JP2006516047A - Transdermal assay with magnetic clamp - Google Patents

Abstract

  The transdermal assay device (600) includes a first (610), a second (650) and a third member (640). The first member (610) has one or more sample surfaces, each configured to receive an upper sample (630). The second member (650) defines one or more reservoirs (654), each having an opening on the surface of the second member. Each sample surface is substantially the same size as each opening. The transdermal assay device also includes a magnet clamp configured to clamp the tissue specimen between the sample surface and the opening. The magnet clamp preferably includes a magnet with a force selected based on the clamping force required between the first member and the second member. Furthermore, the present invention provides a method of using the transdermal assay device.

Description

  The field of the invention relates to tissue barrier assays for screening formulations and chemical compositions.

  In vitro analysis of the movement of compounds (eg drugs) across an epithelial barrier such as the intestinal epithelium or airway epithelium is typically performed using a Ussing-type chamber. In order to perform a tissue barrier assay using a Ussing-type chamber, in a device having a sealed internal hollow chamber in which a piece of tissue is removed from an organism as an intact sheet and the internal chamber is a two-chamber chamber Secure to. Thereafter, a solution compatible with the living body is filled in both chambers, and the target drug is added to one chamber solution. Samples are then taken from the opposite chamber solution at various times to determine the rate of transport of drug across the tissue barrier. This type of tissue barrier assay is not only cumbersome but also inefficient and is limited to a very limited number of independent samples derived from the unit area of the tissue sheet.

  Drug transdermal delivery is a type of tissue delivery that involves the drug being transported from the transdermal drug delivery device through the skin and into the patient's bloodstream. Transdermal drug delivery has many advantages over other drug delivery methods. One obvious advantage is avoidance of the needle and the pain caused by the needle. This is particularly desirable for drugs that are administered repeatedly. Avoiding needle discomfort can improve patient compliance with drug therapy.

  Another advantage of transdermal drug delivery is the potential to provide long-term or sustained delivery that may range from days to weeks. Other delivery methods, such as oral or pulmonary delivery, generally require repeated administration of the drug to maintain a suitable drug concentration in the body. With continuous transdermal delivery, dosage maintenance is automatic over time. This is particularly beneficial for drugs that have a short half-life in the body, such as peptides or proteins.

  The last advantage is that when administered transdermally, drug molecules need only pass through the skin to reach the bloodstream. Drugs administered transdermally avoid first-pass metabolism in the liver and avoid other degradation pathways such as low pH and enzymes in the gastrointestinal tract.

  Skin is the largest organ in the body. The skin is highly impermeable and prevents loss of moisture and electrolytes. The skin is mainly divided into two layers, the outer epidermis and the inner dermis. The epidermis is 50-100 microns thick on the outer layer of the skin (Monteiro-Riviere, 1991; Champion et al., 1992). The dermis is the inner layer of the skin and varies in thickness from 1 to 3 mm. The goal of transdermal drug delivery is to allow the drug to reach this layer of skin, where there are capillaries and allow the drug to be delivered systemically. There are no nerve endings or blood vessels in the epidermis. The main purpose of the epidermis is to create a strong layer of dead cells on the skin surface, thereby protecting the organism from the environment. The outermost layer of the epidermis is called the stratum corneum, and dead cells containing the stratum corneum are called stratum corneum cells or keratinocytes.

  The stratum corneum is usually modeled or described as a brick wall (Elias, 1983; Elite, 1988). This "bricks" are dead and flattened stratum corneum cells. Generally, about 10-15 stratum corneum cells are stacked vertically throughout the stratum corneum (Monteiro-Riviere, 1991; Champion et al., 1992). The stratum corneum cells are wrapped in a sheet of lipid bilayer (“mortar”). The interval between the lipid bilayer sheets is 50 nm or less. In general, there are about 4 to 8 lipid bilayers between each pair of stratum corneum cells. This lipid matrix is mainly composed of ceramides, sphingolipids, cholesterol, fatty acids and sterols, with little water (Lampe et al., 1983 “a”; Lampe et al., 1983 “b”; Elias, 1988) .

  The stratum corneum is the thinnest layer of skin, which is the primary barrier to molecules or microorganisms that invade across the skin. For many molecules, it is very difficult to cross this stratum corneum, which is why transdermal drug delivery means have not been widely used to date. Once molecules pass through the stratum corneum, diffusion occurs rapidly from the epidermis, dermis to blood vessels. Thus, much of the interest in transdermal drug delivery research has focused on transporting molecules and drugs across the stratum corneum.

  The most common form of transdermal drug delivery device is the transdermal drug “patch”, where the drug or pharmaceutical is contained in a reservoir located adjacent to the skin (Schaefer and Redelmeier, 1996). This drug molecule generally passes through the skin by simple diffusion. Transport is governed by the rate of molecular diffusion into and out of the skin and the distribution of the drug into the skin. In general, transdermal drug delivery is limited to those that readily penetrate the skin, such as small lipophilic molecules such as scopolamine, nitroglycerin, and nicotine. Delivery is slow, usually taking time for the drug to pass through the skin, and is only effective when a very small amount of drug is needed to effect this therapeutic biological effect. (Guy and Hadgraft, 1989).

  Because transdermal delivery is time consuming, many substances have been used to facilitate molecular transport rates. These substances are known as chemical or permeation enhancers. Chemical enhancers increase the flux of the drug through the skin by increasing the solubility of the drug in the stratum corneum or increasing the permeability of the drug in the stratum corneum. The choice is further complicated because the number of possible accelerators is large and the combination of accelerators is known to improve drug flux unexpectedly due to the presence of each independent component. ing.

  Transdermal drug delivery devices, such as transdermal patches, generally include an adhesive that serves to keep the device in close contact with the skin, but the adhesive further includes a matrix in which the drug is dissolved or dispersed. It may be formed. There are many different forms of adhesives that can be used, and it is often a very difficult problem to choose which adhesive to use in order to use the drug or drug and accelerator together.

  Currently, the selection of appropriate adhesives and promoters, and their relative proportions with respect to drugs, is determined only by general guidelines from what is considered safe and effective with other drugs. ing. Most drug discovery is done through trial and error experiments.

  Many transdermal transport experiments to date have used relatively large human skin diffusion cells, where a drug solution containing additives is placed on the source side, and typically a saline or dermal model on the sink side Add another solution that you think. A skin membrane separates the cell on two sides, which is often dead skin of the stratum corneum that has been carefully exfoliated from a whole skin sample supplied from a tissue bank. The capacity of the device is typically 5cc or more. Samples are taken periodically from the sink side of the cells to measure the drug flux through the stratum corneum film. The entire process is very laborious and requires the use of a large amount of skin, which is extremely difficult to obtain. Thus, only a relatively small number of the many possible chemical combinations can be analyzed. In addition, the number of formulations that can be tested on the skin of a single donor is limited, and the measurements obtained from responses from different donor skin samples vary further, further comparing the effectiveness of these formulations. It is difficult.

  Accordingly, there remains a need in the art for devices and methods for identifying optimal compositions or formulations for tissue barrier transport of compounds, pharmaceuticals and other ingredients, including transdermal transport. Such a device should be easy to assemble. The device should be sealed where the fastening force and sealing pressure required between the drug or pharmaceutical used to make the model of the dermis, the skin membrane and saline or other solutions are equivalent Does not disturb its function.

SUMMARY OF THE INVENTION The present invention tests a high-throughput system and method for simultaneously preparing multiple component combinations at various concentrations and uniqueness, and the tissue barrier transmission of the components in each combination, such as transdermal delivery. To a high throughput method. According to the method of the present invention, an active ingredient such as a pharmaceutical agent can be used for tissues such as skin, lung tissue, tracheal tissue, nasal tissue, bladder tissue, placenta, vaginal tissue, rectal tissue, stomach tissue, gastrointestinal tissue and eye or corneal tissue. When transported through, it is possible to measure the effects of additional or inert components such as excipients, carriers, accelerators, adhesives and additives. The present invention thus includes high-throughput testing of pharmaceutical compositions or formulations for the purpose of determining an overall optimal composition or formulation to improve tissue transport such as, but not limited to, transdermal transport. Is included. Specific embodiments of the invention are described in detail below.

  In one embodiment, the invention relates to an apparatus for measuring the transport of a component through tissue, the apparatus comprising a support plate, a sample array supported by the support plate, a membrane or tissue specimen covering the sample array, and A storage plate secured to the membrane or tissue specimen opposite the sample array is provided. In one embodiment of the invention, each sample in the array is characterized by a distinct active ingredient, or a component such that different physical states of the active ingredient are present in one or more samples in the sample array. Contains a composition or formulation.

In another aspect of the invention, each sample of the array includes a common component and at least one additional component, each sample comprising:
(I) the identity of the additional components,
It differs from at least one of the other samples with respect to at least one of (ii) the ratio of the common component to the additional component, or (iii) the physical state of the common component.

  The “common component” is a component included in all samples in the sample array. In one embodiment, the common ingredient is an active ingredient, and the active ingredient is preferably a pharmaceutical, a nutritional supplement, a replacement pharmaceutical or a dietary supplement. The sample may have the form of a liquid, solution, suspension, emulsion, solid, semi-solid, gel, foam, paste, ointment or powder.

In another embodiment, the present invention relates to a method for measuring tissue barrier transport of a sample, said method comprising:
(A) preparing a sample array having an active ingredient and at least one additional ingredient, each sample being (i) the identity of the active ingredient (ii) the identity of the additional ingredient (iii) the active ingredient for the additional ingredient Or (iv) different from at least one of the other samples with respect to at least one of the physical states of the active ingredient,
(B) covering the sample array with a tissue specimen;
(C) fixing a storage plate having a storage array corresponding to the sample array to the tissue specimen side opposite to the sample array;
(D) filling the array of reservoirs with a storage medium; and (e) measuring the transport of the active ingredients from each sample through the tissue specimen at one or more time points. Including measuring the concentration.

  In preferred embodiments, the active ingredient is a pharmaceutical, a nutritional supplement, a replacement pharmaceutical, or a dietary supplement. In another embodiment, the tissue specimen is skin.

In another embodiment, the present invention relates to a method for analyzing or measuring a flux of a sample passing through tissue, the method comprising: (a) providing a sample array having a common component and at least one additional component, wherein each sample is (I) the identity of the active ingredient,
(Ii) identity of the additional component (iii) ratio of the common component to the additional component, or (iv) physical state of the common component,
Unlike at least one of the other samples with respect to at least one of
(B) covering the sample array with a tissue specimen;
(C) fixing a storage plate having a storage array corresponding to the sample array to the tissue specimen side opposite to the sample array;
(D) filling the storage array with a storage medium; and (e) measuring the flux of the common component from each sample through the tissue specimen to determine the concentration of the common component in each reservoir as a function of time. Including measuring.

  In an alternative embodiment, the method includes the additional step of cutting the tissue specimen to avoid lateral diffusion between wells. Preferably, the method includes the steps of analyzing the tissue specimen for defects or inhomogeneities and correcting or repairing the defects.

  According to the present invention, a transdermal assay device is provided. The transdermal assay device includes at least a first and a second member. The first member has one or more sample surfaces, each configured to receive a sample thereon. The second member defines one or more reservoirs, each having an opening on the surface of the second member. Each sample surface is substantially the same size as each opening. The transdermal assay device also includes a magnet clamp configured to clamp the tissue specimen between the sample surface and the opening. The magnet clamp also seals the various parts of the device together. The magnet clamp is preferably made of a magnet having a force selected based on a clamping force required between the first member and the second member.

  The present invention also provides methods of using the transdermal assay device. The sample is placed on the sample surface of the first member. The reservoir defined by the second member is filled with a liquid medium, where the reservoir has an opening on the surface of the second member. Next, a tissue specimen is placed between the sample and the opening. The tissue specimen is then clamped between the opening sample and the liquid medium. Periodically, a sample of the liquid medium is removed from the reservoir to measure the sample concentration in the liquid medium. This concentration indicates sample transport through the tissue specimen.

  The devices and methods described above are preferably used for high-throughput screening of active ingredients or drug fluxes through the stratum corneum, where such fluxes are at least partially in the presence of a promoter in the tissue. Allow to be measured by permeability. This permeability is generally governed by at least two factors: the solubility of the active ingredient or drug within the stratum corneum and the diffusibility of the active ingredient or drug within the stratum corneum. These two factors, solubility and diffusivity, can be individually measured by a method of indirectly evaluating the flux through the stratum corneum. Thus, an array of wells containing samples of various compositions of active and inactive compounds is constructed, including, but not limited to, active ingredients / carriers or excipients, active ingredients / Carrier or excipient / accelerator /, active ingredient / adhesive / accelerator / additive. A predetermined amount of stratum corneum is added to each well, and the rate at which the active ingredient or drug is incorporated into the tissue sample is determined by extracting tissue at various time points from similarly prepared wells. If the concentration is measured after sufficient time has passed until the amount of dissolution does not change with time, the equilibrium concentration of the active ingredient or drug in the tissue can be evaluated. The product of this ratio and solubility is proportional to the permeability of the active ingredient or drug.

