effect of the transdermal enhancer can be preliminarily judged according to the permeant with the more increased fluorescence intensity. The results show that the penetration promoting effect of 10% DMSO is better than that of 2% and that of 5% DMSO.
Example 4
Dissolving the fluorescent probe (I) in 0.9ml of physiological saline, adding 100 mu L of ethanol for assisting dissolution to enable the final concentration of the probe to be 10 mu M, respectively adding a certain volume of DMSO (dimethyl sulfoxide) for mixing to prepare test solutions with different concentrations, wherein the concentrations of the DMSO in the solutions are respectively 0%, 2%, 5% and 10%. Respectively smearing 5 mu L of the transdermal enhancer on the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 10 minutes, and evaluating the penetration promoting effect of the transdermal enhancer according to the enhancement degree of fluorescence intensity, namely, the penetration enhancer with the more increased fluorescence intensity can be preliminarily judged to have the better penetration promoting effect. The results show that the penetration promoting effect of 10% DMSO is better than that of 2% and that of 5% DMSO.
Example 5
Dissolving the fluorescent probe (I) in 1ml of physiological saline to obtain a final concentration of 0.1 mu M, adding a certain volume of glycerol respectively, mixing, and preparing test solutions with different concentrations, wherein the concentrations of the glycerol in the solutions are 0%, 2%, 5% and 10% respectively. Respectively applying 0.5 mu L of the transdermal enhancer to the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 1 minute, and evaluating the penetration enhancing effect of the transdermal enhancer according to the degree of fluorescence intensity enhancement, namely, the better the penetration enhancing effect of the transdermal enhancer can be preliminarily judged according to the permeant with the more increased fluorescence intensity. The results show that the penetration promoting effect of 2%, 5% and 10% glycerol is not significant.
Example 6
Dissolving the fluorescent probe (I) in 0.9ml of physiological saline, adding 100 mu L of ethanol for assisting dissolution to enable the final concentration of the probe to be 10 mu M, respectively adding a certain volume of glycerol, and mixing to prepare test solutions with different concentrations, wherein the concentrations of the glycerol in the solutions are respectively 0%, 2%, 5% and 10%. Respectively smearing 5 mu L of the transdermal enhancer on the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 10 minutes, and evaluating the penetration promoting effect of the transdermal enhancer according to the enhancement degree of fluorescence intensity, namely, the penetration enhancer with the more increased fluorescence intensity can be preliminarily judged to have the better penetration promoting effect. The results show that the penetration promoting effect of 2%, 5% and 10% glycerol is not significant.
Example 7
Dissolving the fluorescent probe (II) in 1ml of physiological saline, wherein the final concentration is 0.1 mu M, respectively adding azone with a certain volume, mixing, and preparing test solutions with different concentrations, wherein the azone concentration in each solution is 0%, 2%, 5% and 10%. Respectively smearing 5 mu L of the transdermal enhancer on the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 1 minute, and evaluating the penetration enhancing effect of the transdermal enhancer according to the degree of fluorescence intensity enhancement, namely, the better the penetration enhancing effect can be preliminarily judged as the more the fluorescence intensity is increased. The results show that the penetration promoting effect of 5% and 10% azone are both comparable and better than that of 2% azone.
Example 8
Dissolving the fluorescent probe (II) in 1ml of physiological saline, wherein the final concentration is 10 mu M, respectively adding azone with a certain volume, mixing, and preparing test solutions with different concentrations, wherein the azone concentration in each solution is 0%, 2%, 5% and 10% respectively. Respectively applying 0.5 mu L of the transdermal enhancer to the skin of a nude mouse, carrying out fluorescence imaging (excitation wavelength is 470nm and emission wavelength is 535nm) in a kodak small animal imaging system after 10 minutes, and evaluating the penetration enhancing effect of the transdermal enhancer according to the degree of fluorescence intensity enhancement, namely, the better the penetration enhancing effect of the transdermal enhancer can be preliminarily judged according to the permeant with the more increased fluorescence intensity. The results show that the penetration promoting effect of 5% and 10% azone are both comparable and better than that of 2% azone.
Claims (3)
1. A method for evaluating transdermal penetration enhancer of transdermal administration rapidly, characterized by that through the fluorogenic development evaluation penetration enhancer promotes the transdermal effect of the medicament, wherein the fluorogenic developer used is sensitive to reducing the environmental fluorescent probe, mix fluorogenic developer with penetration enhancer, daub the skin of experimental animal;
after the used fluorescent probe penetrates through the skin epidermis, probe molecules are reduced by a reducing component vitamin C in skin cells and tissues to cause the enhancement of fluorescence intensity;
after a certain time, the penetration promoting effect of the penetration enhancer is rapidly evaluated according to the change degree of the fluorescence intensity through the detection of a fluorescence imaging system;
the used fluorescent probe molecules have structures of a fluorophore and a nitroxide radical, the fluorescence of the fluorophore is quenched by the nitroxide radical, and when the nitroxide radical is reduced by a reducing substance vitamin C, the fluorescence intensity of the probe molecules is enhanced; the fluorescent probes used have the following structural formula (I) (II):
2. a method for rapidly evaluating a transdermal penetration enhancer according to claim 1, wherein the fluorescent probe used is dissolved in a suitable solvent to a final concentration of 0.1 to 10 μ M, mixed with a certain volume of the penetration enhancer, applied to the skin of a nude mouse in an amount of 0.5 to 5 μ L, subjected to fluorescence imaging after 1 to 10 minutes, and the penetration enhancing effect of the transdermal penetration enhancer is evaluated by the degree of enhancement of the fluorescence intensity, i.e., the penetration enhancer with the more increase of the fluorescence intensity is the better.
3. A method for rapidly evaluating a transdermal penetration enhancer according to claim 2, wherein the fluorescent probe (I) is dissolved in a suitable solvent to a final concentration of 0.1 to 1 μ M, mixed with a certain volume of the penetration enhancer, applied to the skin of a nude mouse in an amount of 0.5 to 1 μ L, subjected to fluorescence imaging after 1 minute, and the penetration enhancing effect of the transdermal penetration enhancer is evaluated by the degree of enhancement of the fluorescence intensity, i.e., the more the increase of the fluorescence intensity, the better the penetration enhancing effect.
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Non-Patent Citations (3)
| Title |
|---|
| Saturated Long-Chain Esters of Isopulegol as Novel Permeation Enhancers for Transdermal Drug Delivery;Yang Chen et al.;《Pharmaceutical Research》;20140122;摘要 * |
| 对pH敏感和对氧化还原敏感的荧光分子探针的设计、合成及在肿瘤细胞成像中的应用研究;刘洋;《中国博士学位论文全文数据库 医药卫生科技辑》;20111015(第10期);第E072-9页 * |
| 透皮吸收促进剂评价的新技术;朱学敏等;《中国药学杂志》;20170831;第52卷(第15期);第1301-1307页 * |
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