Method and medical device for analyzing epithelial barrier function

Abstract

The present invention relates to a medical device and method for assessing and monitoring epithelial barrier function in a subject using electrical impedance measurements. The method comprises initiating an impedance measurement phase comprising passing an electrical current through the subject's skin to obtain skin impedance values of a target tissue region, said data comprising at least one impedance value measured in the target tissue region at a different tissue layer. Furthermore, an assessment procedure is applied for analyzing epithelial barrier function in the target tissue region based on the impedance value data set measured in the target tissue region at different tissue layers. The procedure evaluates the obtained impedance value data set to provide a result indicative of the status of epithelial barrier function in the subject.

Description

Technical Field

[0001] This invention generally relates to the field of diagnostics of biological conditions, and specifically to medical devices and methods for noninvasively measuring electrical impedance spectra in tissues of living subjects and using the measured impedance to diagnose the biological conditions of said tissues. More specifically, this invention relates to medical devices and methods for using electrical impedance spectra to analyze and monitor epithelial barrier function and barrier status. The epithelial-skin barrier can be used to detect drug effects and patient responses to drug regimens, for screening purposes (e.g., screening infants with barrier defects), for the diagnosis and treatment of atopic dermatitis, and for planning preventative measures and therapies for patients with skin barrier defects. Background Technology

[0002] Epithelial tissue is composed of tightly packed, multilayered specialized cells, whose primary function is to form physical and chemical barriers between the body and the external environment. The epithelial barrier protects internal tissues from environmental stresses by minimizing water loss and preventing pathogens, pollutants, toxins, and allergens from entering through the skin or mucous membranes (Reference 1). Recent genome-wide association studies have shown that the barrier function of epithelial cells is essential in a variety of allergic diseases (References 2, 3). Barrier defects have been reported in atopic dermatitis, asthma, chronic sinusitis, allergic rhinitis, eosinophilic esophagitis, and colitis (References 4-9). These defects are the starting point for chronic inflammation and allergen sensitization and allow tissue-damaging factors to penetrate deeper into tissues, thereby activating immune and inflammatory responses (References 10, 11).

[0003] The skin possesses two physical barrier structures: the stratum corneum and tight junction proteins (TJs) (Reference 10). The stratum corneum, the outermost layer of the epidermis, is composed of terminally differentiated keratinocytes, called corneocytes, forming a densely packed and extensively cross-linked lipid-protein matrix. Proteins filaggrin, naegrin, and endothelial proteins play crucial roles in skin barrier function through their interaction with intermediate keratin filaments (Reference 12). The most important component of the epithelial barrier is represented by tight junction proteins (TJs), which seal the pericellular space at the apical surface of cells in mucous membranes and at the granular layer level between adjacent epithelial cells in the skin (References 13-15). TJs are associated with epithelial permeability by controlling the pericellular bypass of ions and larger molecules and physically separating two distinct compartments. TJs are essential for proper epithelial cell differentiation and function and are closely related to signal transduction and epithelial cell proliferation and differentiation (References 16-19). They form large complexes in the cell membrane, which is composed of three main types of transmembrane proteins: the claudin family, the tight junction-associated MARVEL family (MAL and related proteins for vesicle transport and membrane junctions), and single-span proteins such as immunoglobulin-like protein-associated adhesion molecules (JAM) and the Coxsackievirus-Adenovirus receptor (CAR). Intracellularly, transmembrane proteins bind to several scaffold proteins, such as the atresia band family (ZO), and thus attach to the actin cytoskeleton (References 16, 20).

[0004] Previously, the epithelial barrier could be assessed in vitro by measuring transepithelial resistance (TEER), which represents the resistance of epithelial cells to the passage of steady currents. For this purpose, epithelial cells were cultured to the air-liquid interface (ALI) in transwell plates. Complete confluence of cells confirmed a sharp increase in TEER, indicating low ion flux and a tight epithelial barrier; while disruption of the junctional complex led to a decrease in TEER. Furthermore, as demonstrated by ALI cultures from different tissues, TEER measurements showed a strong negative correlation with luciferin-dextran channels (References 24, 25).