  The high-throughput combinatorial screening systems and methods of the present invention identify, but are not limited to, optimal compositions or formulations to achieve the desired results for such compositions or formulations including the construction of transdermal delivery devices. To do. In particular, the systems and methods of the present invention include: 1) an optimal composition or formulation comprising one or more active ingredients and one or more inactive ingredients to achieve the desired properties of the composition or preparation; 2) a drug 3) Optimum drug / adhesive / accelerator / additive composition for maximum drug flux through the stratum corneum And 4) may be used to identify the optimal drug / adhesive / promoter / additive composition to minimize cytotoxicity.

The features and advantages of the present invention will be better understood from the following detailed description taken in conjunction with the accompanying drawings.
1 is a schematic diagram of a high throughput apparatus for measuring tissue barrier transport such as transdermal transport according to the present invention. FIG. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput apparatus for measuring tissue barrier transport using a solid source sample according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput apparatus for measuring tissue barrier transport using a solid source sample according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput apparatus for measuring tissue barrier transport using a solid source sample according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput apparatus for measuring tissue barrier transport using a solid source sample according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput device for measuring tissue barrier transport according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a diffusion cell used to measure tissue barrier transport, either alone or as part of a high throughput apparatus according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a diffusion cell used to measure tissue barrier transport, either alone or as part of a high throughput apparatus according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a diffusion cell used to measure tissue barrier transport, either alone or as part of a high throughput apparatus according to the present invention. FIG. 2 is a schematic diagram of an apparatus for filling sample wells in a sample array, such as the sample array in the high throughput apparatus shown in FIG. FIG. 2 is a schematic diagram of an apparatus for filling sample wells in a sample array, such as the sample array in the high throughput apparatus shown in FIG. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput apparatus for measuring or analyzing tissue barrier transport using a solid source sample according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput apparatus for measuring or analyzing tissue barrier transport using a solid source sample according to the present invention. FIG. 6 is a schematic diagram of an alternative embodiment of a high-throughput apparatus for measuring or analyzing tissue barrier transport using a solid source sample according to the present invention. FIG. 6 is an exploded view of another transdermal assay device according to another embodiment of the present invention. FIG. 6 is an exploded view of yet another transdermal assay device according to yet another embodiment of the present invention.

  The present invention relates to high-throughput combination systems and methods that improve tissue barrier transport of active compounds such as, for example, pharmaceuticals or drugs, other compounds, or combinations of compounds. In one embodiment, the systems and methods of the present invention provide optimal components (eg, solvents, carriers, transport enhancers, adhesives, additives, etc.) for pharmaceutical compositions or formulations that are transported to a patient via tissue transport. It may be used to identify other excipients. The pharmaceutical composition or formulation includes, but is not limited to, transdermally (eg, in the form of a transdermal delivery device), topically (eg, in the form of an ointment, lotion, gel, and solution) and ophthalmic. It includes a pharmaceutical composition or formulation that is administered or delivered (eg, in the form of a solution). As used herein, “high throughput” means the number of samples generated or screened as described herein, typically at least 10, more typically at least 50-100, Preferably it refers to 1000 or more samples.

  The high-throughput method of the present invention can be performed using various forms of samples. In general, this method is performed using either a liquid sample or a solid or semi-solid sample.

As used herein, “liquid source” means that the sample or components to be measured or analyzed are in liquid form, including but not limited to liquids, solutions, emulsions, suspensions and the like. All of the above, including solid particulates dispersed in

  As used herein, “solid source” means that the component or components to be measured or analyzed is solid or semi-solid, including but not limited to powders, gels, films, Includes foams, pastes, ointments, adhesives, high viscoelastic liquids, high viscoelastic liquids containing solid particulates dispersed therein, and transdermal patches.

  As used herein, “storage medium” or “liquid medium” refers to a liquid, solution, gel or sponge that is chemically compatible with the sample and tissue components used in the device or method of the present invention. In one embodiment of the invention, the storage medium includes a portion of a specimen removed to measure or analyze transport, flux or diffusion of components across the tissue barrier. The storage medium is preferably a liquid or a solution.

5.1 Overview of Tissue Barrier Transport Measurement Device FIG. 1 is a schematic diagram of a preferred embodiment of a high throughput device 100 for measuring tissue barrier transport of the sample array 112 of the present invention. The apparatus 100 comprises a substrate plate 114 that supports a sample array 112, a tissue specimen 120 and a storage plate 130. In this embodiment, each sample in the sample array 112 is placed in the sample well 116. A base 118 that forms the bottom of each sample well 116 is attached to the bottom of the substrate plate 114. The base 118 may be of any type as long as air is exhausted from the sample well 116 when the sample is filled, and may be a membrane made of a suitable material (for example, a rubber membrane). On the other hand, the base 118 is a rigid, removable substrate plate, such as a plate 214 (described below with respect to FIGS. 2A-2D) that can support an array of solid source samples.

The substrate plate 114 may be a rigid grid or plate capable of supporting several samples. For example, the substrate plate 114 may be a 24, 36, 48, 72, 96 or 384 well plate. The apparatus 100 preferably comprises one or more sample arrays 112, and the number of sample wells 116 in the apparatus 100 is at least 100, preferably at least 1000, more preferably at least 10,000. The size of the sample well 116 is preferably about 1 mm to about 50 mm, more preferably about 2 mm to about 10 mm, and most preferably about 3 mm to about 7 mm. For example, an array of approximately 30,000 samples can be obtained for a 0.25 m ² skin with a 3 mm well type.

  As used herein, the term “array” or “sample array” (eg, array 112) means a plurality of samples used under normal experimentation, each sample containing at least two components, and At least one of the ingredients is an active ingredient. In one embodiment of the invention, one of the components of the sample is a “common component”, and as used herein, this common component refers to the component present in all samples of the array except the negative control.

  Sample array 112 is designed to obtain samples of a plurality of different compositions, and by analyzing the sample array, measuring the optimal composition or formulation to improve the transport of components through tissue 120 Is possible. Each sample in the sample array 112 is preferably, but not necessarily, different from all of the other samples in the array in at least one of the following respects.

(Ii) Identity of active ingredient (ii) Identity of additional ingredient (iii) Ratio of active ingredient or common ingredient to additional ingredient, or (iv) Physical state of active ingredient or common ingredient is 24, 36, 48 96, or more, preferably at least 1,000, more preferably at least 10,000. An array typically comprises one or more sub-arrays. For example, the sub-array can be a plate with 96 sample wells.

  Covering the substrate plate 114 and the sample array 112 is a tissue specimen 120. Tissue 120 is preferably a tissue sheet, for example, skin, lung, trachea, nose, placenta, vagina, rectum, large intestine, gastrointestinal tract, stomach, bladder, or corneal tissue. More preferably, the tissue 120 is a skin tissue or stratum corneum. When using human dead skin as tissue 120, a known method for preparing a tissue specimen is to soak in 60 ° C. water for 2 minutes, then remove the epidermis and store in a humidified chamber at 4 ° C.・ With stripping. Prior to the experiment, a piece of epidermis is removed from the humidification chamber and placed on the substrate plate 114. Tissue 120 may be supported with nylon mesh (Telco) to prevent any damage to the tissue and to mimic the fact that in vivo the skin is supported by a mechanically strong dermis. Alternatively, other types of tissues may be used including live tissue explants, animal tissues (such as rodents, cattle or pigs) or cultured tissue equivalents. Examples of suitable culture tissues include those taught in DERMAGRAFT (Advanced Tissue Sciences) and US Pat. No. 5,266,480, which is incorporated herein by reference.

  As an alternative embodiment of the present invention, the tissue specimen 120 is divided into several segments by grooves 122 between the sample wells 116 to prevent lateral diffusion through the tissue specimen 120 located between adjacent samples. . There are several ways to create the groove 122, such as mechanical engraving or cutting, laser cutting or crimping (eg, between plates 114 and 130, or using a “waffle bake” embossing tool). is there. It is preferable to avoid the use of cutting tools that can distort or damage the tissue specimen 120 due to mechanical pressure and use laser engraving. The laser groove 122 is made into a very small kerf, which allows the use of relatively high density samples and more efficient tissue specimens. The heat affected zone generated by the laser tool can be minimized, thereby reducing damage to the tissue specimen 120.

  A storage plate 130 (eg, a bottomless titer plate) is placed on top of the tissue 120, on the tissue opposite the substrate plate 114. The storage plate includes a plurality of hollow storage parts 132. When fixing the plate 130, each reservoir 132 is aligned over the sample well 116 so that the tissue separates each well 116 and reservoir 132. The storage plate 130 is secured to the substrate plate 114 using clamps, screws, fasteners or other suitable attachment means. Plates 130 and 114 are preferably crimped with sufficient pressure to form a liquid seal around reservoir 132. Each reservoir is filled with a reservoir, such as saline, for receiving sample components or compounds that pass through tissue 120 and diffuse into reservoir 132. In one embodiment, the storage medium is a PBS solution containing about 2% BSA.

  The transport or flux of components from the sample well 116 through the tissue 120 (ie, tissue barrier transport or diffusion) can be analyzed by measuring the component concentration of the sample removed from the reservoir 132. Comparison of various sample / reservoir measurements helps to determine the optimal sample composition for improved tissue transport or diffusion of desirable components (eg, pharmaceuticals).

  Samples are preferably prepared automatically, added to sample wells and mixed. Similarly, specimens from reservoirs 132 containing components that are transported or diffused and their concentrations can be automatically measured and processed. "Automatic" or "automatically" means using computer software and robotics to add, mix and analyze samples, components and specimens or diffusion products.

  The sample is added to the sample wells of the sample array of the present invention using various input or material transfer techniques known to those skilled in the art, for example, sample array 112 of FIG. These techniques include, but are not limited to, manual dosing, pipetting, and other manual or automatic solid or liquid dispensing systems.

  After the ingredients are added to the sample well and mixed, the sample may be processed by known techniques such as heating, filtration and lyophilization. Those skilled in the art will know how to treat samples according to the characteristics to be tested. Samples may be processed individually or in groups, but preferably in groups. Details regarding suitable automatic dispensing sampling devices and methods for formulating solutions or compositions are further disclosed in copending US patent application Ser. No. 09 / 540,462, the entire contents of which are hereby incorporated by reference.

  In short, microarray systems that have been developed by several companies for use in the systems described herein have already been developed, but now they all identify compounds with a certain predetermined activity. It is used as the sole purpose of screening, as opposed to screening components or compounds with known identities to determine the optimal combination of components to achieve the desired result. Such a system may require modification, but is well within the normal technical scope of those skilled in the art. Examples of companies that hold microarray systems include Genelogic of Gaithersburg, Maryland (see US Pat. No. 5,843,767 to Beattie), Luminex, Austin, Texas, and Fullerton, California. Beckman Instruments, MicroFab Technologies, Plano, Texas, Nanogen, San Diego, California, and Hyseq, Sunnyvale, California. These devices test samples based on a variety of different systems. All devices have thousands of microscopic pathways that direct components to test wells, and reactions take place in the test wells. These systems are connected to a computer and data analysis is performed using appropriate software and data sets. The Beckman Instruments system can supply nanoliter samples in 96 or 384 arrays and is well suited for hybridization analysis of nucleotide molecule sequences. Sample supply by the MicroFab Technologies system uses an ink jet printer to evenly distribute separate samples to the wells.

  These and other systems can be adapted as required for use in the present application. For example, the creation of combinations of active ingredients at various concentrations and combinations with various additional or inactive ingredients can be performed using standard formulating software (eg, software available from Mathworks, Natick, Mass.). Matlab). Therefore, the created combination can be downloaded to a spreadsheet such as Microsoft Excel. From this spreadsheet, a sample list can be prepared according to various combinations obtained by the formulation software, so that a work list can be created and commanded to the automatic dispensing mechanism. This work list can be created using standard programming methods according to the automatic distribution mechanism used. By using a so-called work list, the file can be used simply as a processing command rather than as an individually programmed process. This work list combines the formalized output from the formalization program with the appropriate commands in a file format that can be read directly by the automatic distribution mechanism.

