• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Skin models based on artificial membranes with lanolin. The present invention relates to an artificial skin model capable of simulating the behavior of natural skin at the level of permeation and penetration of compounds. This model is formed by a polymeric membrane of different nature on which a lanolin layer is deposited. This artificial skin model is useful for permeation and absorption of pharmacological or cosmetic active ingredients. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2726499A1
    申请号:ES201830343
    申请日:2018-04-06
    公开日:2019-10-07
    发明作者:Merino Cristina Alonso；Vives Victor Carrer；Gelabert Meritxell Marti；NEGRA Mª LUISA CODERCH
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0001] Skin models based on artificial membranes with lanolin
    [0002]
    [0003] The present invention relates to a synthetic skin model based on an artificial membrane of different nature on which a lanolin layer has been applied. This model is useful in permeation and absorption trials of pharmacological or cosmetic active ingredients.
    [0004]
    [0005] BACKGROUND OF THE INVENTION
    [0006]
    [0007] The penetration of an active principle involves several processes: dissolution and release of the formulation, partition within the stratum corneum, diffusion through it, partition from the stratum corneum to the aqueous phase of the epidermis, diffusion through the dermis and access to systemic circulation. In the percutaneous absorption process it is important to consider the processes of penetration, permeation and resorption. The factors involved in percutaneous absorption processes are of two types: biological and physicochemical.
    [0008]
    [0009] There are different models for the study of percutaneous absorption: the in silico, in vitro and in vivo models . In the in silico models an artificial simulation is carried out by means of mathematical equations, in the in vitro the test is carried out in a controlled environment, outside the living organism and in the in vivo models it is performed on the skin of rat, pig or human. Although the ideal model is that of natural skin, it presents difficulties when it comes to achieving it and it is more complicated to maintain its integrity during the testing processes of active compounds, so it is necessary to develop models that simulate the best possible behavior of the natural skin and that facilitate that the results are extrapolated.
    [0010]
    [0011] In the work of Eccleston, G. et al. ( Pharmaceutics 2010, 2, 209-223), a comparative study of the diffusion and permeation of ibuprofen was carried out through various types of synthetic membranes, in which the great flow variability was demonstrated depending on the membrane chosen for the test.
    [0012]
    [0013] The use of lipids of different nature to emulate the intracellular lipids of the stratum Cornea is a common technique to create synthetic skin models. In US2013 / 0000394, a corneal stratum model based on a mixture of lipids (ceramides, palmitic acid and cholesterol) is presented to evaluate the skin roughness that certain compounds can cause.
    [0014]
    [0015] Another compound of a lipid nature is lanolin. It is used to promote the penetration of active ingredients in pharmaceutical formulations as described for example in US 5,492,698. Lanolin forms an occlusive layer that forces the active to penetrate.
    [0016]
    [0017] Therefore, there is a need for artificial skin models that reproduce the absorption and permeation mechanisms in the skin as similarly as possible.
    [0018]
    [0019] DESCRIPTION OF THE INVENTION
    [0020]
    [0021] In a first aspect, the present invention relates to an artificial skin model comprising a polymeric membrane and a lanolin layer deposited on the membrane surface.
    [0022]
    [0023] In a preferred embodiment, the membrane is between 1 and 1000 pm thick.
    [0024]
    [0025] In a preferred embodiment, the membrane is selected from cellulose and derivatives, polyamide, polyacronitrile, polyethersulfone, polysulfone, polycarbonate, polypropylene or PDMS.
    [0026]
    [0027] In a more preferred embodiment, the membrane is made of polycarbonate, such as Nuclepore ™ membranes. In this case, the preferable thickness of the membrane is between 1 to 500 pm.
    [0028]
    [0029] In another preferred embodiment, the membrane is made of polyethersulfone, such as StraM® membranes. In this case, the preferable thickness of the membrane is between 300 to 1000 pm.
    [0030]
    [0031] In another preferred embodiment, the lanolin / membrane weight ratio is between 10 to 200 mg lanolin / membrane.
    [0032]
    [0033] In another preferred embodiment, the lanolin has been extracted with an aqueous phase.
    [0034]
    [0035] In another preferred embodiment, lanolin has been extracted with organic phase.
    [0036]
    [0037] Another aspect of the invention relates to a method of obtaining the artificial skin model as described above comprising the following steps: a) depositing the lanolin solution on the polymeric membrane,
    [0038] b) heat the membrane with lanolin obtained in (a) at a temperature of between 80 and 90 ° C, in a time of 5 to 15 minutes.
    [0039]
    [0040] Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0041]
    [0042] BRIEF DESCRIPTION OF THE FIGURES
    [0043]
    [0044] FIG. 1. Percentage of caffeine permeation through the skin and membranes of Nuclepore ™ without and with 5% lanolines.
    [0045]
    [0046] FIG. 2. Percentage of caffeine permeation through the skin and membranes of Nuclepore ™ without and with 80% lanolines.
    [0047]
    [0048] FIG. 3 . Average values of TEWL and standard deviation for the different membranes used.
