JP2005221442A - Mucopolysaccharide laminated lipid membrane, filter for measuring membrane permeability, membrane permiability measuring instrument, membrane permiability measuring kit, membrane permeability evaluating method and screening method of substance to be inspected - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a phospholipid membrane capable of evaluating elution and membrane permeation at the same time without damaging the low-cost properties and rapidity of the lipid membrane. <P>SOLUTION: This mucopolysaccharide laminated lipid membrane 100 is equipped with the lipid membrane 2 and the hydrophilic filter layer 10, which contains a hydrophilic filter 8 impregnated with a gel-like polymer 6 containing mucopolysaccharide 4, formed on the lipid membrane 2. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a mucopolysaccharide laminated lipid membrane, a membrane permeability measurement filter, a membrane permeability measurement device, a membrane permeability measurement kit, a membrane permeability evaluation method, and a test substance screening method.

  Absorption of a medicinal component (hereinafter referred to as “drug”) from a pharmaceutical preparation is performed through the process of elution of the drug from the preparation and the subsequent permeation of the drug through the digestive tract membrane. In the case of readily water-soluble compounds, oral absorption is mainly determined by gastrointestinal membrane permeability.

  However, in recent drug development, the drug is often a poorly water-soluble compound (Non-patent Document 1). With poorly water-soluble compounds, it is known that in addition to gastrointestinal membrane permeability, elution of drugs in the gastrointestinal tract greatly affects oral absorption (Non-Patent Document 2). Therefore, in the oral absorption evaluation of poorly water-soluble compounds, elution and membrane permeation must be evaluated simultaneously. In particular, simultaneous evaluation of elution and membrane permeation is essential in formulation studies.

  On the other hand, since the cost of drug development has increased and competition for drug development has intensified, low cost and rapidity are also required for oral absorption evaluation. Therefore, there is a need for a method that is low-cost and rapid, and that can simultaneously evaluate elution and membrane permeation.

  As a method capable of simultaneously evaluating the elution process and gastrointestinal membrane permeation, animal experiments and in vitro dissolution-permeability system (Caco-2 DP system) using Caco-2 cell are known (Non-patent Document 3 and 4). However, animal experiments have problems such as costly experiments, difficult data analysis, and undesirable animal ethics. The Caco-2 D-P system also requires cell culture and is not sufficient in terms of cost and speed. A low-cost and rapid solubility measurement method is also known (Non-Patent Document 5), but no method is known that can simultaneously evaluate the membrane permeation process using this method.

  On the other hand, a method using a filter-retaining phospholipid membrane is known as a method capable of rapidly evaluating gastrointestinal membrane permeation at low cost. In particular, parallel artificial membrane permeation assay (PAMPA) is widely used (Non-patent Document 6), and various membranes that can be used for it are also known (for example, Patent Document 1). However, when a poorly water-soluble compound or formulation is evaluated using a filter-carrying artificial phospholipid membrane, there is a problem in that it cannot be evaluated because the drug powder or formulation and the membrane are in direct contact.

In the animal experiment and Caco-2 DP system described above, the contact between the drug powder and the preparation and the cell membrane is prevented by the mucopolysaccharide mucosa layer (Non-patent Document 4). However, since cell culture is required and a mucopolysaccharide mucosa layer separately prepared by covalent cross-linking is used, the cost and speed are not sufficient.
WO01 / 70380 pamphlet Lipinski, CA; J. Pharmacol. Toxicol. Methods, 2000, 44, 235-249 Amidon, GL et al .; Pharm. Res., 1995, 12, 413-420 Kataoka, M. et al .; Pharm. Res., 2003, 20, 1674-1680 Yamashita, Lipid-based formulation symposium, 2000, Yokohama Kerns, EH; J. Pharm. Sci. 2001, 90, 1838-1858 Kansy, Manfred et al .; J. Med. Chem., 1998, 41, 1007-1010

  Accordingly, an object of the present invention is to provide a phospholipid membrane capable of simultaneous elution and membrane permeation without impairing the low cost and rapidity of the lipid membrane.