  The automatic dispensing mechanism supplies each sample well with an active ingredient, such as at least one pharmaceutical agent, and various inert or additional ingredients such as, for example, solvents, carriers, excipients, and additives. An automatic dispensing mechanism that can supply each component in various amounts is preferred. In one embodiment, the automatic dispensing mechanism uses one or more micro solenoid valves.

  Automatic liquid and solid dispensing systems are widely known, such as RTP (Research Triangle Park), North Carolina, Tecan Genesis from Tecan-US. The robotic arm can perform the recovery and distribution of active ingredients and inert ingredients such as solutions, solvents, carriers, excipients, additives, etc. from the stock plate to the sample wells or sites. This process is repeated until the array is complete. The sample is then mixed. For example, the robot arm moves up and down on each well plate a set number of times to ensure proper mixing.

  In use of the apparatus 100 of FIG. 1, the apparatus is described above as containing samples in the sample well 116 of the array 112 above the tissue 120, in the reservoir 132, below the storage medium and the tissue 120. ing. In an alternative embodiment, this position is reversed, the reservoir 132 of the sample array 112 is below the tissue specimen 120, the sample well 116 is above the tissue specimen 120, and the upper plate or upper membrane is the reservoir 132. Over the storage plate 130.

  Another embodiment of the system and method of the present invention is described below, particularly with respect to FIGS.

5.2 Sample Composition Prior to providing additional details of the system and method for assessing tissue barrier transport of the present invention, Applicants describe a sample composition suitable for use in the present invention.

5.2.1 General Composition Terminology The term “component” as used herein means any substance or compound. Ingredients may be active or inactive. The term “active ingredient” as used herein refers to a substance or compound that confers major utility to the composition or formulation when the composition or formulation is used for its intended purpose. Examples of active ingredients include pharmaceuticals, nutritional supplements, alternative medicines, and dietary supplements. The active ingredient may be a sensory compounds, an agrochemical, an active ingredient in a consumer product formula, or an active ingredient in an industrial product formula. As used herein, an “inactive ingredient” is useful or potentially useful for use in a composition or formulation for active ingredient administration, but does not significantly affect the active properties of the active ingredient, Furthermore, it means an ingredient that does not cause the main usefulness of the composition or formulation. Examples of suitable inert ingredients include, but are not limited to, accelerators, excipients, carriers, solvents, diluents, stabilizers, additives, adhesives, and combinations thereof.

  The sample of the array preferably includes active and inactive components. In one embodiment, the active components in the sample of the array may be the same or different, while in another embodiment, the sample in the array includes the active component and the inactive component as common components. A person skilled in the art can make multiple permutations, for example when the active ingredient is a pharmaceutical, nutritional supplement, alternative medicine or dietary supplement, the preferred inactive ingredients are excipients, carriers, solvents, diluents, stabilizers , Accelerators, additives, adhesives and combinations thereof.

  As used herein, “sample” means a mixture of an active ingredient and one or more additional or inactive ingredients. Preferably, the sample contains two or more additional components, more preferably three or more additional components. In general, a sample contains one active ingredient, but can contain a variety of active ingredients. Furthermore, the samples in the sample array may contain one or more common components. The sample can be in any container or holder, or in or on any material or surface, the only requirement is that the sample must be located in a separate location . The sample is preferably contained within a sample well, such as, for example, a 250, 24, 36, 48 or 96 well plate (or filter plate) in a volume of 250 μl commercially available from Millipore, Bedford, Massachusetts. The sample can contain less than about 100 milligrams of active ingredient, preferably less than about 1 milligram, more preferably less than about 100 micrograms, and even more preferably less than 100 nanograms. The total sample volume is preferably about 1-200 μl, more preferably about 5-150 μl, most preferably about 10-100 μl. The sample can be a liquid source or a solid source sample, including, for example, a sample in the form of a solid, semi-solid, film, liquid, solution, gel, foam, paste, ointment, powder, suspension or emulsion.

  In accordance with the invention described herein, the “physical state” of a component is first defined by whether the component is a liquid or a solid. When the component is a solid, its physical state is further defined by the particle size and whether the component is crystalline or amorphous. When the component is crystalline, the physical state is further: (1) whether the crystalline matrix contains a co-adduct or whether the crystalline matrix originally contained a co-adduct, It is divided into (2) crystal habit (3) morphology, ie, crystal habit and particle size distribution, and (4) internal structure (polymorph), whether or not voids remain after removal. In a co-adduct, the crystalline matrix can contain either stoichiometric or non-stoichiometric amounts of the adduct, including, for example, a crystalline solvent or crystal water such as a solvate or hydrate. be able to. Non-stoichiometric solvates and hydrates include inclusions or inclusion compounds in which the solvent or water is trapped in a crystalline matrix, such as a channel, at any interval. A stoichiometric solvate or hydrate is when the crystalline matrix contains a solvent or water at a specific site and in a specific ratio. That is, the solvent or water molecule is the portion of the crystalline matrix that is placed in place. In addition, the physical state of the crystalline matrix can be altered by removing coadducts that are originally present in the crystalline matrix. For example, when solvent or water is removed from a solvate or hydrate, pores are formed in the crystalline matrix, thereby forming a new physical state. A crystal habit is the appearance of an individual crystal. For example, a crystal has a cubic, tetragonal, oblique, monoclinic, triclinic, rhombohedral, or hexagonal crystal form. Processing characteristics are affected by crystal habit. The internal structure of a crystal refers to a crystal form or a polymorph. Certain compounds may exist in different polymorphs, ie as distinct crystal types. In general, different polymorphs of a given compound differ in structure and properties, like crystals of two different compounds. Solubility, melting point, density, hardness, crystal form, optical and electrical properties, vapor pressure and stability all depend on the polymorphic form.

5.2.2. Active Ingredients and Common Ingredients As described above, common ingredients can be either active ingredients such as pharmaceuticals, nutritional supplements, alternative medicines or dietary supplements, or inactive ingredients. In a preferred embodiment of the present invention, the common ingredient is an active ingredient, more preferably a pharmaceutical product. As used herein, the term “medicament” means any substance or compound that has a therapeutic, disease-preventing, diagnosing, or prophylactic effect when administered to an animal or human. The term “pharmaceutical” includes prescription and over-the-counter drugs. Medications suitable for use in the present invention include all known or developed.

  Examples of suitable pharmaceuticals include, but are not limited to, cardiovascular pharmaceuticals such as amlodipine besylate, potassium losartan, irbesartan, diltiazem hydrochloride, clopidogrel bisulfate, digoxin, abciximab, furosemide, amiodarone hydrochloride, beraprost, tocopherol nicotinate, Anti-infective ingredients such as amoxicillin, potassium clavulanate, azithromycin, itraconazole, acyclovir, fluconazole, terbinafine hydrochloride, erythromycin ethyl succinate and acetylsulfixazole, such as sertraline hydrochloride, venlafaxine, bupropion hydrochloride, olanzapine, Such as buspirone hydrochloride, alprazolam, methylphenidate hydrochloride, fluvoxamine maleate and ergoloid mesylate Gastrointestinal products such as lansoprazole, ranitidine hydrochloride, famotidine, ondansetron hydrochloride, granisetron hydrochloride, sulfasalazine and infliximab, such as loratadine, fexofenadine hydrochloride, cetirizine hydrochloride, fluticasone propionate, salmeterol xinafoate and budesonide Respiratory treatments such as atorvastatin calcium, lovastatin, bezafibrate, ciprofibrate, and cholesterol-lowering agents such as gemfibrozil, such as paclitaxel, carboplatin, tamoxifen citrate, docetaxel, epirubicin hydrochloride, leuprolide acetate, bicalutamide, goserelin acetate implant, Irinotecan hydrochloride, gemcitabine hydrochloride and sargramosim (sargramo stim) and other blood preparations such as epoetin alfa, enoxaparin sodium and antihemophilic factors, antiarthritic components such as celecoxib, nabumetone, misoprostol and rofecoxib, such as lamivudine AIDS and AIDS related drugs such as indinavir sulfate, stavudine and lamivudine, for example diabetes and diabetes related therapeutics such as metformin hydrochloride, troglitazone and acarbose, for example biological drugs such as hepatitis B vaccine and hepatitis A vaccine, such as Hormones such as estradiol, mycophenolate mofetil and methylprednisolone, such as tramadol hydrochloride, fentanyl, metamizole, ketoprofen, morphine sulfate, acetylsalicylate lysate Analgesics such as sodium, ketorolac tromethamine, morphine, loxoprofen sodium and ibuprofen, topical products such as isotretinoin and clindamycin phosphate, anesthetics such as propofol, midazolam hydrochloride and lidocaine hydrochloride, e.g. Migraine treatments such as sumatriptan acid, zolmitriptan and rizatriptan benzoate, eg sedative and hypnotics such as zolpidem, zolpidem tartrate, triazolam and butylhycosine bromide, eg iohexol, technetium, TC99M, Imaging components such as sestamibi, iomeprol, gadodiamide, ioversol and iopromide, and for example, alsactide, americium, betazole, histamine, N'nitoru, metyrapone, Petagasutorin (petagastrin), phentolamine, radioactive B12, gadodiamide, gadopentetate, include diagnostic and contrast medium component such as Gadoteridol and perflubron. Other pharmaceuticals for use in the present invention include those listed in Table 1 below, which can be alleviated by developing new compositions or formulations using the systems, arrays and methods of the present invention. Have a problem.

Other examples of suitable pharmaceuticals are “2000 Med News”, No. 19, pages 56-60, and “The Physicians Desk Reference” (Medical Desk Reference), 53rd edition, pages 792-796, Medical Economics ( 1999), both of which are incorporated herein by reference.

  Examples of suitable veterinary drugs include, but are not limited to, vaccines, antibiotics, growth promoting ingredients and anthelmintics. Other examples of suitable veterinary medicines are The Merck Veterinary Manual, 8th edition, Merck, Rahway, NJ, 1998 (1997), The Encyclopedia of Chemical Technology. Encyclopedia) Volume 24, Kirk-Othomer (4th edition, page 826), and ECT (Encyclopedia of Industrial Chemistry), 2nd edition, Volume 21, “Veterinary Drugs”, by ALShore and RJMagee , Published by American Cyanamid.

  Other active ingredients suitable for tissue (or transmembrane) transport analysis using the systems and methods of the present invention include other nutritional supplements, alternative medicines, or dietary supplements.

  As used herein, the term “nutritional supplement” refers to a non-caloric or low-caloric substance that is administered to an animal or human to provide a nutritional effect, or a stable effect that is aesthetically pleasing to the touch, or Means a non-caloric or low-caloric substance administered in food to provide a nutritional effect. Although not limited to nutritional supplements, for example, fat binders such as caducean, plant extracts such as fish oils such as garlic and pepper extracts, vitamins and minerals such as preservatives, acidulants, anti-caking ingredients Antifoaming agent, antioxidant, bulking agent, coloring agent, curing agent, dietary fiber, emulsifier, enzyme, solidifying agent, wetting agent, swelling agent, lubricant, non-nutritive sweetener, food grade solvent, thickener, Alternative fats and food additives such as flavor enhancers and supplements such as, for example, appetite suppressants are included. Examples of suitable nutritional supplements are published in The Encyclopedia of Chemical Technology, Vol. 11, Kirk-Othomer (4th edition, pages 805-833) (1994). Examples of suitable vitamins are (1998) "The Encyclopedia of Chemical Technology" Vol. 25, Kirk-Othomer (4th edition, page 1), "Goodman & Gilman's: The Pharmacological Basis of Therapeutics" (Goodman (Gillman Pharmacology) 9th Edition, Joel G. Harman, edited by Lee E. Limbird, McGrawHill, 1547 (1996), both of which are incorporated herein by reference. Examples of suitable minerals are “The Encyclopedia of Chemical Technology” (Volume 16 of Industrial Chemistry), Kirk-Othomer (4th edition, page 746), and ECT Industrial Chemistry Encyclopedia (3rd edition, Volume 15). Mineral Nutrients "(mineral nutrition), pages 570-603, published by CLRollinson and MGEnig, University of Maryland, both incorporated herein by reference.

  As used herein, the term “alternative drug” refers to a substance administered to a subject or patient, preferably for example a herbal or herbal extract or concentrate, for the treatment of a disease or for physical health or well-being. Means a natural substance, in which case the substance does not require FDA approval. Examples of suitable alternatives include, but are not limited to, ginkgo, ginseng, valerian, oak bark, birch, echinasia, harpagophyti radix, and others, The Complete German Commission E Monographs: Therapeutic "Guide to Herbal Medicine" (all research papers of the German E Committee: Guidebook for Herbal Medicine) Mark Blumenthal et al., Integrative Medicine Communications (1998), incorporated herein by reference.