    [0049]
    [0050] FIG. 4. Percentage of relative recovery of lidocaine in Strat-M® and Nuclepore ™ membranes with and without lanoline surface and in skin.
    [0051]
    [0052] FIG. 5. Percentage of relative recovery of diclofenac sodium in Strat-M® and Nuclepore ™ membranes with and without lanolin surface and skin.
    [0053] FIG. 6. Relative recovery percentage of betamethasone-17.21-dipropionate in Strat-M® and Nuclepore ™ membranes with and without lanoline surface and in skin.
    [0054]
    [0055] FIG. 7 . Relative recovery percentage of the compounds lidocaine, diclofenac and betamethasone-17,21-dipropionate in skin.
    [0056]
    [0057] EXAMPLES
    [0058]
    [0059] The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
    [0060]
    [0061] 1. Optimization of membranes, using TEWL and permeation kinetics “in vitro”
    [0062]
    [0063] Membranes were prepared by applying different types of wool extracts on the Nuclepore ™ artificial membrane. The amount of lipid deposited and the permeability of water across the membranes were evaluated as transepidermal water loss (TEWL). The permeation kinetics of an active substance, in this case caffeine, was performed on a skin or synthetic membrane placed on a Hanson type vertical diffusion cell (1.77 cm2 area) and a diffusion system in order to determine the amount of the compound detected in the receiving fluid after an exposure of 1, 2, 3, 4, 5, 7, 10, 13, 16 and 20 hours. These studies follow FDA-SUPAC SS procedures.
    [0064]
    [0065] 1.1 Formation of the membranes / skin models of the invention
    [0066]
    [0067] Dermatomized swine skin approximately 500 pm thick was used as reference, preparing skin discs of 2.5 cm in diameter, vacuum packed and frozen until used.
    [0068]
    [0069] The membrane used was Nuclepore ™ , with a diameter of 25 mm (surface 5cm2) a pore size of 0.05 pm and a thickness of 25 pm. Three types of lipid mixtures from wool were deposited with this artificial membrane. On the one hand, Internal Wool Lipids (IWL) were deposited, which are extracted from the clean wool with chloroform / methanol. Two lanolin fractions were also deposited, one corresponding to lanolin extracted with aqueous baths and surfactant (LanA) and another corresponding to that extracted with hexane solvent (LanD) from the LIFE Eco-efficient Dry Wool Scouring (WDS) project.
    [0070]
    [0071] The methodology described by Pullmannová et al. Was adapted for the preparation of the Nuclepore ™ membrane. ( Biophys Chem. 2017; 224: 20-31). The membranes were hydrated with hexane / ethanol 96% (2: 1) and allowed to dry at room temperature on a steel surface. The 5% and 80% lanolins solution was applied with lipids from wool in hexane / 96% ethanol (2: 1) (3x 100 pL) under an atmosphere of N 2 . These membranes were introduced in a vacuum desiccator and kept between 2 and 6 ° C for 24 hours. After the time, the membranes were placed in an oven at 85 ° C in the case of lanolines and 100 ° C in the case of the internal lipids of the wool (transition temperatures of the lipids) for 10 minutes. They were then left at room temperature for 3 hours and finally kept in incubation at 32 ° C for 24 hours.
    [0072]
    [0073] Thus, for each type of lipid tested IWL, LanA and LanD the membranes were formed in triplicate depositing 300 pL of solutions of these lipids either 5% or 80% in hexane / ethanol 96% (2: 1) as just detail. Once the sample application and fixation process is finished, they are weighed to determine the amount of surface lipid adhered to the membrane. (Table 1)
    [0074]
    [0075] 1.2 Preparation of the Franz type diffusion cells and TEWL Evaluation
    [0076]
    [0077] The cell consists of two parts: the upper compartment (giver) where the formulation is applied and the lower one (receiver) where the permeate compound is collected, separated by the membrane. The receiving part (7 mL) is filled with MilliQ water that is kept moving by constantly stirring a magnet. The cell has a jacket where water circulates thanks to an external bath and maintains an adequate temperature so that the surface of the membrane is 32 ° C. In the case of the skin, swine skin discs were previously thawed. Before proceeding to assemble the Franz diffusion cell, the thickness of the skin was measured with a digital micrometer. The thickness should be 500 ± 50 pm.
    [0078]
    [0079] Cells were prepared with swine skin or synthetic membranes in the bathroom thermostated so that the membranes (skin / artificial) reach a temperature of 32 ± 1 ° C. After conditioning the cells, 1 hour, the temperature and the TEWL value of the skin and membranes was measured with a probe. The skin / membrane temperature should be 32 ± 1 ° C and the TEWL value of the skin less than 15 g / hm2, since at higher values the integrity of the skin barrier may be compromised. TEWL values will be shown below.
    [0080]
    [0081]
    [0082]
    [0083]
    [0084] Table 1. Weight of lipid on membrane and transepidermal loss of water, on the skin, on the Nuclepore ™ membrane and membranes with different lanolines at two concentrations.