  The present inventor has intensively studied to achieve the above object. As a result, a mucopolysaccharide-containing gel film that can be easily mounted by sol-gel transition by mixing a polymer having sol-gel transition and mucopolysaccharide. Found that it can be created. Moreover, it discovered that the film | membrane which can achieve the said objective was obtained by forming and forming this film | membrane on a lipid membrane.

  That is, the present invention provides a mucopolysaccharide laminated lipid membrane comprising a lipid membrane and a hydrophilic filter layer comprising a hydrophilic filter impregnated with a gel polymer containing mucopolysaccharide formed on the lipid membrane. Is. The hydrophilic filter layer is preferably formed on one side of the lipid membrane.

  As described above, the hydrophilic layer is provided on the lipid membrane, the mucopolysaccharide is contained in the hydrophilic layer, and the mucopolysaccharide is held in a gel polymer and further combined with a hydrophilic filter. By using the mucopolysaccharide laminated lipid membrane of the invention, the elution process and gastrointestinal membrane permeation can be simultaneously evaluated at low cost and rapidly.

  The hydrophilic filter layer is obtained by impregnating the hydrophilic filter with a mixture containing a polymer capable of sol-gel transition and mucopolysaccharide in a sol state, and transferring the sol polymer to a gel polymer. It is preferable that the layer be formed. By forming the hydrophilic filter layer in this manner, the thickness of the hydrophilic filter layer on the lipid membrane can be reduced, and peeling from the lipid membrane can be prevented even when the thickness is thin. .

  The mixture can be an aqueous solution, and agarose is suitable as the polymer that can undergo sol-gel transition. When the mixture is an aqueous solution, the hydrophilic filter can be easily and reliably impregnated, and a hydrophilic filter layer can be formed at a relatively low temperature by using agarose as the polymer that can undergo sol-gel transition. it can.

  The mucopolysaccharide can be selected from the group consisting of mucin, chondroitin sulfate, hyaluronic acid, sialic acid polysaccharide, heparin, hepatylline sulfate, keratosulfate, and chitin, and the hydrophilic filter comprises a paper filter, a cellulose filter, and a cellulose filter derivative. You can choose from a group. The lipid membrane is preferably an artificial phospholipid membrane. By using such a material, the above-described effects can be exhibited more remarkably.

  The above lipid membrane can be applied as a filter. That is, a filter for measuring membrane permeability comprising a mucopolysaccharide laminated lipid membrane is provided. Moreover, a membrane permeability measuring apparatus can be produced using a lipid membrane. Such a membrane permeability measuring apparatus includes a mucopolysaccharide laminated lipid membrane, and a test substance containing a test substance that is disposed on one side of the mucopolysaccharide laminated lipid membrane and contacts the mucopolysaccharide laminated lipid membrane. Examples include a membrane permeability measuring device including a container and a permeate container that is disposed on the other surface of the mucopolysaccharide laminated lipid membrane and contains a permeate from the mucopolysaccharide laminated lipid membrane.

  Furthermore, a mucopolysaccharide laminated lipid membrane, a test substance containing container for containing a test substance disposed on one side of the mucopolysaccharide laminated lipid membrane and contacting the mucopolysaccharide laminated lipid membrane, and a mucopolysaccharide laminate There is provided a kit for measuring membrane permeability comprising at least a permeate containing container for containing a permeate from the mucopolysaccharide laminated lipid membrane disposed on the other surface of the lipid membrane. The mucopolysaccharide laminated lipid membrane may be prepared in advance or may not be prepared. If the mucopolysaccharide laminated lipid membrane has not been prepared in advance, for example, the material used as the raw material of the mucopolysaccharide laminated lipid membrane and a document showing the preparation method are attached so that the kit user can prepare it when necessary. It only has to be.

  The present invention is also arranged such that a solution containing a test substance is in contact with one side of the mucopolysaccharide laminated lipid membrane, and a solution containing no test substance is in contact with the other side of the mucopolysaccharide laminated lipid membrane. And measuring the membrane permeability of the test substance by measuring the amount of the test substance in the solution on the other side and / or the amount of the test substance in the solution on the one side after a predetermined time. Provide a sex assessment method.