As used herein, the term “dietary supplement” means a food or food product that has both caloric value and pharmaceutical or therapeutic properties. Examples of dietary supplements include garlic, pepper, bran fiber and health drinks. Examples of suitable dietary supplements are MCLinder's “Nutritional Biochemistry and Metabolism with clinical Applications”, Elsevier, New York (1985), Pszczola et al. (1998) “Food technology”. (Food Technology) Vol. 52 , pages 30-37, and Shukla et al. (1992) “Cereal Foods World”, Volume 37 , pages 665-666.

When the active ingredient is a pharmaceutical, nutritional supplement, alternative medicine or dietary supplement, it is preferred that at least one of the additional ingredients is an excipient. As used herein, the term “excipient” refers to an inert substance used in the preparation of a pharmaceutical product as a result of processing or manufacture, or a pharmaceutical product, nutritional supplement, alternative pharmaceutical product for administration to an animal or human, and By inert substance used by those skilled in the art to formulate dietary supplements. Excipients that are approved or considered safe for human and animal administration are preferred. Examples of suitable additives include, but are not limited to, acidulants such as lactic acid, hydrochloric acid, and tartaric acid, solubilizers such as nonionic, cationic and anionic surfactants such as bentonite, cellulose and kaolin. Absorbents, e.g. alkalizing components such as diethanolamine, potassium citrate and sodium bicarbonate, e.g. anti-caking components such as tricalcium phosphate, magnesium trisilicate and talc, e.g. benzoic acid, sorbic acid, benzyl alcohol, Antibacterial components such as benzethonium chloride, bronopol, alkylparabens, cetrimide, phenol, phenylmercuric acetate, thimerosal and phenoxyethanol, such as ascorbic acid, alpha tocopherol, propyl gallate and sodium metabisulfite Oxidizing agents such as acacia, alginic acid, carboxymethylcellulose, hydroxyethylcellulose, dextrin, gelatin, guar gum, magnesium aluminum silicate, maltodextrin, povidone, starch, vegetable oils and zein, such as sodium phosphate, malic acid and Buffering components such as potassium citrate, for example chelating components such as EDTA, malic acid and maltol, such as
Coating components such as addition sugar, cetyl alcohol, polyvinyl alcohol, carnauba wax, lactose maltitol, titanium dioxide, sustained release excipients such as microcrystalline wax, white wax and yellow wax, such as calcium sulfate Detergents such as detergents such as sodium lauryl sulfate such as calcium phosphate, sorbitol, starch, talc, lactitol, polymethacrylates, sodium chloride and glyceryl palmitostearate such as colloidal silicon dioxide, cloth Disintegrants such as carmellose sodium, magnesium aluminum silicate, polacrilin potassium and glycol starch sodium, such as poloxamer 386 and polyoxyethylene fatty esters ( Dispersible components such as cetearyl alcohol, lanolin, mineral oil, petrolatum, cholesterol, isopropyl myristate and lecithin, such as anionic emulsifying wax, monoethanolamine and medium chain triglycerides Emulsifying ingredients such as ethyl maltol, ethyl vanillin, fumaric acid, malic acid, maltol and menthol and other wetting agents such as glycerin, propylene glycol, sorbitol and triacetin, e.g. calcium stearate, canola oil, palmi Lubricants such as glyceryl tostearate, magnesium oxide, poloxamer, sodium benzoate, stearic acid and zinc stearate, for example alcohols, benzyl phenylformate , Vegetable oil, diethyl phthalate, ethyl oleate, glycerol, glycofurol, indigo carmine, polyethylene glycol, sunset yellow, tartadine, solvents such as triacetin, such as cyclodextrin, albumin, xanthan gum For example, tonic components such as lyserol, dextrose, potassium chloride and sodium chloride, and mixtures thereof. Excipients include those that alter the absorption rate, bioavailability, or other pharmacokinetic properties of a pharmaceutical product, nutritional supplement, alternative pharmaceutical product, or dietary supplement. Examples of suitable excipients, such as binders and fillers, are other examples of Remington's Pharmaceutical Sciences, 18th edition, edited by Alfonso Gennaro, Mack Publishing, Easton, Pa. (1995), and Handbook of Pharmaceutical excipients "(handbook of pharmaceutical excipients) 3rd edition, edited by Arthur H. Kibbe, American Pharmacists Association, Washington D. C. (2000), both of which are incorporated herein by reference.

  Excipients that are commonly used in the formation of transdermal delivery devices and are therefore particularly useful in formulating samples of the present invention are permeation enhancers, adhesives and solvents. Each of these is described in more detail below.

5.2.3. Permeation enhancers Various types of permeation enhancers may be used to facilitate transdermal delivery of drugs. Permeation enhancers can be divided into chemical accelerators and mechanical accelerators, each of which is described in detail below.

5.2.3.1 Chemical Accelerators Chemical accelerators promote the transport rate of molecules through tissues or membranes by various mechanisms. In the present invention, chemical promoters are preferably used to reduce the barrier properties of the stratum corneum. Drug interaction changes to a more permeable drug (prodrug), which is then metabolized in the body back to its original form (6-fluorouracil, hydrocortisone) (Hadgraft, 1985) or Increasing drug solubility (ethanol, propylene glycol). Despite numerous studies (more than 200 compounds studied) (Chattaraj and Walker, 1995), there is still no universally applicable mechanical theory for chemical enhancement of molecular transport. Much of the published work on chemical accelerators is mostly based on experience and trial and error (Johnson, 1996).

  Many different classes of chemical accelerators have been identified, such as cationic, anionic, and nonionic surfactants (sodium dodecyl sulfate, polyoxamers), fatty acids and alcohols (ethanol, oleic acid, Lauric acid, liposomes), anticholinergic agents (benzylonium bromide, oxyphenonium bromide), alkanones (n-heptane), amides (urea, NN-diethyl-m-toluamide), fatty acid esters (n- Butyrate), organic acids (citric acid), polyols (ethylene glycol, glycerol), sulfoxides (dimethyl sulfoxide) and terpenes (cyclohexene) (Hadgraft and Guy 1989, Walters 1989, Williams and Barry 1992, Chattaraj and Walker 1995). Many of these enhancers interact with either skin or drugs. These enhancers that interact with the skin are referred to as “lipid permeation enhancers” in the present application and have an interaction with the skin. For the interaction with the skin, the stratum corneum is distributed by the enhancer, Includes splitting the stratum (azone, ethanol, lauric acid), binding and splitting proteins in the stratum corneum (sodium dodecyl sulfate, dimethyl sulfoxide), or hydration of the lipid bilayer (urea, benzylonium bromide) . Other chemical enhancers increase drug solubility (hereinafter referred to as “dissolution enhancers”) in the medium to facilitate increased transdermal delivery of the drug. The lipid permeation enhancers, dissolution enhancers, and combinations of enhancers (also referred to as “binary systems”) are described in further detail below.

5.2.2.3.1.1. Lipid penetration enhancers Chemical substances that promote permeability to lipids are known and commercially available. For example, ethanol increases drug solubility up to 10,000-fold and estradiol flux by 140-fold, while unsaturated fatty acids increase lipid bilayer fluidity (Bronaugh and Maibach, Marcel Dekker 1989 ”1-12). Examples of fatty acids that disrupt the lipid bilayer include linoleic acid, caprylic acid, lauric acid and neodecanoic acid, which may be mixed in a solvent such as ethanol or propylene glycol. Evaluation of public permeation data using lipid bilayer splitting agents is in good agreement with the particle size dependent observation of lipophilic compound permeation enhancement. Permeation enhancement of three bilayer splitting compounds in propylene glycol, caprylic acid, lauric acid, and neodecanoic acid is reported in Aungst et al., Pharm. Res., 7: 712-718 (1990). They tested the permeability of four lipophilic compounds: benzoic acid (122 Da), testosterone (288 Da), naloxone (328 Da) and indomethacin (359 Da) that pass through human skin. The permeation facilitation of each promoter for each drug was calculated according to E _{c / pg} = P _{e / pg} / P _pg , where _{Pe / pg} is the drug permeability according to the promoter / propylene glycol formulation and P _pg is Permeability by propylene glycol alone.

  The main mechanism by which unsaturated fatty acids such as linoleic acid are believed to promote skin permeability is by disrupting the order of intercellular lipid domains. For example, detailed structural studies of unsaturated fatty acids such as oleic acid have been performed using differential scanning analysis (Barry J., Controlled Release (Volume 6) 85-97 (1987)) and Using infrared spectroscopy (Ongpipattanankul et al., Pharm. Res., 8: 350-354 (1991), Mark et al., J. Control. Rd., 12: 67-75 (1990). It has been discovered that oleic acid can disrupt the order of the highly ordered SC lipid bilayer and form a separate oily phase within the intercellular region. SC lipid bilayers in which fatty acids or other bilayers are disordered by disruptors may be essentially similar to liquid phase lipid bilayers.

  The separated oil phase should have properties similar to the bulk oil phase. Much is known about the transport of fluid bilayers and bulk oil phases. Specifically, liquid phase diffusion coefficients, such as dimyristoylphosphatidylcholine (DMPC) bilayers, Clegg and Vaz, “Progress in Protein-Lipid Interactions” (Progress in Protein-Lipid Interactions), Watts (Elsevier, New York 1985) 173-229, Tocanne et al., “FEB” 257, 10-16 (1989), and Perry et al., “Perry's Chemical Engineering Handbook” (McGraw-Hill, Perry's Chemical Engineering Handbook) NY (1984)), these diffusion coefficients exceed that of SC and, more importantly, exhibit a particle size dependence that is significantly lower than that of SC transport. “Prodrugs: Topical and Ocular Delivery” by Kasting et al., Sloan edited by Marcel Dekker, New York, 1992, pages 117-161, “Pharm. Res.” By Ports and Guy, 9,663 -339 (1992), Willschut et al., "Chemosphere", 30, 1275-1296 (1995). As a result, the diffusion coefficient of a given solute is greater in a fluid bilayer such as DMPC or in a bulk oil phase than in SC. Due to the strong particle size dependence of SC transport, diffusion in SC lipids is much slower with larger compounds, while transport in fluid DMPC bilayers and bulk oil phases is moderate even with larger compounds. Only late. The difference between the diffusion coefficient in the SC and the diffusion coefficient in the fluid DMPC bilayer or bulk oil phase is larger as the solute is larger, and the difference is smaller as the compound is smaller. Thus, the ability of the bilayer to disrupt the order of a compound that can convert an SC lipid bilayer into a fluid bilayer phase, or a compound to which a separate bulk oil phase can be added, has low permeability enhancement for small compounds, large compounds Shows the particle size dependency with high permeability enhancement.

  A comprehensive list of lipid bilayer splitting agents is described in European Patent Application No. 43,738 (1982), which is hereby incorporated by reference. An exemplary compound is shown by the following formula.

R—X, wherein R is a straight chain alkyl of about 7 to 16 carbon atoms, a non-terminal alkenyl of about 7 to 22 carbon atoms or / and a branched alkyl of about 13 to 22 carbon atoms. And X is —OH, —COOCH ₃ , —COOC ₂ H ₅ , —OCOCH ₃ , —SOCH ₃ , —P (CH ₃ ) ₂ O, COOC ₂ H ₄ OC ₄ H ₄ OH, —COOCH (CHOH) ₄ CH ₃ OH, —COOCH ₂ CHOHCH ₃ , COOCH ₂ CH (OR ″) CH ₂ OR ″, — (OCH ₂ CH ₂ ) _m OH, —COOR ′ or —CONR ′ ₂ (where R ′ is H, − CR ₃ , —C ₂ H ₅ , —C ₂ H ₇ or —C ₂ H ₄ OH, R ″ is —H, or a non-terminal alkenyl consisting of about 7-22 carbon atoms, and m is 2 ~ 6, R "is alkenyl and X is -O Or if COOH, at least one double bond cis form.)]
5.2.2.3.1.2 Solubilizer Another way to increase transdermal delivery of drugs is to use chemical solubilizers that increase drug solubility in the vehicle. This can be achieved either by changing the drug-vehicle interaction by introducing different excipients or by changing the drug crystallinity (Flynn and Weiner, 1993).

  Solubilizers include, for example, water-diols such as propylene glycol and glycerol, mono-alcohols such as ethanol, propanol and higher alcohols, DMSO, dimethylformamide, N, N-dimethylacetamide, 2-pyrrolidone N- (2-hydroxyethyl) pyrrolidone, N-methylpyrrolidone, 1-dodecylazacycloheptan-2-one and other n-substituted-alkyl-azacycloalkyl-2-ones.