    [0085]
    [0086] Note the low permeation of the skin versus the high permeation of the Nuclepore ™ membrane. The addition of IWL decreases TEWL to 56 g / h.m2 for the 80% concentration. Lanolines have a greater effect on the impermeability of the membrane reaching values <15 g / h.m2 for the two lanolines at 80% and for LanD for 5%.
    [0087]
    [0088] 1.3 "In vitro" permeation kinetics
    [0089]
    [0090] To study the permeation properties of the different membranes, the behavior of caffeine, commonly used in dermatology, was evaluated.
    [0091]
    [0092] A solution of 10 mg / mL of caffeine in H2O / MeOH (1: 1) was prepared and 1 mL was deposited on the skin or membranes mounted on Franz cells. Aliquots were taken 700pL of receiving fluid at intervals of 1, 2, 3, 4, 5, 7, 10, 13, 16 and 20 hours after being replaced by the same amount of receiving solution at the same temperature.
    [0093]
    [0094] The extracted samples and the formulations were analyzed by high performance liquid chromatography with diode detector (HPLC-DAD). Table 2 shows the conditions of the analysis. The analytical method used for the evaluation of the different compounds was validated in terms of the calibration curve, detection limit and quantification limit following the guidelines of the International Council for Harmonization (ICH) 2005. The calibration curve was made from the standard solutions of caffeine at different concentrations, obtained from an initial solution of 100 pg / mL. The detection limit value (LDD) is the lowest concentration that can be detected, but not necessarily quantified. It was obtained by the ratio of the standard deviation of the response and the slope of the calibration line at low concentrations of caffeine multiplied by 3. The quantification limit (LDQ) was obtained by the ratio of the standard deviation of the response and the slope of the calibration line at low concentrations of caffeine multiplied by 10.
    [0095]
    [0096] Caffeine Compound
    [0097] LiChrocart ® 125-4
    [0098] Column
    [0099] LiChrosphere® 100 RP-185pm
    [0100] Wavelength 271 nm
    [0101]
    [0102] Volume injected 20 pL
    [0103] 75% H 2 O MilliQ
    [0104] Mobile phase
    [0105] 25% MeOH
    [0106]
    [0107] MeOH preparation solvent: H2O (1: 1)
    [0108]
    [0109] Retention time 8 min
    [0110]
    [0111] Calibration line Y = 15876 [Caffeine] +2136.9
    [0112]
    [0113] LDQ / LDD (pg / mL) 2.20 / 0.66
    [0114]
    [0115] Table 2. Chromatographic conditions in the quantification of caffeine, straight line Calibration and quantification limit.
    [0116]
    [0117] After analyzing the aliquots with the specified method, the permeated quantity is evaluated to obtain the permeation graph. Thus, for each experiment, Kp (the membrane permeability coefficient, which determines the speed at which the substance crosses the membrane), AUC (the area under the curve, which defines the total amount of permeated substance) and Cmax is obtained (the maximum concentration of permeation of the substance) and the flow (amount of compound that crosses the skin or membrane surface per unit of time). The means of three experiments for each type of membrane are detailed in Table 3. The caffeine permeation curves can be visualized in Figure 1, for Nuclepore ™ skin and membranes without and with 5% lanolines, and in Figure 2, for Nuclepore ™ skin and membranes without and with 80% lanolines.
    [0118]
    [0119]
    [0120]
    [0121]
    [0122] Table 3. Parameters of caffeine permeation through the skin, membranes without lanolin and membranes with different lanolines at two concentrations.
    [0123]
    [0124] In the permeation kinetics, the aforementioned permeation parameters of caffeine in skin, Nuclepore ™ and Nuclepore ™ with IWL (internal wool lipids) and lanolin of two types, LanA which is the normal commercial aqueous and the LanD are evaluated. It is extracted with organic solvent. The skin has very low permeation values, especially when compared to the Nuclepore ™ membrane alone, so that the lower the permeation parameters, the more they will resemble the skin results. It is observed that permeation decreases somewhat when apply IWL, but especially decreases when applying the two lanolines, making the latter very similar to the skin. When preparing the membranes, two different concentrations of lanolin solution, 5% and 80%, have been deposited. While LanA needs 80% to obtain a skin-like impermeability (Figure 2), it can be seen that 5% LanD has very low permeability values, very similar to those of the skin (Figure 1).
    [0125]
    [0126] In conclusion of the optimization of the type of lipid and its amount deposited on the membrane, it is worth noting the significant decrease in water permeability in the case of the two lanolines at 80% and LanD at 5%. Likewise, the permeation kinetics of caffeine indicates a greater impermeability of the LanD, especially at the lowest concentration tested. Therefore, 5% LanD was the formulation chosen to be deposited on Nuclepore ™ and another type of membrane (Strat-M®) and evaluate the penetration of assets with different physical-chemical properties compared to pig skin.