[0005] Several in vivo non-invasive methods exist for assessing epithelial barrier function. One of these is the quantification of transepidermal water loss (TEWL) across the stratum corneum. Although TEWL increases proportionally with the degree of damage, it is also influenced by environmental factors such as humidity, temperature, season, and skin hydration. Other non-invasive methods used include stratum corneum hydration, colorimetry, skin surface pH assays, skin moisture tests, and oil tests (U. Heinrich, U. Koop et al.: Multicenter comparison of skin hydration based on physical, physiological, and product-related parameters using capacitance methods (Moisture Testing Technique CM 825), *International Journal of Aesthetic Science*, 2003, 25, 45-51). These provide information about different skin characteristics and / or conditions but do not directly measure barrier function (Reference 26).

[0006] Therefore, there is a need for improved and more accurate tools that can be used to analyze and assess epithelial-skin barrier function in vivo to overcome the above-mentioned shortcomings. Summary of the Invention

[0007] One object of the present invention is to provide an improved medical device and method for in vivo analysis and evaluation of the epithelial skin barrier.

[0008] Another objective of the present invention is to provide an improved medical device and method for detecting drug effects in a patient and determining in vivo the patient's response to the epithelial-skin barrier of drug delivery.

[0009] Another objective of the present invention is to provide an improved medical device and method for screening epithelial skin barrier function in vivo.

[0010] Another objective of this invention is to provide improved medical devices and methods for in vivo analysis and evaluation of the epithelial-skin barrier, with increased accuracy and reliability.

[0011] Another objective of the present invention is to provide an improved medical device and method for in vivo analysis and evaluation of the epithelial skin barrier, which is stable relative to the influence of environmental factors.

[0012] Another objective of the present invention is to provide an improved medical device and method that reduces the burden on patients and users and facilitates measurement procedures when analyzing and evaluating the epithelial skin barrier.

[0013] These and other objectives of the invention are achieved by the apparatus and methods described in the independent claims. Other embodiments are defined in the appended claims.

[0014] This invention is based on a deeper understanding of the dielectric properties of various tissues, making it possible to establish a method for assessing epithelial barrier function in vivo that is relatively stable relative to the influence of environmental factors. The method of this invention can be used as a diagnostic instrument for skin inflammations with barrier defects, such as atopic dermatitis (AD). Electrical impedance (EI) mapping is a relatively new technique previously used to characterize skin tumors. Current is transmitted through the skin at several different depths and frequencies, and the impedance response is measured, which is influenced by certain tissue integrity properties. Typically, when tissue structure and cellular composition change, an imprint related to the type of tissue alteration appears in the electrical impedance spectrum (Reference 27). In some diseases, such as melanoma, tissue EI measurements have been used to diagnose, assess disease progression, and evaluate treatment efficacy. The inventors have now discovered that EI spectroscopy can be used in skin and mucosal diseases where it is necessary to monitor epithelial barrier dysfunction and the effects of certain treatments on the epithelial barrier (References 4-7) and… Structural differences and reactions with skin and oral mucosa The relevant bioelectrical impedance “The Annals of the New York Academy of Sciences, April 20, 1999, Vol. 873, pp. 221-6 and Comparison of clinically normal atopic skin and non-atopic skin as seen by electrical impedance, Nicander, Ingrid; Ollmar, Stig, Skin Research and Technology, August 2004, Vol. 10(3), pp. 178-183.

[0015] Therefore, according to the present invention, the function or integrity of the epithelial skin barrier can be evaluated / quantified. Impaired skin barrier function is a precursor to many conditions, such as atopic dermatitis. Therefore, changes in the skin barrier can be detected by the present invention and conditions such as atopic dermatitis can be predicted at early stages, such as infancy, adolescence, and adulthood. Furthermore, the efficacy of various treatments for said diseases can be assessed. Quantifying the sensitivity of the skin, oral cavity, bronchi, esophagus, stomach, duodenum, small and large intestine, vagina, and genitourinary system to allergens and toxic / irritant substances is a further application of the present invention. Monitoring the treatment of skin diseases such as psoriasis (excluding eczema) and assessing lesions such as lichenification in the skin and oral cavity are other conceivable applications of the present invention. Additionally, the present invention can be used to assess periodontitis, for example, to quantify the risk of tooth loss.