  By applying this membrane permeability evaluation method, it is possible to screen a test substance. That is, a measurement process for measuring the membrane permeability of a plurality of test substances by the above-described membrane permeability measurement method, and a desired test substance from a plurality of test substances based on the membrane permeability data obtained in the measurement process. It is possible to provide a screening method for a test substance, which includes a selection step of selecting a substance.

  By using the mucopolysaccharide laminated lipid membrane of the present invention, it is possible to simultaneously evaluate the elution and membrane permeation of drugs and their preparations at low cost and rapidly, and the development of useful pharmaceuticals becomes possible.

  Hereinafter, a cover material and a patch with a cover material according to a preferred embodiment will be described with reference to the drawings. In addition, the dimension ratio of drawing does not necessarily correspond with the thing of description.

  FIG. 1 is a cross-sectional view schematically showing a mucopolysaccharide laminated lipid membrane according to the first embodiment. A mucopolysaccharide laminated lipid membrane 100 according to the first embodiment shown in FIG. 1 includes a lipid membrane 2 and a hydrophilic filter 8 formed on the lipid membrane 2 and impregnated with a gel polymer 6 containing the mucopolysaccharide 4. And a hydrophilic filter layer 10. In FIG. 1, the mucopolysaccharide 4 is shown in a granular form, but the mucopolysaccharide 4 may be dispersed in the gel polymer 6 as described above, or may be dissolved or swollen in the gel polymer 6. In FIG. 1, the hydrophilic filter 8 is shown to exist only on the lipid membrane 2 side of the hydrophilic filter layer 10, but the hydrophilic filter 8 may be disposed on the entire hydrophilic filter layer 10.

  FIG. 2 is a cross-sectional view schematically showing a mucopolysaccharide laminated lipid membrane according to the second embodiment. The mucopolysaccharide laminated lipid membrane 110 according to the second embodiment shown in FIG. 2 includes a lipid membrane 2 and a gel polymer 6 containing the mucopolysaccharide 4 formed on the lipid membrane 2 as in the first embodiment. Although the hydrophilic filter layer 10 including the impregnated hydrophilic filter 8 is provided, the lipid membrane 2 is different from the first embodiment in that it is supported by the support 12. Thus, by supporting with the support body 12, the shape stability of the lipid membrane 2 can be ensured, and membrane permeability evaluation and screening of a test substance can be performed more accurately.

  FIG. 3 is a cross-sectional view schematically showing a membrane permeability measuring apparatus 200 incorporating the mucopolysaccharide laminated lipid membrane 110 according to the second embodiment shown in FIG. The membrane permeability measuring apparatus 200 shown in FIG. 2 includes a mucopolysaccharide laminated lipid membrane 110 and a test substance that is placed on one side of the mucopolysaccharide laminated lipid membrane 110 and contains a test substance so as to be in contact with the mucopolysaccharide laminated lipid membrane 110. A test substance storage container 20 and a permeate storage container 22 that is disposed on the other surface of the mucopolysaccharide laminated lipid membrane 110 and that contains a permeate from the mucopolysaccharide laminated lipid membrane 110 are provided.

  FIG. 4 is a cross-sectional view schematically showing a state in which the membrane permeability of the test substance is measured using the membrane permeability measuring apparatus 200 shown in FIG. FIG. 4 shows that the solution 30 containing the test substance 32 is in contact with one side of the mucopolysaccharide laminated lipid membrane 110, and the solution 34 not containing the test substance 32 is placed on the other side of the mucopolysaccharide laminated lipid membrane 110. It is arranged so as to be in contact with each other, and shows a state where a predetermined time has elapsed.

  In FIG. 4, the test substance 32 initially contained in the solution 30 stored in the test substance storage container 20 is reduced, and a part of the test substance 32 passes through the mucopolysaccharide laminated lipid membrane 110 and passes through. It has reached the solution 34 in the container 22. In the state shown in FIG. 4, the membrane permeability of the test substance 32 is measured by measuring the amount of the test substance 32 in the solution 34 on the other side and / or the quantity of the test substance 32 in the solution 30 on the one side. Can be measured.