    [0127]
    [0128] 2. Optimization of membranes, by means of TEWL and percutaneous absorption "in vitro"
    [0129]
    [0130] Membranes were prepared by applying the LanD extract at a concentration of 5% on two types of membrane, Nuclepore ™ and Strat-M®. The amount of deposited lipid and the permeability of water across membranes, such as transepidermal water loss (TEWL), was evaluated. Percutaneous absorption of different assets, such as lidocaine, sodium diclofenac and betamethasone-17.21-dipropionate with different permeability properties, was determined in a skin or synthetic membrane with or without lanolines, placed on Franz-type vertical diffusion cells ( 3 mL, 1.86 cm2 area). With these diffusion cells it is possible to determine the amount of compound that has diffused through the skin. OECD procedures are followed in these studies.
    [0131]
    [0132] 2.1. Formation of the membranes / skin models of the invention
    [0133]
    [0134] Dermatomized swine skin approximately 500 pm thick was used as reference, preparing skin disks of 2.5 cm in diameter, vacuum packed and frozen until used.
    [0135] In this case, two types of artificial membranes have been used: Strat-M® (Merck, Millipore) and Whatman®Nuclepore ™ (Merck, Millipore). The lanolin used LanD comes from the LIFE Eco-efficient Dry Wool Scouring (WDS) project. LanD lanolin was extracted with hexane in a pilot plant. In the laboratory the solvent was evaporated to dryness in a rotary evaporator at 35 ° C and the lanolin moisture was removed in a desiccator with phosphorus pentoxide until a constant weight was obtained. This lanolin proved to have a more polar lipid content than the lanolin obtained in aqueous medium and to be more effective in topical applications because it is more similar to the lipids present in the stratum corneum of human skin.
    [0136]
    [0137] The lanolin that was applied to the membranes was a 5% solution of LanD in hexane / ethanol 96% (2: 1). The pH assessment of lanolin was performed on a flat plastic surface on which a thin layer of WDS lanolin was applied and the pH value was measured with a pH meter. The pH value of the WDS lanolin was 6.1.
    [0138]
    [0139] For the preparation of the Strat-M® membrane, the 5% lanolin solution in hexane / 96% ethanol (2: 1) was prepared first. 100 pL of said solution was deposited 3 times on the membrane surface under a flow of N2. The membranes were then brought to a temperature of 85 ° C for 10 minutes for optimal lipid fixation to the membrane. The methodology described in 1.1 was carried out for the preparation of the Nuclepore ™ membrane.
    [0140]
    [0141] Thus, for each type of Nuclepore ™ and Strat-M® membrane, the membranes were formed in triplicate by depositing 300 pL of 5% LanD solutions in hexane / ethanol 96% (2: 1) as detailed above. Once the sample application and fixation process is finished, they are weighed to determine the amount of surface lipid adhered to the membrane. (Table 4)
    [0142]
    [0143] 2.2 Preparation of the Franz type diffusion cells and TEWL Evaluation
    [0144]
    [0145] The in vitro study was performed with swine skin membranes and artificial membranes, placed in Franz type vertical diffusion cells (3 mL, 1.86 cm2 area). With these diffusion cells it is possible to determine the amount of compound that has diffused through the skin. Franz's cell consists of two parts, the upper chamber (giver) and the lower chamber (receiver). Between them the membrane is placed (leather / artificial) and the two chambers are held together by a clamp. The sample under study is applied to the donor part and the liquid, in this case receiving fluid, is located in the receiving part. The lower chamber has a lateral sampling arm where the receiving fluid is collected and subsequently analyzed. The cell is submerged in a thermostated bath to keep the temperature constant throughout the experiment. After the end of the exposure time, the membrane (skin / artificial) is processed to determine the amount of compound that has penetrated into each of them.
    [0146]
    [0147] In the case of the skin, swine skin discs were previously thawed. Before proceeding to assemble the Franz diffusion cell, the thickness of the skin was measured with a digital micrometer. The thickness should be 500 ± 50 pm.
    [0148]
    [0149] The cells were prepared with swine skin or synthetic membranes in the thermostated bath so that the membranes (skin / artificial) reached a temperature of 32 ± 1 ° C. After conditioning the cells, 1 hour, the temperature and the TEWL value of the skin and membranes was measured with a probe. The skin / membrane temperature should be 32 ± 1 ° C and the TEWL value of the skin less than 15 g / hm2, since at higher values the integrity of the skin barrier may be compromised. The TEWL values are shown below in Table 4 and in Figure 3.
    [0150]
    [0151]
    [0152]
    [0153]
    [0154] Table 4. Lanolin weight on membrane and transepidermal water loss, on the skin, on the Nuclepore ™ membrane and membranes with different lanolines at two concentrations.
    [0155]
    [0156] The low water permeability of the skin is observed in contrast to the large membrane permeability, especially Nuclepore ™. The addition of LanD equivalent to about 17 mg / membrane for StratM and 14 mg / membrane for Nuclepore ™ causes a decrease in permeability to values of 15 g / h.m2.
    [0157]
    [0158] 2.3 Percutaneous absorption "in vitro"
    [0159]
    [0160] To study the similarities and differences of the different membranes, the permeability of three compounds commonly used in dermatology was studied: lidocaine, diclofenac sodium and betamethasone-17.21-dipropionate.