[0016] EI spectroscopy can also be used to predict infantile atopic dermatitis (AD), thereby identifying disease risk and potentially enabling preventative measures. Furthermore, EI spectroscopy can be used to track skin lesions to obtain information about the effectiveness of local or systemic treatments and to collect additional information to monitor the stage and severity of lesion progression. Additionally, it is a useful, non-invasive, and cost-effective tool for overall clinical assessment and patient tracking without requiring complex barrier assessment analyses, such as analysis for filaggrin mutations from DNA. As a follow-up to these initial data in different mouse models, EI spectroscopy is still being further investigated in human subjects with AD and / or other skin inflammations. Measurements can be compared between diseased and non-diseased skin to detect any differences in skin permeability and electrical responses due to inflammatory conditions. Furthermore, comparisons of EI measurements between AD patients and healthy volunteers will indicate whether patients have perceptible and detectable defects in their skin electrophysiological behavior.

[0017] According to one aspect of the invention, a method is provided for assessing and monitoring epithelial barrier function in a subject using EI measurements. The method includes initiating an impedance measurement phase, comprising passing an electric current through the subject's skin to obtain skin impedance values ​​for a target tissue region, the data comprising impedance values ​​measured in at least one target tissue region at different tissue layers. Furthermore, an evaluation procedure is applied to analyze the epithelial barrier function in the target tissue region based on the dataset of impedance values ​​measured in the target tissue region at different tissue layers. The procedure evaluates the obtained impedance value dataset to provide results representing the state of the subject's epithelial barrier function.

[0018] According to another aspect of the invention, a method is provided for determining a patient's response to a drug using electrical impedance measurement. The method includes initiating an impedance measurement phase, comprising passing an electric current through the subject's skin to obtain skin impedance values ​​for a target tissue region, the data comprising impedance values ​​measured in at least one target tissue region at different tissue layers. Furthermore, an evaluation procedure is applied to analyze the epithelial barrier function in the target tissue region based on the dataset of impedance values ​​measured in the target tissue region at different tissue layers. The obtained impedance value dataset is evaluated to provide results representing the state of the subject's epithelial barrier function. The patient's response to the drug is determined based on the state of epithelial barrier function and clinical data, including drug prescription.

[0019] According to another aspect of the invention, a method is provided for screening the epithelial barrier function of a large number of subjects using impedance measurement. The method includes a phase of performing impedance measurements on the subjects, including passing an electric current through the skin of each subject to obtain skin impedance values ​​for a target tissue region, the data comprising impedance values ​​measured for each subject in at least one target tissue region at different tissue layers. An evaluation procedure is applied to analyze the epithelial barrier function in the target tissue region based on the dataset of impedance values ​​measured for each subject in the target tissue region at different tissue layers. Furthermore, the obtained impedance value dataset is evaluated to provide results representing the epithelial barrier function status of each subject.

[0020] According to another aspect of the present invention, a medical device is provided for assessing and monitoring the epithelial barrier function of a subject using electrical impedance measurement. The device includes an impedance measurement unit configured to pass an electric current through the subject's skin to obtain skin impedance values ​​for a target tissue region, the data comprising at least one impedance value measured in the target tissue region at different tissue layers; and an evaluation unit configured to apply an evaluation procedure to analyze the epithelial barrier function in the target tissue region based on the dataset of impedance values ​​measured in the target tissue region at different tissue layers and to evaluate the obtained impedance value dataset to provide results representing the state of the subject's epithelial barrier function. The medical device of the present invention is preferably used in the method of the present invention.

[0021] In embodiments of the invention, the medical device includes a probe for measuring the electrical impedance of a subject's tissue. The probe comprises a plurality of electrodes adapted for direct contact with the subject's skin and connectable to an impedance measurement circuit adapted to apply a voltage and measure the resulting current to determine an impedance signal. In a preferred embodiment, the probe further includes a switching circuit that selectively activates an electrode pair by connecting at least two electrodes to the impedance measurement circuit and disconnecting the remaining electrodes from the impedance circuit, wherein a voltage is applied to two electrodes and the current obtained between the at least two electrodes is measured. The switching circuit is adapted to receive a control signal instructing it to activate the electrode pair according to a predetermined activation scheme, the predetermined activation scheme including progressively scanning the subject's tissue at a first tissue depth by activating adjacent electrodes in a sequential manner, thereby obtaining a series of impedance signals from the selected tissue depth.

[0022] According to an embodiment, the probe is configured with electrodes having an elongated rectangular shape and arranged in parallel within the probe. However, several alternative designs are available. For example, the electrodes may be arranged in concentric rings or a square. The electrodes may be arranged with microspiked spikes, wherein each electrode contains at least one spike. The spikes are laterally spaced from each other and have a length sufficient to penetrate at least the stratum corneum in the case of skin measurements. In an alternative embodiment, the electrodes are non-invasive, and each electrode has a generally flat surface suitable for placement against the subject's tissue. It is also possible to combine electrodes with microspiked spikes with non-invasive electrodes.