  Since the membrane permeability measuring apparatus 200 shown in FIGS. 3 and 4 uses the mucopolysaccharide laminated lipid membrane 110 having the hydrophilic filter layer 10 on the lipid membrane 2, it occurs in Non-Patent Literature 5 and Patent Literature 1. The problem that the evaluation cannot be performed due to the direct contact between the drug powder or the preparation and the film is solved. Further, unlike the structure of Non-Patent Document 4, the mucopolysaccharide laminated lipid membrane 110 is provided with a hydrophilic filter layer 10 on the lipid membrane 2, and the mucopolysaccharide 4 is contained in the hydrophilic filter layer 10 so that the mucopolysaccharide is contained. Since 4 is held by the gel polymer 6 and further combined with the hydrophilic filter 8, the elution process and gastrointestinal membrane permeation can be simultaneously evaluated at low cost and rapidly.

Hereinafter, preferred embodiments will be described for each of the components applied to the present invention.
Specifically, mucopolysaccharide refers to mucin, chondroitin sulfate, hyaluronic acid, sialic acid polysaccharide, heparin, hepatylline sulfate, keratosulfate, chitin, and mixtures thereof.

  A sol-gel transitionable polymer is a polymer whose viscoelasticity changes in response to a physical stimulus (temperature, light, ionic strength, pH, etc.), and is a natural polymer as long as it achieves the purpose. Or a synthetic polymer.

  Examples of natural polymers include agarose, gelatin, carrageenan, mannan and the like. Furthermore, those chemical decorations and chemical treatments can be mentioned.

  Synthetic polymers include poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, polyvinyl methyl ether, polyvinyl alcohol partial oxides, polyethylene oxide-polypropylene oxide-polyethylene oxide block polymers (PEO-PPO-PEO: trade name Pluronic, authorization) The grades are F68 and F127), and polypropylene oxide-polyethylene oxide-polypropylene oxide block polymers (PPO-PEO-PPO: trade name Pluronic®) are known as similar polymers. Polyethylene oxide-polylactic acid / polyglycolic acid copolymer-polyethylene oxide block polymer (PEO-PLGA-PEO: trade name Regel) using a polylactic acid / polyglycolic acid copolymer instead of PPO in the hydrophobic part Alternatively, polylactic acid / polyglycolic acid copolymer-polyethylene oxide-polylactic acid / polyglycolic acid copolymer block polymer (PLGA-PEO-PLGA: trade name Regel) is also known to have a similar temperature-responsive function. (International Publication WO99 / 18142).

  Among these, a polymer whose viscoelasticity changes in response to temperature is preferable from the viewpoint of simplicity in use, and among these, a natural polymer is particularly preferable from the viewpoint of cost.

  The hydrophilic filter is a filter through which an aqueous solution can easily penetrate, and examples thereof include a paper filter, a cellulose filter, and a filter subjected to other hydrophilic treatment. Specific examples include filter paper.

  The lipid membrane is not particularly limited as long as it is an artificial lipid membrane used for evaluating membrane permeability, but is preferably an artificial phospholipid membrane. Specifically, a membrane composed of a lipid and an organic solvent, a membrane in which membrane components collected from a living body (rat) are dissolved in n-decane (INUI, Ken-ichi et al .; Journal of Pharmacy and Pharmacology, 1977, 29, 22-26), membranes using dodecane, phosphatidylcholine, 1,9-decadiene, membranes containing fatty acids (Kansy, Manfred et al .; J. Med. Chem., 1998, 41, 1007-1010), carbon number Examples include a membrane using 7 to 9 unsaturated hydrocarbons (International Publication No. WO01 / 70380). Preferred is a membrane containing an unsaturated hydrocarbon having 7 to 9 carbon atoms, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and cholesterol. The lipid membrane may further include a support capable of supporting it. Here, examples of the support include porous sheets or membranes such as filter paper, and preferably hydrophobic such as hydrophobic PTFE (polytetrafluoroethylene) and hydrophobic PVDF (polyvinylidene difluoride). Examples thereof include those made of a conductive material (hereinafter also referred to as a hydrophobic filter).