    [0161]
    [0162]
    [0163]
    [0164]
    [0165] Table 5: Compounds studied in percutaneous absorption
    [0166]
    [0167] Table 5 details two physicochemical properties of great influence on diffusion across membranes: the octanol / water distribution coefficient (Log D) and molecular weight. Log D offers information about the lipophilicity of a molecule at a certain pH. Log D at pH 5.5 (skin pH) and at pH 7.4 (physiological pH) were considered of special interest. In this particular case, the assets to be absorbed into the skin must diffuse through the lipid matrix present in the stratum corneum. On the other hand, molecular weight also influences the absorption of molecules in the skin, making it difficult for those with more molecular weight.
    [0168]
    [0169] The extracted samples and the formulations were analyzed by HPLC-DAD. Table 6 shows the conditions of the analysis. The analytical method used for the evaluation of the different compounds was validated in terms of the curve of
    [0170] calibration, detection limit and quantification limit. The calibration curves are
    [0171] made from the standard dissolutions of assets to different
    [0172] concentrations, obtained from an initial solution of 100 pg / mL. The value of
    [0173] detection limit (LDD) is the lowest concentration that can be detected, but
    [0174] Not necessarily quantify. It was obtained visually from the chromatograms.
    [0175] The quantification limit (LDQ) was obtained from the percentage representation
    [0176] of Relative Standard Deviation (% DSR) of the concentration areas plus
    [0177] low standard, injected three times. A DSR value of less than
    [0178] 5%.
    [0179]
    [0180] Diclofenac Betamethasone-17.21-Lidocaine Compound
    [0181] sodium dipropionate
    [0182] LiChrocart ®
    [0183] LiChrocart ® 125
    [0184] 250-4 LiChrocart ® 250-4 4
    [0185] Lichrosphere 100 RP column LiChrosphere®-LiChrosphere®
    [0186] ® 100 RP-18 185pm
    [0187] 100 RP-185pm
    [0188] 5 pm
    [0189]
    [0190] Wavelength 205 nm 254 nm 239 nm
    [0191]
    [0192] Volume injected 20 pL 20 pL 20 pL
    [0193]
    [0194] 70% Buffer 66% MeOH
    [0195] 73% MeOH
    [0196] Phosphate mobile phase pH = 7 34% H3PO4
    [0197] 27% H2O
    [0198]
    [0199]
    [0200] Solvent
    [0201] ACN-TFA 0.5 preparation / extraction MeOH MeOH
    [0202]
    [0203] on
    [0204]
    [0205] Retention time 18 min 16 min 11 min
    [0206]
    [0207] Area =
    [0208] Area = 414046 [with Area = 181141 [with] +477 Calibration line 80050 [with] -
    [0209]
    [0210]
    [0211] R2
    [0212] 0.9999 0.9997 0.9999
    [0213] LDQ / LDD (pg / mL) 0.384 / 0.080 0.784 / 0.080 0.380 / 0.080
    [0214]
    [0215] Table 6. Chromatographic conditions in the quantification of the compounds, calibration line and quantification limit.
    [0216]
    [0217] Once the skin and membranes were in the necessary conditions (32 ± 1 ° C and TEWL <15 g / hm2 and 32 ± 1 ° C respectively), 20 pL of the solution of the compound to be studied on the surface was applied of the membrane (skin / artificial) with a micropipette. This solution was left for 24 hours in contact with the skin and membranes. After the time, the disassembly of the cells was carried out. The receiving fluid was collected with a Pasteur pipette through the arm of the cell receiving chamber and introduced into a 5 mL flask.
    [0218]
    [0219] In the case of the skin, the skin surface was washed in triplicate with a 0.2% aqueous solution of texapon (500 pL), adding the solution and collecting it three times without changing the tip of the pipette and drying subsequently the skin with cotton bud. All pipette tips, wash aliquots, cotton swabs and the top of Franz's cell were collected in a canister for subsequent extraction and analysis of the compound. For the separation of the stratum corneum, 8 "strippings" of the skin surface were performed with adhesive strips. The first disk was added to the boat where the washing fractions were located. The remaining 7 "strips" were divided into groups of 2-4 and 5-8 and were introduced in two vials for subsequent extraction of the compound. To obtain the dermis and epidermis, the skin biopsy was placed after stripping on a heating plate at 80 ° C for ten seconds. After this, with some tweezers, the epidermis was separated from the dermis. The epidermis and dermis were stored in separate vials for the extraction of the compound and its analysis.
    [0220]
    [0221] The extraction of the active ingredients of the membranes and of the different layers of the skin was performed with methanol in the case of lidocaine and betamethasone and with 0.5% ACN-TFA in the case of diclofenac (Table 6). The amount of extractor solvent used for washing the skin or membrane surface was 10 mL, for the stratum corneum 2 mL, for the epidermis 1 mL and finally for the dermis 1 mL. After staying overnight in contact with the extracting solvent, the samples are they waved for 30 minutes and samples of the skin compartments were sonicated for 15 minutes. The extracts were filtered with Nylon filters with a pore size 0.45 pm in vials and analyzed by HPLC-DAD. The conditions used are indicated in table 6.