[0023] WO 01 / 52731 discloses an exemplary medical electrode for detecting bioelectric potential generated in a living subject. The electrode comprises a plurality of microspins adapted to penetrate the skin. The microspins are long enough to reach and penetrate at least into the stratum corneum, and their surfaces are conductive and interconnected to form an array. EP 1 437 091 discloses a device for diagnosing biological conditions using impedance measurements on organic and biological materials. The device comprises a probe including a plurality of electrodes, each electrode having a plurality of microspins, each microspin being long enough to penetrate at least into the stratum corneum. The microspins according to EP 1 437 091 are also “nail-shaped,” meaning they have a generally circular cross-section, a body with a constant or gradually decreasing diameter, and a tip portion having a generally spherical or spike-like tip.

[0024] In embodiments of the present invention, the probe may have a spherical shape, that is, the probe surface with electrodes is spherical.

[0025] According to embodiments of the present invention, the method and medical device can be used for moisturizing cream treatment selection, wherein EIS measurement is used to determine the most suitable moisturizing cream for an individual (typically AD) patient.

[0026] According to embodiments of the present invention, EIS monitoring of outbreak areas can be used to predict and preventatively treat with corticosteroids or moisturizing creams.

[0027] According to embodiments of the present invention, the method and medical device can be used to determine the appropriate time to discontinue the use of topical corticosteroids.

[0028] According to embodiments of the present invention, the method and medical device can be used to determine the extent to which inflammation spreads from an eczema outbreak / rash to an external subcritical level.

[0029] According to embodiments of the present invention, the method and medical device can be used to identify infants whose barrier function has deteriorated and / or who have inflammation before the onset of AD symptoms.

[0030] As those skilled in the art will understand, the method steps of the present invention and its preferred embodiments are suitable for implementation in the form of a computer program or a computer-readable medium.

[0031] Unless otherwise expressly defined herein, all terms used in the claims and specification should generally be understood in accordance with their general meaning in the art. Unless otherwise expressly described, all references to “a / the [element, device, assembly, unit, component, step, etc.]” ​​should be openly interpreted as referring to at least one of the stated elements, devices, assemblies, units, components, steps, etc. Unless otherwise expressly defined herein, the steps of any method disclosed herein are not necessarily performed in the exact order disclosed.

[0032] Other objectives and advantages of the invention will be discussed below through exemplary embodiments. Attached Figure Description

[0033] Exemplary embodiments of the invention will now be described with reference to the accompanying drawings, in which:

[0034] Figure 1 This is a schematic block diagram of one embodiment of the medical device of the present invention;

[0035] Figure 2 , Figure 2 (Continued): Topical application of increased concentrations of papain damages the epithelial barrier, leading to a dose-dependent decrease in EI and an increase in TEWL in the skin;

[0036] Figures 3a-c: Papain downregulates the expression of molecules involved in epithelial barrier function;

[0037] Figures 4a-c: Trypsin shows a similar effect on the skin epithelial barrier, leading to a decrease in EI and an increase in TEWL;

[0038] Figures 5a-b: Tape peeling leads to a decrease in EI and an increase in TEWL in the skin;

[0039] Figure 6 Cholera toxin reduces the amount of EI (extracellular irradiation) in the skin.

[0040] Figures 7a-b: After papain treatment and tape peeling, the electrical impedance measured in the surface layer showed a more dramatic decrease compared to the deeper layers;

[0041] Figure 8 The Nyquist plot shows the effect of papain on EI, represented as a vector on the Nyquist plot, where the two components of EI (real and imaginary parts) are plotted on the X and Y axes.

[0042] Figures 9a-b: AD patients showed significantly reduced EI values ​​compared to healthy subjects, and compared to non-lesioned skin, their lesion characteristics were reduced EI and increased TEWL;

[0043] Figures 10a-b: During the 21-day treatment period, the affected skin showed an increase in EI and a decrease in TEWL. Detailed Implementation

[0044] The following is a description of exemplary embodiments of the present invention. This description should not be construed as limiting, but is merely for the purpose of illustrating the general principles of the invention. Although specific types of probes, including microinvasive and non-invasive probes, will be described, the invention is also applicable to other types, such as invasive probes.