In the present invention, the polymer capable of sol-gel transition and mucopolysaccharide are preferably prepared as a mixed solution and impregnated in the hydrophilic filter layer. The amount of the mixed lysate is not particularly limited, but in order to quantitatively evaluate the membrane permeability of the test substance, it is necessary to impregnate the entire hydrophilic filter layer and cover the lipid membrane almost uniformly. It is. Specifically, about 10 μL is sufficient for the film having a surface area of 0.266 cm ² , but an excessive amount may be used.

  A hydrophilic filter layer is formed on the lipid membrane, but the hydrophilic filter layer may be attached and / or held on the lipid membrane. Here, the adhesion and / or retention specifically means that a hydrophilic filter layer impregnated with a mixed solution of a polymer capable of sol-gel transition and mucopolysaccharide is adhered and / or retained on a lipid membrane. Refers to the state of being. At this time, the hydrophilic filter layer impregnated with the mixed solution of the polymer capable of sol-gel transition and mucopolysaccharide and the lipid membrane are not directly bonded (by a covalent bond or the like).

  At this time, the hydrophilic filter layer impregnated with the mixed solution of the polymer capable of sol-gel transition and mucopolysaccharide may cover the entire lipid membrane, and may adhere to and / or adhere to one side of the lipid membrane. If it is retained, its purpose can be achieved. In consideration of the similarity to the living body, the amount of each raw material used, the labor required for the preparation of the membrane, etc., it is preferably attached and / or held on one side of the lipid membrane.

  If the thickness of the lipid membrane including the hydrophilic filter layer impregnated with a mixed solution of a polymer capable of sol-gel transition and mucopolysaccharide impregnated with sol-gel transition is less than 100 μm, stable utilization is difficult. Since the thickness of the gastrointestinal mucopolysaccharide layer (about 100 to 500 μm) is significantly exceeded if it is thicker than 500 μm, the membrane permeability of the test substance to be evaluated is that in vivo. There is a high risk that they will not match. Accordingly, the thickness of the entire film including the hydrophilic filter layer impregnated with the mixed solution of the polymer capable of sol-gel transition and mucopolysaccharide and having a thickness of 100 μm to 500 μm is preferable. The lipid membrane itself can usually be prepared in the range of 50 to 200 μm.

  The mucopolysaccharide laminated lipid membrane of the present invention can be prepared, for example, as follows. A mucopolysaccharide gel is prepared by adding mucopolysaccharide and a sol-gel transitionable polymer to the buffer. The pulverized hydrophilic filter is dispersed in a volatile organic solvent, added to the hydrophobic filter and then filtered, and the organic solvent is evaporated to join the hydrophilic filter layer on the hydrophobic filter. The mucopolysaccharide gel is made into a sol state by physical stimulation (temperature, light, ionic strength, pH, etc.), then added to the hydrophilic filter layer, and gelled and fixed by physical stimulation. A lipid component is added to the hydrophobic filter to form a lipid membrane.

  At this time, the mucopolysaccharide laminated lipid membrane of the present invention is prepared on a multi-hole membrane filter such as a 96-hole membrane filter (donor plate), and the same kind of multi-hole membrane filter (acceptor) on which the lipid membrane is not formed. If it is mounted on a plate), the membrane permeability of the test substance can be easily measured.

  The membrane permeability of the test substance is measured by, for example, placing a solution containing the test substance on one side of the mucopolysaccharide laminated lipid membrane of the present invention and not containing the test substance on the other side of the mucopolysaccharide laminated lipid film. A solution is disposed, and the amount of the test substance permeated to the other side and / or the amount of the test substance remaining on the one side may be measured after a predetermined time has elapsed. You may use for the screening of a test substance combining the process of measuring the membrane permeability of such a test substance, and the process of selecting the test substance which has predetermined | prescribed membrane permeability.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples of the present invention. However, the present invention is not limited to these examples, and various modifications can be made without departing from the technical idea of the present invention. Can be changed.