    [0222]
    [0223] The results presented below are based on the evaluation of the transdermal loss of water from the skin and membranes and the evaluation of percutaneous absorption using the "in vitro" methodology with Franz cells as well, through the skin or the membranes, of the three compounds lidocaine, diclofenac sodium and betamethasone -17,21-dipropionate.The following table shows the theoretical and real concentrations obtained for the preparation of the propylene glycol formulations.
    [0224]
    [0225] Compound Lidocaine Diclofenac Betamethasone- sodium 17,21-Dipropionate Concentration
    [0226] theoretical 2.00% 0.50% 1.00%
    [0227]
    [0228] Actual concentration 1.84% 0.49% 0.79%
    [0229]
    [0230] Table 7. Results of the extractions of the three compounds.
    [0231] These actual concentrations were used to evaluate the permeation of the compounds in both artificial membrane and skin.
    [0232]
    [0233] 2.3.1 2% lidocaine permeation results
    [0234]
    [0235]
    [0236]
    [0237] Table 8. Percentage of absolute recovery and pg recovered from lidocaine in skin.
    [0238]
    [0239]
    [0240]
    [0241] Table 9. Absolute recovery percentage and pg recovered from lidocaine in Strat-M® membrane alone or with lanolin
    [0242]
    [0243]
    [0244]
    [0245] Table 10. Absolute recovery percentage and pg recovered from lidocaine in Strat-M® membrane alone or with lanolin
    [0246]
    [0247] Comparing the skin and artificial membranes, it can be seen that they are obtained very different values (Figure 4). In the case of the skin, lidocaine penetrates a lot, obtaining a high percentage in the receiving fluid. In the case of membranes, the results are very different. In the Strat-M® membrane we observe a permeation that increases with the addition of lanolin on its surface. In contrast, the Nuclepore ™ membrane allows an almost total permeation of lidocaine that decreases slightly with the presence of lanolin.
    [0248]
    [0249] 2.3.2. Permeation results of diclofenac sodium 0.5%
    [0250]
    [0251]
    [0252]
    [0253]
    [0254] Table 11. Percentage of absolute recovery and pg recovered from diclofenac sodium in skin.
    [0255]
    [0256]
    [0257] _____ _____
    [0258] Table 12. Percentage of absolute recovery and pg recovered from diclofenac sodium in Strat-M® membrane alone or with lanolin
    [0259]
    [0260]
    [0261]
    [0262] Table 13. Absolute recovery percentage and pg recovered from diclofenac sodium in Nuclepore ™ membrane alone or with lanolin
    [0263]
    [0264] The results show that permeation of diclofenac in skin is similar in Strat-M®, Strat-M® + lanolin and Nuclepore ™ + lanolin, while Nuclepore ™ is highly permeable (Figure 5). The incorporation of lanolines into Strat-M® and Nuclepore ™ has modified absorption values especially in the latter. By adding lanolines, the permeability of diclofenac is reduced by equating it to the values obtained in skin. There are two possible reasons that can favor the high permeability of diclofenac sodium. On the one hand its high water solubility and on the other its low molecular weight, properties that improve its affinity towards the receiving fluid.
    [0265]
    [0266] 2.3.3. Permeation results of betamethasone-17.21-dipropionate 1%
    [0267]
    [0268]
    [0269]
    [0270] Table 14. Recovery percentage and pg recovered from betamethasone 17,21-dipropionate in skin.
    [0271]
    [0272]
    [0273]
    [0274] Table 15. Absolute recovery percentage and pg recovered from betamethasone 17,21-dipropionate in Strat-M® membrane alone or with lanolin
    [0275]
    [0276]
    [0277]
    [0278] Table 16. Absolute recovery percentage and pg recovered from betamethasone 17,21-dipropionate in Nuclepore ™ membrane alone or with lanolin
    [0279] In this case it is important to highlight the low permeation of betamethasone in all the membranes used (Figure 6). Betamethasone is the most hydrophobic substance and of greater molecular weight of the three selected. Both properties make this substance have a low affinity for the receptor fluid, which can make it difficult to diffuse through the membranes. By adding lanolin to Nuclepore ™ and Strat-M® membranes, absorption is slightly increased compared to membranes without lanolin. This phenomenon may be due to the contents of polar lipids that can act as a cosolvent of betamethasone, helping its solubilization and greater affinity in the receptor fluid.
    [0280]
    [0281] 2.4. Discussion of the results of membrane optimization, using TEWL and percutaneous absorption "in vitro"
    [0282]
    [0283] The loss of water through the skin is an indicative value of the integrity of the membrane and its permeability. In this example (2.1), the TEWL values have been determined for all the membranes used as the average of the values of all of them (Figure 3). Table 4 shows that a low standard deviation was obtained for all types of membranes studied. This fact is important, since it indicates both a homogeneity with respect to the integrity of the skin biopsies used, as well as a homogeneity and stability of the commercial membranes (Nuclepore ™ and Strat-M®) and the proposals in this work (Nuclepore ™ + lanolin and Strat-M® + lanolin).