5.2.2.3.1.3. Combination of accelerators (binary system)
US Pat. No. 4,537,776 to Cooper outlines the use of a specific binary system for permeation enhancement. Similarly, European Patent Application No. 43,738 was selected as a solvent with a broad category of compounds that disrupt the order of the cell-envelope for the delivery of pharmacologically active compounds with lipophilicity. The use of diols is described. A binary system for enhancing methaclopramide permeation is disclosed in British Patent Application GB 2,153,223A, which is a C8-32 aliphatic monocarboxylic acid (unsaturated and / or branched if C is 18-32). Monohydric alcohol esters or C6-24 aliphatic monoalcohols (unsaturated and / or branched when C is 14-24) and N-cyclic compounds such as, for example, 2-pyrrolidone or N-methylpyrrolidone Consists of.

For example, to facilitate transdermal delivery of steroids such as progestogens and estrogens, a combination of an accelerator consisting of diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate and methyl laurate is disclosed in US Pat. No. 4,973. 468. A dual accelerator consisting of glycerol monolauric acid and ethanol for transdermal drug delivery is described in US Pat. No. 4,820,720. Many accelerators are listed, fatty alcohol ethers of these accelerators fatty acid esters, or alkane diols C ₂ -C ₄ for transdermal drug administration in U.S. Patent No. 5,006,342 In this case, the number of carbon atoms of each ester / ether fatty acid / alcohol is about 8-22. U.S. Pat. No. 4,863,970 discloses a permeation enhancing composition for topical use, which composition comprises a specific amount of one or more extracellular membrane disordered compounds, such as oleic acid, oleyl Contains an active diluent contained in permeation enhancing excipients including glycerol esters of alcohol and oleic acid, inert diluents such as C ₂ or C ₃ alkanols and water.

  Although not necessarily involved in the binary system, other chemical accelerators are dimethyl sulfoxide (DMSO) or an aqueous solution of DMSO (eg, US Pat. No. 3,551,554 to Herschler, US to Herschler) Patent No. 3,711,602 and U.S. Pat. No. 3,711,606 to Herschler) and azones (n-substituted alkylazacycloalkyl-2-ones) (eg, granted to Cooper U.S. Pat. No. 4,557,943). PCT / US96 / 12244 by Massachusetts Institute of Technology shows the results of passive experiments using polyethylene glycol dilaurate 200 (PEG), isopropyl myristate (IM), and glycerol trioleate (GT) Corticosterone flux enhancement values for passive flux by itself are 2, 5 and 0.8. However, 50% ethanol and LA / ethanol significantly increase the passive flux of corticosterone by a factor of 46 and 900.

  Some chemical accelerators can have side effects such as toxicity and dermatitis. US Pat. No. 4,855,298 discloses a composition for reducing dermatitis caused by a chemical promoter-containing composition having dermatitis properties with an amount of glycerin sufficient to provide an anti-inflammatory effect. Is disclosed. The present invention allows testing of the effect of a number of accelerators on tissue barrier transport of compounds, pharmaceuticals, or other components such as transdermal transport.

5.2.3.2. Mechanical accelerators for transdermal delivery For convenience, mechanical accelerators are defined to include all exogenous accelerators, such as ultrasound, mechanical or osmotic pressure, electric fields (electroporation or iontophoresis) Or a magnetic field etc. are included.

There have been many reports on the use of ultrasound (generally in the range of 20 kHz to 10 MHz) to facilitate transdermal delivery. Ultrasound is used alone and in combination with other chemical and / or mechanical promoters. For example, as reported in Massachusetts Institute of Technology PCT / US96 / 12244, by using therapeutic ultrasound (1 MHz, 1.4 W / cm ² ) and a chemical accelerator together, PBS, PEG, IM, And corticosterone flux by GT produces a flux that is 1.3 to 5.0 times greater than passive flux by the same promoter. By combining ultrasound and 50% ethanol, corticosterone transport is increased by a factor of 2 compared to the passive case, but transport by LA / ethanol is increased by a factor of 14 with a flux of 0.16 mg / cm ² / hr, PBS A flux that is 13,000 times greater than alone is produced.

  Pressure gradients can also be used to facilitate fluid transport across the skin. The pressure can be applied by a vacuum or positive pressure device. On the other hand, osmotic pressure may be used to cause transdermal transport.

Similarly, electrical current has been applied to facilitate transdermal drug transport and blood analyte extraction. Such currents facilitate transport by different mechanisms. For example, the use of an electric field provides a driving force for the transport of charged molecules through the skin, and, secondly, the use of an electric field refers to the convection of ion movement across the skin, referred to as electroosmosis. Can be induced. This mechanism is thought to play a major role in the transdermal transport of neutral molecules during iontophoresis. Iontophoresis requires the use of current, preferably DC or AC, and current densities greater than zero and up to about 1 mA / cm ² . In order to achieve transdermal extraction of glucose, enhancement of skin permeability using electric current is described in Tamada et al., Proceed Intern. Symp. Control. Rel. Bioact. Mater., Vol. 22, pp. 129-130 (1995). It has been reported.

  To transport the magnetically active species through the skin, a magnetic field can be used on the skin, either pre-treating the skin or in combination with other permeation enhancers. For example, polymer fine particles loaded with magnetic particles can be transported through the skin.

5.2.4 Adhesive Devices for delivery of active ingredients or drugs across the tissue barrier, particularly transdermal delivery devices such as transdermal patches, generally include an adhesive. The adhesive often forms a matrix in which the active ingredient or drug dissolves or disperses, and of course the device remains in intimate contact with tissue such as the skin. The compatibility of the active ingredient or drug with the adhesive is affected by their solubility in the adhesive. The supersaturation conditions that occur during storage or use are all usually very stable against precipitation of the active ingredient or drug within the adhesive matrix. In order to increase the motive force for penetrating the tissue and improve the stability of the device, it is desirable that the adhesive has a high solubility.

  Several classes of adhesives are used, each of which has many possible adhesive forms. These classes include polyisobutylene, silicone and acrylic adhesives. Acrylic adhesives are often used in the form of derivatives. Thus, selecting an adhesive that appears to be optimal for use with a particular drug and promoter is often a challenge. In general, all components present in the device are dissolved in a solvent and cast or coated onto a plastic substrate. When the solvent evaporates, a drug-containing adhesive film is produced. The present invention allows rapid and efficient testing of the effects of various types and amounts of adhesives in a sample composition or formulation.

5.2.5 Solvent The solvent for the active ingredient, carrier or adhesive is selected based on the biocompatibility as well as the solubility of the material being dissolved and the location where the appropriate interaction with the active ingredient or drug is transported. The For example, relaxation due to an active ingredient or drug dissolved in a solvent and adverse effects of the solvent on the active ingredient or drug being transported are factors to consider in selecting a solvent. A water soluble solvent can be used to produce a matrix formed from a water soluble polymer. Organic solvents are usually used to dissolve hydrophobic and several hydrophilic polymers. Preferred organic solvents are those that are volatile or have a relatively low boiling point, or that can be removed under vacuum, and those that are tolerated in humans such as methylene chloride. Other solvents may also be used, such as ethyl acetate, ethanol, methanol, dimethylformamide (DMF), acetone, acetonitrile, tetrahydrofuran (THF), acetic acid, dimethyl sulfoxide (DMSO), chloroform, and combinations thereof. . Preferred solvents are those published in "Federal Register" Vol. 62, No. 85, pages 24301-24309 (May 1997) and are rated as Class 3 residual solvents by the Food and Drug Administration. The solvent for the drug is usually distilled water, buffered saline, lactated Ringer's solution or other pharmaceutically acceptable carrier.

5.3 Sample Preparation and Screening Methods The high-throughput screening methods of the present invention include, for example, 1) one or more active ingredients and one or more active ingredients to achieve the desired characteristics of such a composition or formulation. Optimal composition or formulation comprising an inert ingredient, 2) optimal adhesive / accelerator / excipient composition for compatibility with active ingredient or drug, and 2) maximum drug flux through the stratum corneum Optimal active ingredient or drug / adhesive / promoter / additive composition, and 3) optimal active ingredient or drug / adhesive / promoter / additive composition to minimize cytotoxicity, etc. Confirm.

  The basic requirements for sample preparation, processing and screening are a dispensing mechanism and a test or screening mechanism. The dispensing mechanism adds components to sites that are separated on the array plate, eg, sample wells. Preferably, the dispensing mechanism is automated, controlled by computer software, and may change at least one additional variable condition such as, for example, identity of component (s) and / or component concentration. More preferably, two or more variable conditions can be changed. For example, filling or adding samples of pharmaceutical ingredients and excipients (such as accelerators and adhesives) to sample wells includes material manipulation techniques and robotics well known to those skilled in the pharmaceutical process manufacturing art. Of course, individual components can be manually placed into the appropriate wells of the array as needed. This pick and place technique is also well known to those skilled in the art. The test mechanism is preferably used to test one or more properties of each sample, such as drug concentration over time. The test mechanism is preferably automated and driven by a computer.

  In one embodiment, the system further includes a processing mechanism for processing the sample after component addition. For example, after assembling the device after adding components to the sample well, the sample is agitated and milled using methods and devices well known to those skilled in the art before placing the tissue specimen in a specific location on the sample well. Can be processed by filtration, centrifugation, emulsification or solvent removal (such as lyophilization) and reconstitution. The sample is preferably processed automatically and simultaneously.

  As noted above, preferred uses of the tissue barrier transport device of FIG. 1 are based on the presence, absence or concentration of a component (eg, a pharmaceutical agent) that diffuses directly or indirectly through tissue 120 into the reservoir 132 of the array. With decision. Such measurements may be made by various means well known to those skilled in the art. For example, it is known that spectroscopic techniques can be used to determine the presence, absence or concentration of common components. Suitable measurement methods include, but are not limited to, spectroscopy, infrared spectroscopy, near infrared spectroscopy, Raman spectroscopy, NMR, X-ray diffraction, neutron diffraction, powder X-ray diffraction, radioactive labeling and radioactivity. included.

  In one exemplary embodiment, without limitation, passive permeability of an active ingredient (eg, drug) that passes through human skin can be measured using a trace amount of radiolabeled active ingredient or drug. In accordance with known methods, the radiolabeled compound or drug is subjected to a rotary evaporator to remove all of the solvent that transports the compound or drug and, conversely, all of the tritium transferred into it. The radiolabeled compound or drug is then re-dissolved in various composition formulations containing accelerators, carriers, additives, adhesives and / or other additives as described below to a general concentration of 1 μChi / ml, For example, to a sample well, such as sample well 116 of array 112 of FIG. A passive transmission experiment is then performed. For example, in the storage section such as the storage section 132 of FIG. 1, phosphate buffered saline (PBS, phosphate concentration = 0.01M, NaCl concentration = 0.137M) (Sigma Chemical Co.), etc. It is preferable to add. Other receiving solutions may be used, as will be known to those skilled in the art. The concentration of the radiolabeled component or drug in the sample and reservoir compartment is measured using a scintillation counter (eg, model 2000 CA, Packaxd Instruments). Duplicate formulations may be used for several samples and / or repeated experiments may be performed to optimize measurement reliability.

The transmission value can be calculated from the relation P = (dN _r / dt) / (AC _d ) under steady conditions. Where A is the specific surface area of the tissue accessible to the sample, C _d is the concentration of the component or drug in the sample, and N _r is the amount of component or drug that has permeated into the receptor reservoir. Williams et al., “Int. J. Pharm.”, Vol. 86, pages 69-77 (1992), reported a 40% change in human skin permeability between subjects. Passive permeability enhancement, E _p, is calculated from the relationship with PBS passive permeability according to Equation 1 below.

In the formula, P (accelerator) is drug permeability by a predetermined accelerator, and P (PBS) is drug permeability by PBS. The flux J ^sat from the saturated solution is calculated from J ^sat = PC ^sat , where C ^sat is the drug solubility of the prescription drug. Flux acceleration, E _j, is calculated using Equation 2 below.

^Where J ^sat (accelerator) and J ^sat (PBS) are the flux of the drug from the accelerator and a saturated solution of PBS, respectively.

5.4 Correction or Repair of Microscopic Defects in Skin Tissue Sample The present invention includes a method for repairing and / or correcting microscopic defects in a tissue specimen such as skin. For example, devices or diffusion cells used for studies of transdermal delivery of active ingredients (eg, pharmaceuticals or drugs) require skin samples that are free of defects that can serve as a diffusion fast transport route. There are several types of such defects, ranging from a few millimeters to tens of microns. Physical tears and hair follicles are only two types of defects that can be compromised in the interpretation of transport or diffusion data. Inhomogeneous tissue segments, ie segments containing an abnormal amount of defects, lead to inaccurate and incorrect diffusion measurements, particularly when using relatively small tissue samples in the present invention. Rapid identification of the location of defects on a given tissue sample surface can be achieved by image analysis, and it is preferred to use video microscopy or photomicrography to rapidly microinspect each tissue segment.