Example 1 Production of Mucopolysaccharide Layer-Artificial Phospholipid Membrane A mucopolysaccharide laminated lipid membrane according to Example 1 was produced according to the process diagram shown in FIG.
First, chondroitin sulfate (purchased from Sigma) and agarose (low melting point) (purchased from Sigma) were added to a pH 6.0 phosphate buffer, dissolved by heating, and a gel containing 1% of each (hereinafter referred to as mucopolysaccharide). Gel).

  A paper filter (Advantec 5A (manufactured by TOYO)) was put in water, crushed with a mixer, and further crushed with a histron crusher. The obtained crushed material was dispersed in ethanol to prepare a filter paper dispersion (filter paper 3 mg / EtOH).

To a Millipore 96-well membrane filter (hydrophobic PVDF (polyvinylidene difluoride), pore 0.45 μm, area 0.266 cm ² ) (purchased from Nihon Millipore), 200 μL of filter paper dispersion was added and filtered with suction. A hydrophilic filter layer was constructed on the hydrophobic filter by evaporating at 30 ° C. for about 30 minutes. 10 μL of heat-melted mucopolysaccharide gel was added to the hydrophilic filter layer and cooled at 5 ° C. for 30 minutes. Further, 5 μL of a phospholipid organic solvent solution consisting of phosphatidylcholine 2.0% (w / w) and dodecane 98.0% (w / w) is added to the hydrophobic filter, so A membrane (mucopolysaccharide layer-artificial phospholipid membrane: mucopolysaccharide laminated lipid membrane) on which phospholipid membranes were superimposed was produced. The appearance of the produced mucopolysaccharide laminated lipid membrane is shown in FIGS. 6 (a) and 6 (b).

(Comparative Example 1)
In Example 1, mucopolysaccharide gel was added without forming a hydrophilic filter layer. The external appearance in that case is shown in FIG. When 10 μL of mucopolysaccharide gel was added, it became droplets due to the surface tension with the hydrophobic filter. In order to add uniformly, 75 μL was required (FIG. 7B). The film thickness of the mucopolysaccharide gel when 75 μL was added was about 3000 μm.

(Example 2)
300 μL of a pH 7.4 phosphate buffer containing 30% DMSO was added to a 96-well Teflon (registered trademark) acceptor plate (purchased from Nihon Millipore). On the acceptor plate, the same method as in Example 1 (however, phosphatidylcholine 0.8% (w / w), phosphatidylethanolamine 0.8% (w / w), phosphatidylserine 0) .2% (w / w), Phosphatidylinositol 0.2% (w / w), Cholesterol 1.0% (w / w), 1,7-octadiene 97.0% (w / w) A plate having a mucopolysaccharide layer-artificial phospholipid membrane prepared at the bottom (using a phospholipid organic solvent solution) was attached (donor plate). When the mucopolysaccharide layer-artificial phospholipid membrane was prepared, the mucopolysaccharide layer-artificial phospholipid membrane was prepared by changing the amount of mucopolysaccharide gel added from 7.5 μL to 75 μL. At the same time, as a control, a Millipore 96-hole membrane filter, phosphatidylcholine 0.8% (w / w), phosphatidylethanolamine 0.8% (w / w), phosphatidylserine 0.2% (W / w), phosphatidylinositol 0.2% (w / w), cholesterol 1.0% (w / w), 1,7-octadiene 97.0% (w / w) An artificial phospholipid membrane having no mucopolysaccharide layer prepared by adding only a solvent solution was also prepared.

  Hydrocortisone, Ketoprofen, Propronol (all purchased from Sigma) were used as test substances, and 100 μL of their 0.5 mM pH 6.0 buffer was added to each well. After incubating at 30 ° C. for 2 hours, the amount permeated to the acceptor side was measured by UV (Spectramax, manufactured by Molecular Devices). The measurement results are shown in FIG. From FIG. 8, the membrane transmittance is the ratio of the actual UV absorption amount to the theoretical UV absorption amount when no film is present. It was suggested that the membrane permeability decreased as the mucopolysaccharide layer became thicker.