    [0284]
    [0285] The TEWL values obtained in the skin membranes demonstrate the integrity of the skin barrier according to the specifications of the OECD and its aptitude for testing in Franz cells. Regarding the two artificial membranes, it can be seen that they have a lower barrier function, especially for the Nuclepore ™ membrane.
    [0286]
    [0287] The objective of adding 5% WDS lanolin on the surface of commercial membranes is to increase its barrier function. By incorporating lanolin, the lipid matrix present in the stratum corneum is emulated. The result has been a significant reduction in TEWL values, a fact that indicates an increase in the barrier function. This is achieved for the two membranes with lanolin, an average value of «15 g / h.m2 that is acceptable for use as a membrane model in percutaneous absorption.
    [0288]
    [0289] When comparing the three active ingredients applied to the skin, a high permeability for lidocaine, an intermediate permeability for diclofenac sodium, followed by low permeability for betamethasone. Considering the physicochemical properties and that the skin's pH is 5.5, we can see how the compounds penetrate more according to a decrease in their molecular weight and Log D value at that pH (Figure 7).
    [0290]
    [0291] The lidocaine molecule, at pH 5.5, has a greater affinity towards the receptor fluid due to its hydrophilicity. At this pH, diclofenac sodium is the next in permeability, it is more hydrophobic than lidocaine, with up to 10% found in the epidermis and dermis. Betamethasone is the most hydrophobic compound of the three and its largest molecular size causes this compound to be found only in the stratum corneum and in the epidermis.
    [0292]
    [0293] Comparing the skin with the two types of membranes without lanolines, it should be noted that the Strat-M® membrane is more similar to the skin (Figures 4,5,6) since in the Nuclepore ™ membrane, much higher permeabilities are obtained for diclofenac. sodium and lidocaine (Figures 4,5). However, the two membranes discriminate well the betamethasone compound obtaining a lower permeability to the other two active ingredients.
    [0294]
    [0295] As we have seen, the hydrophilicity of the substances and their affinity to the receptor fluid is especially important. When the Nuclepore ™ and Strat-M® membranes without lanolines are used, the compounds were applied in a medium of pH 7.4 since said membranes were hydrated with the receiving fluid at this pH. Therefore, the log D value at pH 7.4 of the compounds should be considered, at this pH the most hydrophilic compound being diclofenac and the most lipophilic is betamethasone. In this way we can see that the most hydrophilic substance is the most absorbed and the most lipophilic is the least absorbed, just like the results observed in skin. In the case of Nuclepore ™, these differences between the lipophilicities of lidocaine and diclofenac are hardly noticeable because the permeability values are higher.
    [0296]
    [0297] In the case of lanolin membranes, the most permeated substance in both models (Strat-M® + lanolin and Nuclepore ™ + lanolin) is lidocaine, followed by diclofenac sodium and finally betamethasone. This change with respect to Strat-M® and Nuclepore ™ without lanolines may be due to the change in pH caused by the incorporation of lanolines. Lanolines are substances that contain fatty acids that slightly acidify the environment in which the active substances are found. This change in pH leads to significant changes in the lipophilicity of non-neutral compounds: lidocaine and diclofenac. At acidic pH the log D of lidocaine (log D = 0.61) is lower than that of diclofenac sodium (log D = 2.75) and betamethasone (log D = 3.96). As we have seen so far, the substances with the highest penetration are the most hydrophilic (smallest log D), this behavior is also maintained now because the most absorbed substances in the Nuclepore ™ + lanolin and Strat-M® + lano membrane is the lidocaine followed by Diclofenac and betamethasone. In this way, by adding lanolin, we obtain permeability ranges for the three active ingredients much more similar to those obtained in skin.
    [0298]
    [0299] Thus, the inclusion of lanolin in the membranes modulates permeation for the active substances. In the Strat-M® membrane, the permeability of lidocaine and betamethasone is increased without modifying that of sodium diclofenac. For the Nuclepore ™ membrane, the permeability of the more hydrophobic and higher molecular weight compound, betamethasone, increases slightly and the permeation for the rest of the active ingredients decreases. The permeability of the Strat-M® membrane that is already thought of as a skin model is favored by the addition of lanolin by increasing the permeability of lidocaine and betamethasone without modifying that of diclofenac.
    权利要求:
    Claims (11)
    [1]
    1. Artificial skin model comprising a polymeric membrane and a lanolin layer deposited on the surface of the membrane.
    [2]
    2. Model according to claim 1 wherein the membrane is between 1 and 1000 pm thick.
    [3]
    3. Model according to any of claims 1 or 2, wherein the membrane is selected from cellulose and derivatives, polyamide, polyacronitrile, polyethersulfone, polysulfone, polycarbonate, polypropylene or PDMS.