  In accordance with a preferred embodiment of the present invention, diffusion data regarding heterogeneous tissue segments may be discarded to avoid inaccurate measurements. On the other hand, if the effect of a defect in a tissue segment can be characterized and / or quantified, the associated diffusion measurement can mathematically adjust for that defect.

  In another embodiment of the present invention, a defect in a tissue specimen is repaired by supplying the defect location to an inkjet printer that is commanded to print wax to cover the defect location. . The pattern to be printed is slightly overlapped on the defect-free area so that the entire defect is covered. The wax print head prints molten wax that solidifies as soon as it strikes the tissue. This solid wax is water resistant and serves as a seal to ensure that the repaired area does not affect the diffusion flux during subsequent testing. The droplet arrangement preferably has sufficient overlap to form a seal.

5.5 Alternative Embodiment for Solid Source Sample (FIGS. 2A-D)
2A-D are schematics of an alternative high throughput apparatus 200 and method for measuring tissue barrier transport using a solid source sample. Device 200 is similar to device 100 of FIG. 1, but for testing solid source samples, such as, for example, a composition containing a semi-solid material such as an adhesive, a relatively flat transdermal patch or a film-like sample. It differs in that it is designed. The substrate plate 214 is a dense plate, such as a plastic or glass plate, that supports an array 212 of samples 216. Each sample contains a combination of one active ingredient (eg, a pharmaceutical product) and an ingredient that includes at least one inactive ingredient. Examples of suitable components are described above with respect to FIG.

  The first step of the method involves creating an array 212 of regions of different compositions (ie, sample 216) on a dense substrate 214. There are a variety of ways to make this array, but one simple method is to use a combinatorial distributor to make a solution containing all the components in a simple solvent. Suitable dispensing devices and methods for formulating solutions or compositions have been described previously and are also disclosed in US patent application Ser. No. 09 / 540,462, the entire contents of which are incorporated herein by reference.

  In a preferred embodiment, the formulated solution comprises a sample array 112 consisting of sample wells 116 (FIG. 1), a well of a microtiter plate similar to the substrate plate 114, and a separable, dense bottom rather than a base 118. Contained in plate 214. The solvent is then vaporized and each sample in the well is dried, resulting in a film at the bottom. This vaporization method mimics the manufacturing method used to manufacture various tissue transport devices such as transdermal patches. The top plate may then be removed to obtain the array shown in FIG. 2A. Sample 216 may take any form, but generally circular is preferred as shown in FIG. 2A.

  It should be noted that this type of plate can be used for the purpose of precipitating active ingredients such as pharmaceuticals or drugs, for example to evaluate the stability of a drug / adhesive / promoter solution composition or formulation. . Whether or not precipitation has occurred can be determined by optical inspection of each film. This is because when the sample is exposed to light, precipitates cause an increase in light scattering. Crystallization can also be detected visually by observing birefringence (of a birefringent compound). If the film is already sufficiently opaque to eliminate this scattering method in advance, alternative means may be used. One such method is the second harmonic generation method (SHG) that easily detects the presence of crystals in the film. It is also possible to use microfocus X-ray diffraction to detect the presence of crystals.

  As shown in FIG. 2B, the next step in the method of the present invention is to prepare a tissue specimen 220 for use in the study. A specimen 220, such as a stratum corneum specimen, may be prepared or obtained by conventional methods as described above. However, whatever plate type is used for the study, it is most convenient if the sample specimen 220 is large enough to cover it. For example, make it large enough to cover a 96-well microtiter plate. Accordingly, separate tissue samples are prepared for each research plate 214. The tissue is then placed on the plate 214 so as to cover each of the sample areas, as shown in FIG. 2B. Care is taken to ensure that there are no air pockets under the tissue 220. One method places tissue 220 progressively across the surface of the plate beginning at one end of plate 214. The air is pushed beyond the tissue / plate contact line.

  As shown in FIG. 2C, in one embodiment of the present invention, in order to ensure that no lateral diffusion occurs between adjacent samples, the region of tissue 220 on each sample region is now Segments 224 are created by physically cutting or separating from neighboring regions. As mentioned above, this can be done by any method, for example, mechanical nicking or cutting, laser cutting or crimping along cut 222.

  In turn, each tissue segment 224 on each plate 214 may be imaged and characterized by video microscopy. Automatic image recognition can be used to identify and record these damaged or inhomogeneous tissue segments. As previously mentioned, damaged or heterogeneous tissue segments 224 may be replaced, repaired or ignored. On the other hand, data relating to damaged or inhomogeneous segments 224 may be adjusted for defects. Optionally, tissue 220 may be imaged, replaced or repaired prior to segmentation. In yet another alternative, the tissue segment 224 is placed on the sample 216 after the tissue 220 has been cut and / or imaged.

  In FIG. 2D, the next step in the method of the present invention is to place a storage plate 230 or bottom-opening titer plate similar to the storage plate 130 of FIG. 1 over the tissue segment 224 as shown. . The storage plate 230 includes a plurality of hollow storage parts 232. Each reservoir 232 lines up on the sample and tissue such that the tissue segment 224 separates each sample from the reservoir 232 when the plate 230 is secured in place. The storage plate 230 is secured to the substrate plate 214 using clamps, screws, fasteners, or other suitable attachment means. Plates 230 and 214 are preferably crimped with sufficient pressure to create a liquid seal around reservoir 232. Each reservoir is filled with a storage medium, preferably a liquid or solution such as saline, to contain sample compounds that diffuse across the tissue segment 224 into the reservoir 232. In one embodiment, the storage medium is a PBS solution containing about 2% BSA.

  Incubation of the apparatus 200 with automatic periodic sampling and preparation of the solution in the reservoir 232 is used to perform a permeability assessment of the active ingredients for all samples of the combinatorial study.

  While the embodiments of the present invention described herein focus on the transport of compounds across tissue, the systems and methods of the present invention study the transport of compounds across membranes or all other barriers. Is also appropriate.

5.6 Alternative Embodiment Using Indirect Measurement (Figure 3)
In FIG. 3, in an alternative embodiment of the present invention, the apparatus 300 relates to an apparatus and method for high-throughput screening of active ingredient fluxes through tissue specimens such as stratum corneum, such fluxes at least in part, It is observed that it is measured by the permeability of the active ingredient (pharmaceutical or drug) in the tissue in the presence of a promoter. This permeability is generally governed by at least two factors, the solubility of the active ingredient within the tissue (eg, stratum corneum) and the diffusivity of the active ingredient within the tissue specimen. These two factors, solubility and diffusivity, are individually measured by a method that indirectly assesses the flux through the tissue specimen.

  In FIG. 3, an array 312 is constructed consisting of wells 316 containing samples (eg, solution 338) of different compositions consisting of active and inactive ingredients (eg, pharmaceutical / adhesive / accelerator / additive). A predetermined amount of tissue segment 340, such as stratum corneum, is added to each well. On the other hand, a tissue segment is placed over each well 316 (similar to the arrangement shown in FIGS. 1, 2C and 2D) such that each segment is in contact with the sample solution 338. Extract the tissue 340 at different times from a well 316 similarly prepared to measure the rate (rate) at which a component (eg, drug or pharmaceutical) is taken into a tissue sample and measure the presence, absence, or concentration of that component You may measure by doing. By taking a sufficiently long time so that the amount of dissolution does not change over time, the concentration, ie, the component equilibrium concentration in the tissue 340 can be evaluated by measuring the concentration. The product of ratio and solubility is proportional to the permeability of the components.

5.7 Alternative device for tissue barrier transport (FIGS. 4A-C)
As shown in FIG. 4A, an alternative embodiment of the apparatus of FIG. The diffusion cell 400 includes a sink plate 410, a source plate 430, and a tissue specimen 420 disposed between the sink plate 410 and the source plate 430. The sink plate 410 includes a sink well 412 for holding a storage medium, as described above with respect to FIG. Although the sink well 410 is illustrated as a cylindrical shape with an open end, the sink well may have a rectangular, hexagonal, spherical, elliptical, or any other shape. The sink plate 410 includes at least one access port 416 in fluid communication with the sink well 412 along the end of the sink well 412. The sink plate 410 also preferably includes a surface feature 414 configured to mate with the source plate 430 and forms a seal with the tissue specimen 420.

  In one preferred embodiment, the tissue specimen 420 is skin tissue, but may be any tissue or membrane as described above with respect to the tissue specimen 120 of FIG. The tissue specimen 420 is cut, formed or cut to specific dimensions to cover the sink well 412 and the surface features 414. The tissue specimen 420 is preferably placed so as not to completely cover the access port 416.

  As shown in FIG. 4C, the source plate 430 includes a source reservoir or well 432 that includes an open end and aligns with the sink well 412 when the source plate 430 is placed on the tissue specimen 420. . The path 436 passes through the source plate 430 and does not communicate with the source well 432 but is substantially adjacent. Path 436 is configured to align with access port 416 to access the storage medium in sink well 412 without removing source plate 430.

  As shown again in FIG. 4A, the source plate 430 is also preferably configured to mate with the surface feature 414 of the sink plate 410 and cut to a specific size and includes a surface feature 434, and the sink well. A seal is formed with tissue specimen 420 around 412 and source well 432. For example, in one embodiment, surface feature 414 is a convex ring extending from surface sink plate 420 around the open boundary of sink well 412 and surface feature 434 is configured to mate with surface feature 414, A concave ring formed in the source plate 430.

  In another embodiment of the present invention, several diffusion cells 400 are assembled or formed together to create an array of diffusion cells similar to the array 112 of FIG.

  The exemplary use of the apparatus of FIGS. 4A-C is identical to the exemplary use described above with respect to FIG. 1, but without access to source plate 430 or tissue 420, by access port 416 and path 436. The storage medium can be added or removed from the sink well 412. The storage medium used for diffusion cell 400 is preferably a liquid or a solution. In an alternative use of the diffusion cell 400, the storage medium and sample arrangement may be the reverse of FIG. For example, a storage medium may be placed over the tissue specimen 420 in the source well 432 and the sample held in the sink well 412. In such embodiments, sample can be added or removed through path 436 and access port 416.

5.8 Method of filling or adding samples (Figures 5A and B)
5A and B are schematic diagrams of an apparatus 500 for use when adding or filling a sample 530 into a sample well 522 in a sample array, such as the sample array 112 shown in FIG. In that case, the generation of air pockets or bubbles between the sample 530 and the tissue 524 is avoided. In the sample array, tissue 524 includes, for example, sample wells 522 located in a substrate plate, such as substrate plate 114 shown in FIG. 1, and a reservoir located in a storage plate, eg, storage plate 130 shown in FIG. 526. In the filling method of the present invention, a supply cannula 510 comprising a sample source 514 and an exhaust space 512 punctures a basement membrane 520 that covers one side of a sample well 522 for filling with a sample 530.

  Sample source 514 is then extended into sample well 522 until it contacts tissue 524. Sample 530 is then supplied to sample source 512 and when sample 530 begins to fill sample well 522, air is forced out of sample well 522 through exhaust space 512 in supply cannula 510. The When the desired amount of sample 530 is loaded into sample well 522, sample source 512 and supply cannula 510 are completely withdrawn from basement membrane 520 and sample well 522.

  In a preferred embodiment of the filling method of the present invention, the sample source 514 is sampled at a rate that is synchronized with the fill rate at which the sample 530 enters the sample well 522 whenever the sample 530 is sent to the sample well 522. Retracted into the well 522, the outlet of the sample source 514 is inside the sample 530 protruding into the sample well 522. When the desired amount of sample 530 is loaded into sample well 522, both sample source 512 and supply cannula 510 are completely withdrawn from basement membrane 520 and sample well 522.

  In a preferred embodiment, the basement membrane 520 is a rubber membrane.

  The filling method of the present invention can be carried out manually or using automatic dispensing means. The sample wells in the sample array are then automatically distributed, allowing the same or different samples to be distributed to multiple sample wells in one or more sample arrays in a fast, accurate and controlled manner Filled using the device.

  The sample 530 dispensed according to the filling method of the present invention is preferably a liquid source sample.

5.9 Alternative Embodiment of Solid Source Sample (FIGS. 6-9)
FIG. 6 is an exploded schematic view of a preferred embodiment of a high throughput apparatus 600 for measuring tissue barrier transport in an array 630 of solid source samples in accordance with the present invention. The apparatus 600 includes a base plate 610 that supports a spacer plate 620, an array of solid source samples 630, a tissue specimen 640, a storage plate 650 that includes an array of donor reservoirs 654, and a stepped screw 660 having a strip 662, for example. Tightening means are provided.