Example 3 Application to Evaluation of Absorption from Drug Suspension 300 μL of pH 7.4 phosphate buffer containing 30% DMSO was added to an acceptor plate made of 96-well Teflon (registered trademark). On the acceptor plate, the same method as in Example 1 (however, phosphatidylcholine 0.8% (w / w), phosphatidylethanolamine 0.8% (w / w), phosphatidylserine) 0.2% (w / w), phosphatidylinositol 0.2% (w / w), cholesterol 1.0% (w / w), 1,7-octadiene 97.0% (w / w) A plate (donor plate) with a mucopolysaccharide layer-artificial phospholipid membrane prepared at the bottom was used as a control when creating the mucopolysaccharide layer-artificial phospholipid membrane. , Phosphatidylcholine 0.8% (w / w), Phosphatidylethanolamine 0.8% (w / w), Phosphatidylserine 0.2% (w / w) Only phospholipid organic solvent solution consisting of 0.2% (w / w) of phosphatidylinositol, 1.0% (w / w) of cholesterol, 97.0% of 1,7-octadiene (w / w) is added An artificial phospholipid membrane having no mucopolysaccharide layer was also prepared.

  Donor plates were treated with fast and satiety simulated gastrointestinal fluid (Galia, E .; Nicolaides, E .; Horter, D .; Lobenberg, R .; Reppas, C. et al., Pharm. Res. 1998, 15, 698-705.) Was added in an amount of 95 μL. Furthermore, 5 μL (0.4 mg / mL) of a sodium carboxymethylcellulose suspension of danazol, which is a poorly water-soluble drug, was added. After incubation at 30 ° C. for 15 hours, the amount permeated to the acceptor side was measured by HPLC. HPLC conditions were Alliance (manufactured by Waters), mobile phase 60% acetonitrile 0.1% TFA, column Cadenza CD C18 50 × 3 mm (manufactured by Imtakt), column temperature 40 ° C., flow rate 1 mL / min, detection wavelength 229 nm. It was. In the same manner, the measurement was also performed without the mucopolysaccharide layer. As shown in Table 1 below, the permeation amount when using the fasted simulated gastrointestinal fluid and the ratio when using the satiety simulated solution were defined as the fasting / saturation ratio. Absence of mucopolysaccharide did not reflect clinical absorption, but it did reflect the presence of mucopolysaccharide. In Table 1, “a” means the ratio of permeability when using a fasting gastrointestinal simulated solution and a satiety gastrointestinal simulated solution, and “b” is absorption of fasting administration and satiety administration in humans. Mean ratio (Charman, WN; Rogge, MC; Boddy, AW; Berger, BMJ Clin. Pharm. 1993, 33, 381-386.).

It is sectional drawing which shows typically the mucopolysaccharide laminated lipid membrane which concerns on 1st Embodiment. It is sectional drawing which shows typically the mucopolysaccharide laminated lipid membrane which concerns on 2nd Embodiment. It is sectional drawing which shows typically the membrane-permeability measuring apparatus 200 incorporating the mucopolysaccharide laminated lipid membrane 110 which concerns on 2nd Embodiment. It is sectional drawing which shows typically the state which is measuring the membrane permeability of a test substance using the membrane permeability measuring apparatus 200. FIG. 2 is a cross-sectional view schematically showing a process for producing a mucopolysaccharide laminated lipid membrane according to Example 1. FIG. (A) is a side view of the mucopolysaccharide laminated lipid membrane of Example 1, and (b) is a top view of the mucopolysaccharide laminated lipid membrane of Example 1. (A) is a side view showing a state in which mucopolysaccharide gel is added without forming a hydrophilic filter layer, and (b) is a state in which mucopolysaccharide gel is uniformly added without forming a hydrophilic filter layer. FIG. It is a figure which shows the result of the membrane permeability evaluation in Example 8.