    [4]
    4. Model according to claim 3, wherein the membrane is polycarbonate.
    [5]
    5. Model according to claim 4, wherein the polycarbonate membrane is between 1 and 500 pm thick.
    [6]
    6. Model according to claim 3, wherein the membrane is polyethersulfone.
    [7]
    7. Model according to claim 6, wherein the polyethersulfone membrane has a thickness of between 300 to 1000 pm.
    [8]
    8. Model according to any of the preceding claims wherein the lanolin / membrane weight ratio is between 10 to 200 mg lanolin / membrane.
    [9]
    9. Model according to any of the preceding claims wherein the lanolin has been extracted with aqueous phase.
    [10]
    10. Model according to any of claims 1 to 8 wherein the lanolin has been extracted with organic phase.
    [11]
    11. Method of obtaining the artificial skin model according to claim 1 comprising the following steps:
    a) deposit the lanolin solution on the polymeric membrane,
    b) heat the membrane with lanolin obtained in (a) at a temperature of between 80 and 90 ° C, in a time of 5 to 15 minutes.
    类似技术:
    公开号 | 公开日 | 专利标题
    Björklund et al.2013|Glycerol and urea can be used to increase skin permeability in reduced hydration conditions
    Leveque et al.2004|Comparison of Franz cells and microdialysis for assessing salicylic acid penetration through human skin
    Björklund et al.2010|A water gradient can be used to regulate drug transport across skin
    Mura et al.2000|Evaluation of transcutol as a clonazepam transdermal permeation enhancer from hydrophilic gel formulations
    Megrab et al.1995|Oestradiol permeation across human skin, silastic and snake skin membranes: the effects of ethanol/water co-solvent systems
    Holmgaard et al.2012|Comparison of open-flow microperfusion and microdialysis methodologies when sampling topically applied fentanyl and benzoic acid in human dermis ex vivo
    CN102579287B|2015-01-28|Cosmetic composition for repairing corneous layer of skin
    Milewski et al.2011|Vehicle composition influence on the microneedle-enhanced transdermal flux of naltrexone hydrochloride
    Čuříková et al.2017|Simplified stratum corneum model membranes for studying the effects of permeation enhancers
    Nguyen et al.2018|Qualitative and quantitative analysis of lateral diffusion of drugs in human skin
    ES2726499B2|2020-04-16|LEATHER MODELS BASED ON ARTIFICIAL MEMBRANES WITH LANOLINE
    Gomaa et al.2012|Flux of ionic dyes across microneedle-treated skin: Effect of molecular characteristics
    ES2750303T3|2020-03-25|Polymeric membranes with permeability properties similar to human skin and uses thereof
    Alonso et al.2020|Permeation kinetics of active drugs through lanolin-based artificial membranes
    Farah et al.2019|Heat enhanced follicular delivery of Isotretinoin to the skin
    Mary et al.1998|Assessment of the recovery of three lipophilic psoralens by microdialysis: an in vitro study
    Almeida et al.2016|Tissue-based in vitro and ex vivo models for dermal permeability studies
    CN104721138A|2015-06-24|Glabridin freeze-dried liposome and preparation method thereof
    Ilbasmis-Tamer et al.2017|Carbon nanotube membranes to predict skin permeability of compounds
    ES2326692T3|2009-10-16|PROCEDURE FOR ANALYSIS OF LIPOSOMES.
    Bentzel et al.1978|Permeability changes in Necturus proximal tubule during volume expansion
    d'Arpino et al.2003|Influence of vehicles on the in vitro percutaneous absorption of piroxicam to optimise the formulation of patch tests in dermatology
    Neethling2006|Effect of Brij 97 in the presence and absence of carrageenan on the transdermal delivery of 5-Fluorouracil
    BR102016028713A2|2018-07-17|use of coffee grounds oil and pharmaceutical composition
    Akhter et al.1986|Permeation of drugs through human skin: method and design of diffusion cells for in vitro use
    同族专利:
    公开号 | 公开日
    ES2726499B2|2020-04-16|
    WO2019193234A1|2019-10-10|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5196190A|1990-10-03|1993-03-23|Zenith Technology Corporation, Limited|Synthetic skin substitutes|
    法律状态:
    2019-10-07| BA2A| Patent application published|Ref document number: 2726499 Country of ref document: ES Kind code of ref document: A1 Effective date: 20191007 |
    2020-04-16| FG2A| Definitive protection|Ref document number: 2726499 Country of ref document: ES Kind code of ref document: B2 Effective date: 20200416 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201830343A|ES2726499B2|2018-04-06|2018-04-06|LEATHER MODELS BASED ON ARTIFICIAL MEMBRANES WITH LANOLINE|ES201830343A| ES2726499B2|2018-04-06|2018-04-06|LEATHER MODELS BASED ON ARTIFICIAL MEMBRANES WITH LANOLINE|
    PCT/ES2019/070232| WO2019193234A1|2018-04-06|2019-04-05|Skin models based on artificial membranes with lanolin|
    [返回顶部]