  Base plate 610 is preferably made of aluminum, and spacer plate 620 and storage plate 650 are preferably made of transparent plastic or polycarbonate.

  The base plate 610 has a threaded hole 612 that is drilled to mate with the strip 662 of the stepped screw 660 so that when the screw 660 passes through the device and is threaded into the screw hole 612 and tightened, the device It is tightened. When the device is clamped, the storage plate 650 and the tissue specimen 640 are sealed. The number of screw holes 612 located around the edge of the base plate 610 can be any number, but the number of screw holes 612 is preferably at least 4, more preferably between 4 and 8. In a preferred embodiment, the base plate 610 further includes an array of guide marks 614 that form any array, for example, 2 × 2, 4 × 4, 6 × 6, and 8 × 12. It may also be used to assist in aligning the various components of the device 600 during assembly.

  The threaded holes 622 and threaded holes 652 in the spacer plate 620 and storage plate 650 are each drilled and the steps and strips 622 of the stepped screw 660 can pass smoothly (FIGS. 7 and 8 respectively). Stepped screws 760 and 860), the head of the stepped screw 660 cannot pass. There may be any number of screw holes 622 and screw holes 652 around the ends of the spacer plate 620 and the storage plate 650, but the number of screw holes 622 and screw holes 652 is preferably at least four, More preferably, the number is 4 to 8. In a preferred embodiment, there is at least one screw hole at each corner of both the spacer plate 620 and the storage plate 650.

  In an alternative embodiment, the apparatus 600 further comprises an upper plate positioned over the storage plate 650, which is manufactured from the same material as the base plate 610 (eg, aluminum) and the screw holes 652 in the storage plate 650. Either an open frame that includes threaded holes that fit with or a solid plate that includes an array of threaded holes and reservoirs identical to the donor reservoirs 654 on the threaded holes 652 and the reservoir plate 650.

  The apparatus 600 is assembled by first placing the spacer plate 620 on the top surface of the base plate 610 and aligning the screw holes 622 in the spacer plate 620 with the screw holes 612 in the base plate 610. An array of solid source samples 630 is made on the spacer plate 620 in a pattern corresponding to the pattern of the donor reservoir 654 in the storage plate 650, and the guide marks 614 on the base plate 610 indicate that each sample 630 is an upper plate. Used to ensure alignment with donor reservoir 654 in 650. The size of sample 630 corresponds to the size of donor reservoir 654.

  Each sample 630 includes a combination of one active ingredient (eg, pharmaceutical) and an ingredient that includes at least one inactive ingredient. Examples of suitable components are described above with respect to FIG.

  The tissue specimen 640 is placed on the sample array 630 in a manner that avoids the formation of air pockets between the tissue specimen 640 and the sample 630. A storage plate 650 comprising an array of donor reservoirs 654 is then placed on the skin such that the screw holes 652 on the top plate 650 align with the corresponding screw holes 622 in the spacer plate 620.

  The resulting assembly device 600 is then clamped, which includes sliding a stepped screw 660 having strips 622 through the aligned screw holes 652 of the assembled device 600 and storing plate 650 and tissue specimen. Each stepped screw 660 is tightened so that the space between 640 is closely attached. A stepped screw 660 is preferably used at least in each of the four corners of the assembled device 600.

  The storage medium is added to the donor reservoir 654 of the assembled device 600 and samples are taken from the donor reservoir 654 at timely or various time intervals and in the sample 630 passing through the tissue specimen 640, eg, activity Used to measure transport or flux of components such as components and common components. If multiple samples are taken, remove the contents of the specimen from the donor reservoir 654 and then add an equal amount of storage medium to the same donor reservoir 654.

  The size of donor reservoir 654 is about 1 mm to about 50 mm, more preferably about 2 mm to about 10 mm, and most preferably about 3 mm to about 7 mm.

  FIG. 7 is a compression schematic of a high throughput apparatus 700 for measuring tissue barrier transport of an array of solid source samples in accordance with the present invention. Device 700 is similar to device 600, except that the array of donor reservoirs 754 is a total of 96 arrays of 8 × 12. In that case, each reservoir does not exceed 6 mm in diameter. The apparatus 700 includes a base plate 710 that supports a spacer plate 720, an array of solid source samples (eg, sample 630 shown in FIG. 6), a tissue specimen (eg, tissue specimen 640 shown in FIG. 6), a donor reservoir 754. A storage plate 750 comprising an array of and a clamping means such as a stepped screw 760, for example.

  FIG. 8 is a compression schematic of a high throughput apparatus 800 for measuring tissue barrier transport in an array of solid source samples in accordance with the present invention. Apparatus 800 is similar to apparatus 600 and apparatus 700, except that the array of donor reservoirs 854 is an array of 16 × 24 total 384 donor reservoirs 854. In that case, each reservoir does not exceed 3 mm in diameter. The apparatus 800 includes a base plate 810 that supports a spacer plate 820, an array of solid source samples (eg, sample 630 shown in FIG. 6), a tissue specimen (eg, tissue specimen 640 shown in FIG. 6), a donor reservoir 854. A storage plate 850 comprising an array of: a clamping means such as a stepped screw 860.

  7 and 8 are the same as those described in FIG. 6, and other variations and exemplary uses of the devices of FIGS. 6-8 are applicable if applicable. Identical to that previously described with respect to FIG.

  FIG. 9 is an exploded view of another transdermal assay device according to another embodiment of the present invention. The transdermal assay device 900 is similar to the devices 600, 700, and 800 described above because the device 900 is also used to measure sample transport through a tissue barrier or specimen. The sample is preferably a solid source sample, but may be other suitable samples.

  The transdermal assay device 900 includes first and second members 902 and 904, respectively. The first and second members are preferably made of stainless steel and are suitably coated to obtain the desired chemical resistance. Also, these members may be formed from a transparent plastic such as polycarbonate, other plastics or glass. The first member 902 includes opposing surfaces 905 and 902. These opposing surfaces are preferably substantially parallel to each other and are substantially flat surfaces. One surface 905 includes one or more sample surfaces 906 configured thereon to receive one or more samples 908. As described above, the sample surface 906 is preferably circular and arranged in an array.

  The second member 904 also includes opposing surfaces 910 and 914 that are substantially parallel to each other and are preferably substantially flat surfaces. The second member 904 defines one or more storage portions 912 therein. The size of each reservoir 912 is similar to that described above in connection with FIGS. Each storage unit includes an opening 934 in the surface 910 of the second member 904. In the preferred embodiment, each opening 934 is substantially the same size as each sample surface 906.

  In use, each sample surface 906 is configured to substantially align with a corresponding opening 934 in a direction substantially perpendicular to the sample surface 906. Thus, in use, each sample in the plurality of sample arrays is configured to substantially align with an opening 934 corresponding to the array of openings. In one embodiment, the reservoir 912 extends through the second member from the surface 910 to the opposing surface 914 in a manner similar to that described above.

  The transdermal assay device 900 preferably includes a third member 916. Preferably, the third member 916 is made of aluminum, but may be made of many other materials such as, for example, steel, brass, plastic, ceramic, etc., in a portion that provides the desired shape and dimensional stability. It may be manufactured. The third member 916 also includes opposing surfaces 918 and 920 that are substantially parallel to each other and are preferably substantially flat surfaces. Third member 916 preferably includes a cavity 924 for receiving magnet 926 therein. In another embodiment, the magnet 926 is configured to be non-removable within the third member 916, coupled to the third member, or the third member 916 itself is a magnet 926, i.e. a third The member is made of a magnetic material. The magnet 926 may be a permanent magnet, an electromagnet, or the like.

  Magnet 926 forms part of a magnetic clamp that is used to clamp first member 902, second member 904, and third member 916 together. The other part of the magnetic clamp preferably comprises one or more inserts 928 secured within the second member 904. In a preferred embodiment, these inserts 928 are made of a ferrous material that is attracted to the magnet 926. In another embodiment, the insert 928 is a magnet insert that is attracted to the magnet 926. That is, the magnet insert and the magnet 926 are arranged so as to have a counter electrode with the other facing the magnet insert. These magnet inserts are preferably permanent magnets, but may instead be electromagnets. In a preferred embodiment, the first and third members are made of iron or a magnetic material (eg, stainless steel). Thereafter, these magnetic members are held by the magnets in the third member to ensure a desired tightening force.

  The third member preferably includes at least one alignment pin 30 configured to engage with the complementary alignment hole 932 in the first member 902, but preferably includes two or more. The alignment pin is preferably made of a ferrous material to promote attraction between the magnet 926 and the insert 928 and to hold the magnet 926 in place in the cavity 924.

  It should be appreciated that in other embodiments, the magnet clamp may be performed in a variety of other types. For example, the magnet clamp may comprise one or more magnets and / or iron inserts disposed in, on or near the first member 902, the second member 904, and / or the third member 916. In addition, these members can be wholly or partly made of iron and / or magnetic material. Furthermore, the magnetic force of the magnet 926 or the gap between the magnet and the insert or magnetic material is preferably selected to ensure the desired clamping force between the members. In other words, replacing magnet 926 with a stronger or weaker magnet can change the resulting clamping force and optimize the seal between members for device 900.

  In a preferred method of use, the magnet 926 is coupled to the third member 916. A sample 908 is disposed on each sample surface 906 of the first member 902 so that each sample surface 906 is aligned with a corresponding opening 934. The size of the sample 908 corresponds to the size of each opening 934. Each sample 908 includes a combination of ingredients including an active ingredient (eg, pharmaceutical) and at least one inactive ingredient. Examples of suitable components are described above with respect to FIG.

  The second member 902 is then placed on the third member 916 such that the alignment pin 930 engages the alignment hole 932. Thereby, the second member 902 is aligned at a predetermined position on the third member 916. The tissue specimen 936 is placed on the sample 908 in a manner that avoids the formation of air pockets between the tissue specimen 936 and the sample 908.

  The second member 904 is then placed over the tissue specimen 936, thereby exposing the tissue specimen 936 to each reservoir 934. The magnetic force between the magnet 926 and the insert 928 preferably occurs along the alignment pin 930, but aligns the second member 904 and the first member 902 so that each sample 908 is under the corresponding opening 934. Align. This magnetic force between the magnet 926 and the insert 928 also creates a clamping force that clamps the first, second and third members together, thereby creating a tissue specimen between each sample 908 and each opening 934. tighten.

  A liquid medium is added to the reservoir 912 of the assembled device 900, and a sample of the liquid medium is taken from the reservoir 912 at timely or at various time intervals and in a sample 908 passing through the tissue specimen 936, for example, Used to measure the transport or flux of ingredients such as active ingredients and common ingredients. When a plurality of specimens are collected, an equal amount of liquid medium is added to the same reservoir 912 after a sample amount is taken from the reservoir 912.

  FIG. 10 is an exploded view of a transdermal assay device 1000 according to still another embodiment of the present invention. The transdermal assay device 1000 is similar to the transdermal assay device 900 (FIG. 9) described above. Unlike the transdermal assay device 900 (FIG. 9), the transdermal assay device 1000 includes a sample plate and an elastic magnet as described in detail below. Further, the sample is preferably a solid source sample, but may be other suitable samples.

  The transdermal assay device 1000 includes a first member 1010 and a second member 1002. These members are preferably made of suitably covered stainless steel to obtain the desired chemical resistance. These members may also be made of a transparent plastic such as polycarbonate, other plastics or glass. In addition, these members preferably have opposing flat surfaces and are substantially parallel to each other. The second member 1002 defines one or more reservoirs 1014 therein, each of which has an opening, as described above with respect to FIG.

  In use, an elastic magnet 1008 is disposed between the first and second plates. The sample array is preferably placed on the sample plate 1006, but is placed between the elastic magnet 1008 and the second plate. Similarly, the tissue specimen 1004 is disposed between the sample plate 1006 and the second plate 1002.

  Sample plate 1006 comprises one or more sample surfaces 1012 configured to receive one or more samples thereon. As described above, the sample surface 1012 is preferably circular and arranged in an array. In use, each sample surface 1012 is formed in a direction substantially perpendicular to the sample surface 1012 to align with an opening substantially corresponding to each reservoir 1014. Thus, in use, each sample of the sample array is configured to align with an opening substantially corresponding to the array opening. In one embodiment, the reservoir 1014 extends through the second member 1002, similar to the aspects described above.

  The elastic magnet 1008 forms part of a magnet clamp that is used to clamp the first member 1010 and the second part 1002 together. The second plate 1002 is preferably made of an iron material that is attracted to the elastic magnet 1008. In another embodiment, the iron insert is disposed within the second member 1002.

  The transdermal assay device 1000 is used as described above.