Explanation of symbols

  2 ... lipid membrane, 4 ... mucopolysaccharide, 6 ... gel polymer, 8 ... hydrophilic filter, 10 ... hydrophilic filter layer, 12 ... support, 20 ... Test substance storage container, 22 ... Permeate storage container, 30, 34 ... Solution, 32 ... Test substance, 100 ... Mucopolysaccharide laminated lipid membrane according to the first embodiment, 110 -Mucopolysaccharide laminated lipid membrane according to the second embodiment, 200 ... membrane permeability measuring device.

Claims (14)

Lipid membranes,
A mucopolysaccharide laminated lipid membrane comprising: a hydrophilic filter layer including a hydrophilic filter formed on the lipid membrane and impregnated with a gel polymer containing mucopolysaccharide.   The mucopolysaccharide laminated lipid membrane according to claim 1, wherein the hydrophilic filter layer is formed on one surface of the lipid membrane. The hydrophilic filter layer is
A layer formed by impregnating the hydrophilic filter with a mixture containing a polymer capable of sol-gel transition and mucopolysaccharide in a sol state and transferring the sol polymer to a gel polymer. The mucopolysaccharide laminated lipid membrane according to claim 1 or 2, wherein   The mucopolysaccharide laminated lipid membrane according to claim 3, wherein the mixture is an aqueous solution.   The mucopolysaccharide laminated lipid membrane according to claim 3 or 4, wherein the polymer capable of sol-gel transition is agarose.   The mucopolysaccharide laminated lipid film according to claim 1, wherein the mucopolysaccharide is at least one selected from the group consisting of mucin, chondroitin sulfate, hyaluronic acid, sialic acid polysaccharide, heparin, hepatylline sulfate, keratosulfate, and chitin. .   The mucopolysaccharide laminated lipid membrane according to any one of claims 1 to 6, wherein the hydrophilic filter is at least one selected from the group consisting of a paper filter, a cellulose filter, and a cellulose filter derivative.   The mucopolysaccharide laminated lipid membrane according to any one of claims 1 to 7, wherein the lipid membrane is an artificial phospholipid membrane.   The mucopolysaccharide laminated lipid membrane according to any one of claims 1 to 8, wherein the hydrophilic filter layer has a thickness of 100 µm to 500 µm.   A filter for measuring membrane permeability comprising the mucopolysaccharide laminated lipid membrane according to any one of claims 1 to 9. Mucopolysaccharide laminated lipid membrane according to any one of claims 1 to 9,
A test substance storage container for storing a test substance so as to be disposed on one side of the mucopolysaccharide laminated lipid film and to be in contact with the mucopolysaccharide laminated lipid film;
A membrane permeability measuring device, comprising: a permeate containing container that is disposed on the other surface of the mucopolysaccharide laminated lipid membrane and contains a permeate from the mucopolysaccharide laminated lipid membrane. Mucopolysaccharide laminated lipid membrane according to any one of claims 1 to 9,
A test substance storage container for storing a test substance so as to be disposed on one side of the mucopolysaccharide laminated lipid film and in contact with the mucopolysaccharide laminated lipid film;
A membrane permeability measurement kit comprising at least a permeate containing container disposed on the other surface of the mucopolysaccharide laminated lipid membrane and containing a permeate from the mucopolysaccharide laminated lipid membrane. It arrange | positions so that the solution containing a test substance may contact | connect one side of the mucopolysaccharide laminated lipid membrane as described in any one of Claims 1-9, and the said test substance is put on the other side of the said mucopolysaccharide laminated lipid membrane Arrange the solution so that it does not contain
After a predetermined time has elapsed, the membrane permeability of the test substance is measured by measuring the amount of the test substance in the solution on the other side and / or the amount of the test substance in the solution on the one side. Membrane permeability evaluation method. A measurement step of measuring the membrane permeability of a plurality of test substances by the membrane permeability measurement method according to claim 13;
And a selection step of selecting a desired test substance from the plurality of test substances based on the membrane permeability data obtained in the measurement step.

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