6). Examples The following examples further illustrate the methods and arrays of the present invention. Of course, the present invention is not limited by the specific details of the following examples.

Example 1 Nicotine Permeation through Human Dead Skin Human dead skin epidermis is isolated, which first separates the skin from the underlying fat, then uses standard techniques at 60 ° C. By performing a heat treatment for 90 seconds to separate the epidermis.

  NICODERMCQ® Nicotine Step 1 (21 mg / 24 hours) transdermal patch (GlaxoSmithKline, Research Triangle Park, North Carolina, USA) with a 5/16 inch diameter circular hole. Hold the support and release liner over the resulting perforated sample until the perforated sample is placed in the test apparatus.

  The device was assembled as described in FIG. In that case, each plate in the device is rectangular and measures 5.030 inches (127.76 mm) x 3.365 inches (85.48 mm). First, a transparent polycarbonate spacer plate 620 having a thickness of 1/8 inch (3.175 mm) is placed on the upper surface of the aluminum base plate 610, and then screw holes 622 in the spacer plate are formed in the base plate. The device was assembled by aligning with screw holes 612. A 4 × 4 sample array was then fabricated on the spacer plate 620 as described in Table 2 below.

Place the perforated samples one by one on the 4 × 4 array spacer plate 620 of Table 2 and ensure that each array sample is aligned with the donor reservoir 654 in the storage plate 650. In order to do so, the location of each array sample was selected using guide marks 614 on the base plate 610. The 96 guide marks 614 on the base plate 610 having a depth of 0.020 inches (0.508 mm) are 11.24 mm inward from the long side end of the base plate 610 and 14.38 mm from the short side end. Arranged in a 12 × 8 array located inside. Upon placement, the release liner on each sample was removed to expose the sample drug reservoir / adhesive.

  In array samples A4 and D1, the drug reservoir of the sample patch was peeled from the support along with the release liner. Array positions B3, C1 and D3 are no sample controls to measure the potential impact of lateral diffusion when measuring transdermal transport using this device.

  After all samples were placed in the array, a heat-peeled human dead skin piece larger in size than the sample array was gently and slowly placed on the sample to avoid air pockets between the skin and the sample. The skin was adjacent to the sample along with the stratum corneum. Next, a 1/4 inch (6.35 mm) thick clear polycarbonate storage plate 650 with a 4 × 4 array of 1/4 inch (6.35 mm) diameter donor reservoirs 654 was placed on all of the storage plates 650. The screw holes 652 were placed on the skin so that they aligned with the screw holes 622 of the corresponding spacer plate 620.

  The resulting assembly device 600 is clamped, which slides a stepped screw 660 with ridges 622 through the aligned screw holes 652 at each of the four corners of the assembled device, and the storage plate 650 and skin. Each stepped screw 660 was tightened so that the gaps were in close contact with each other. The screw holes 612 on the base plate 610 have 10-24 taps ranging from 0.250 inches (6.35 mm) to 0.188 (4.775 mm), and when the screws are tightened, the thread 660 strips. Catch 662 and tighten the device.

  75 ml Dulbecco's mild phosphate sanitary saline (PBS) was added as a storage medium to each donor reservoir in the array.

  Two hours later, 50 ml of the test sample of the storage medium was collected from each donor reservoir 654, and 50 ml of PBS was added to each donor reservoir 654 at that time. Place each of the 2-hour test specimens in an HPLC vial and add 450 ml of 50 mM potassium phosphate adjusted to pH 3.0 with phosphoric acid and acetonitrile = 50/50 (v / v) to 500 ml. Dilute to

  The above method was repeated after 3 hours, 4 hours and 5 hours. At the end of the experimental sampling phase, each donor reservoir 654 in the array resulted in four (4) 50 ml test specimens diluted as indicated above, but only for the B3 donor reservoir. Test specimens collected after 3 hours were diluted to 950 ml instead of 500 ml.

  The nicotine content in each test specimen was then determined by HPLC analysis.

  The components of the HPLC system used to analyze the test specimens are Waters 2790 Separations Module®, Waters Photodiode Array Detector Model 996® and Waters Millennium 32v3.2 Chromatography Software® (Waters) Milford, Massachusetts).

  The HPLC analysis was performed using a platinum EPSC18 column (registered trademark: Alltech Associates, Muskegan, Michigan) having a size of 250 mm × 4.6 mm and a particle size of 5 mm. The mobile phase was 50 mM potassium phosphate (pH adjusted to 3.0 with phosphoric acid) / acetonitrile = 50/50 (v / v), and the flow rate was 1.0 ml / min. Detection was performed by measuring ultraviolet absorbance at a wavelength of 260 nm. The run time was 4 minutes. The injection volume was 10 ml. The column temperature was ambient.

  The quantification of nicotine content in each test specimen was performed by comparison to a calibration curve obtained using a set of nicotine standards (Sigma). The amount of nicotine was found to be linear in the range of 1-100 mg / ml. Using this method, potential chromatographic interference with other components in the skin (eg fat, protein) was ignored by direct analysis.

  The results of HPLC analysis are shown in Tables 3 and 4 below.

The accumulation of nicotine in each donor reservoir was calculated according to the following formulas (3) to (6).

The results of nicotine accumulation calculations for each donor reservoir in the array are shown in Table 6A and Table 6B below.

As can be seen from the previous results, the active ingredient, nicotine, was detected in the donor reservoir and therefore nicotine passed through the skin barrier. These results also indicate that the detection or measurement method used was sufficiently sensitive to detect transported nicotine. Transdermal transport of the active ingredient, nicotine and many samples clearly showed similar transport rates.

  In addition, lateral diffusion of nicotine into adjacent “wells” was shown to be sufficiently slow compared to direct transdermal delivery, but very low amounts of nicotine were detected in donor reservoirs not located on the sample It was. Thus, this experiment clearly demonstrates the potential of this device for measuring the transport of components across tissue barriers.

The specific embodiments of the present invention detailed above have been given for purposes of illustration and description. They are clearly not intended to limit the invention to the precise form disclosed, but obviously can be improved and varied in light of the above techniques. The examples are chosen to illustrate the best principles of the principles of the invention and its substantive application, and thus allow those skilled in the art to make various modifications to suit the invention and the particular use discussed. Thus, various embodiments can be used to the maximum. Further, the procedures in the method need not necessarily be performed in the order described. The scope of the invention should be determined by the claims and their equivalents. Several references have been described, the entire contents of which are incorporated herein by reference.

Claims (41)

A first member having a sample surface configured to receive a sample on the surface;
A transdermal assay device comprising: a second member defining a reservoir having an opening on a surface; and a magnet clamp configured to clamp a tissue specimen between the sample surface and the opening.   The transdermal assay device according to claim 1, wherein the sample surface and the opening have substantially the same size.   The transdermal assay device according to claim 1, wherein the first member includes a plurality of sample surfaces, and the second member includes a plurality of storage portions each having an opening on the surface of the second member.   2. The transdermal assay device of claim 1, wherein in use, the sample surface and the opening are substantially aligned with each other along a line substantially perpendicular to the sample surface.   The transdermal assay device of claim 1, wherein the reservoir is configured to accommodate at least one liquid medium therein. The magnet clamp is
The transdermal assay device according to claim 1, comprising at least one magnet connected to the first member and at least one iron insert connected to the second member. The magnet clamp is
The transdermal assay device of claim 1, comprising at least one magnet coupled to the first member and at least one magnet insert coupled to the second member. The magnet clamp is
The transdermal assay device according to claim 1, comprising at least a part of the second member made of iron material and at least one magnet connected to the first member. The magnet clamp is
The transdermal assay device according to claim 1, comprising at least a part of the first member made of a magnetic material and at least a part of the second member made of an iron material. The magnet clamp is
The transdermal assay device according to claim 1, comprising at least a part of the first member made of a magnetic material and at least a part of the second member made of a magnetic material.   The transdermal assay device of claim 1, further comprising a third member configured to clamp the first member between the third member and the second member. The magnet clamp is
The transdermal assay device of claim 11, comprising at least one magnet coupled to the third member and at least one iron insert coupled to the second member.   13. The transdermal assay device of claim 12, wherein the third member includes a cavity therein that houses at least one magnet. The magnet clamp is
The transdermal assay device of claim 11, comprising at least one magnet coupled to the third member and at least one magnet insert coupled to the second member. The magnet clamp is
The transdermal assay device according to claim 11, comprising at least a part of the second member made of an iron material and at least one magnet connected to the third member. The magnet clamp is
The transdermal assay device according to claim 11, comprising at least a part of the third member made of a magnetic material and at least a part of the second member made of an iron material. The magnet clamp is
The transdermal assay device according to claim 11, comprising at least a part of the third member made of a magnetic material and at least a part of the second member made of a magnetic material. The first member includes at least one alignment hole;
The transdermal assay device of claim 11, wherein the third member includes at least one alignment pin configured to engage the at least one alignment hole.   The transdermal assay device according to claim 1, wherein the third member is made of a material selected from the group consisting of aluminum, steel, brass, plastic, ceramic, and any combination described above.   The transdermal assay device of claim 1, wherein the magnet clamp comprises a permanent magnet.   The transdermal assay device of claim 1, wherein the magnet clamp includes an electromagnet.   The transdermal assay device according to claim 1, wherein the magnet clamp includes a magnet having a force selected based on a clamping force required between the first member and the second member.   The transdermal assay device according to claim 1, wherein the first and second members are made of a material selected from the group consisting of stainless steel, plastic, polycarbonate, glass and any combination of the foregoing. Furthermore, the sample further comprises a plurality of samples, the sample comprising a common component and at least one additional component, each sample comprising:
The transdermal assay according to claim 1, wherein (i) the identity of the additional component (ii) the ratio of the common component to the additional component or (iii) at least one of the physical states of the common component differs from at least one of the other samples. apparatus.   The transdermal assay device according to claim 24, wherein the common component is a pharmaceutical, a nutritional supplement, a substitute pharmaceutical, or a dietary supplement.   The transdermal assay device according to claim 24, wherein the additional component is an adhesive, an accelerator, an additive, a solvent, an excipient, or a combination thereof.   27. The transdermal assay device of claim 26, wherein the enhancer is a chemical enhancer, a lipid permeation enhancer, a dissolution enhancer or a combination of enhancers.   The transdermal assay device according to claim 24, wherein the adhesive is polyisobutylene, silicone and acrylic adhesive.   The transdermal assay device of claim 1, wherein the sample is a solid source sample or a liquid source sample.   The transdermal assay device according to claim 1, wherein the tissue specimen comprises skin tissue.   The transdermal assay device according to claim 30, wherein the skin tissue is composed of an epidermis or a stratum corneum.   The transdermal assay device according to claim 30, wherein the skin tissue is human skin tissue, animal skin tissue or cultured skin tissue.   The transcutaneous assay device according to claim 1, wherein the tissue specimen is divided into a plurality of segments by mechanical cutting, scoring, laser cutting, cutting or crimping. Place the sample on the sample surface of the first member,
Filling the reservoir defined by the second member with a liquid medium, the reservoir comprising an opening on the surface of the second member;
Use of a transdermal assay device comprising placing a tissue specimen between the sample and the opening, and clamping the tissue specimen between the sample and the liquid medium at the opening by a magnetic clamping force Method.   35. The method of using a transdermal assay device according to claim 34, wherein the tightening includes attracting the first and second members to each other using a magnetic force between a magnet and a ferrous material.   The method of using a transdermal assay device according to claim 34, wherein the tightening includes attracting the first and second members using a magnetic force between at least two magnets facing each other and having counter electrodes. 35. The method of claim 34, further comprising collecting a specimen of the liquid medium and measuring the sample content in the liquid medium, the content indicating transport of the sample through the tissue specimen. How to use a transdermal assay device. Prepare the sample,
Placing the sample on the sample surface of the first member;
Filling the reservoir defined by the second member with a liquid medium, the reservoir comprising an opening on the surface of the second member;
Placing a tissue specimen between the sample and the opening;
At the opening, the tissue specimen between the sample and the liquid medium is clamped with a magnetic clamping force, and the presence of the sample in the reservoir is measured to determine transport of the sample through the tissue specimen. A method of measuring transdermal transport of a sample.   39. The method of claim 38, wherein the tightening includes attracting the first and second members in the direction of each other utilizing a magnetic force between a magnet and a ferrous material.   40. The method of claim 38, wherein the tightening includes attracting the first and second members in the direction of each other utilizing a magnetic force between at least two magnets facing each other and having counter electrodes. The measurement is
39. The method of claim 38, wherein a specimen of the liquid medium is taken and the content of the sample in the liquid medium is measured, the content indicating transport of the sample through the tissue specimen.