[0045] Therefore, preferred embodiments of the invention will now be described with reference to the accompanying drawings for illustrative purposes, wherein like reference numerals denote like elements throughout the drawings. It should be understood that the invention covers other exemplary embodiments comprising the feature combinations described below. Furthermore, other exemplary embodiments of the invention are defined in the appended claims.

[0046] First refer to Figure 1 The following will provide a general description of the medical device of the present invention. Device 10 includes an impedance measurement circuit or unit 2 and an analysis or evaluation unit 4, which is used to analyze the epithelial barrier function in the target tissue region based on a dataset of impedance values ​​measured in the target tissue region at different tissue layers and to evaluate the obtained impedance value dataset to provide results representing the epithelial barrier function status of the subject. The impedance measurement unit 2 is used to obtain impedance data of the target tissue region of the subject's tissue. It should be understood that the impedance data of the target tissue region includes at least one impedance value obtained at different tissue depths (or layers), for example at four different depths, and at a certain frequency range, for example at 35 different frequencies in the range of 1 kHz to 2.5 MHz.

[0047] Impedance data of a target tissue region can be obtained by measuring tissue impedance using probe 8 integrated within the medical device 10 or by probes external to and connected to the medical device 10. Whether external or integrated, the probe may contain multiple electrodes 14 adapted to contact the tissue to be analyzed (typically the subject's skin). Tissue impedance can be measured by applying an AC voltage to a pair of electrodes and measuring the current through that pair. In an embodiment, a two-point measurement method is used by applying a voltage to a pair of electrodes and measuring the current. The remaining electrodes may be grounded or free-floating. In embodiments of the invention, probe 8 contains, for example, seven or five electrodes, such as rectangular electrode rods. The electrodes are adapted to be placed in direct contact with the skin.

[0048] In one embodiment, the adjacent electrodes are spaced approximately 0.3 mm apart and are approximately 5 mm long, a configuration that has proven practically useful for detecting disease conditions such as malignant melanoma, in terms of both lateral and depth spatial resolution. Thus, the probe covers approximately 5 × 5 mm or approximately 25 mm. 2 The probe covers a skin area and, at high frequencies above approximately 100 kHz, reaches a deepest tissue layer of approximately 2.5 mm, which has proven to be a clinically relevant depth. To cover a larger skin area, the probe can be moved to adjacent skin sites. However, as those skilled in the art will understand, the probe may include more or fewer than seven electrodes, such as three, four, five, or nine electrodes. Furthermore, other electrode sizes, geometries, and other dimensions between adjacent electrodes are also conceivable, such as electrodes approximately 4 mm wide and approximately 8 mm long.

[0049] By selecting adjacent electrode pairs, the top layer of skin can be scanned progressively, and by selecting spaced-apart electrodes—that is, electrode pairs with one or more intermediate electrodes—the resulting current path allows for the measurement of deeper layers of skin. The ability to measure the top layer of skin in small, continuous sections (particularly by measuring the spacing between adjacent electrodes and the frequency of the applied current) is particularly important, as it enables the detection of small anomalies in the skin and tissue. Each electrode of the probe can be set to four different states: injection (the electrode is set to inject a measuring current into the tissue), measurement (the current obtained from the tissue is measured by the electrode), grounding (the electrode is grounded to prevent superficial current leakage when measuring with other electrodes), and floating (the electrode is disconnected).

[0050] The assessment unit 4 may include a storage unit (not shown) for storing, for example, impedance data obtained from the patient. The diagnostic unit 4 may also include processing circuitry 5, included in this embodiment, adapted to process the obtained impedance data to reduce the number of variables by linearly or nonlinearly projecting the impedance data to a lower subspace to remove inconspicuous variables. In a preferred embodiment of the invention, principal component analysis (PCA) is used. An alternative method is parallel factor analysis (PARAFAC). Furthermore, classification rules determined, for example, by linear discriminant analysis (LDA) or cluster-independent soft model analysis (SIMCA) can be used to improve the assessment.

[0051] Additionally, the assessment unit 4 can be connected to a display component, for example, for displaying the state of the epithelial barrier. The assessment unit 4 applies an assessment program to analyze the epithelial barrier function in the target tissue region based on a dataset of impedance values ​​measured in different tissue layers, and evaluates the obtained impedance value dataset to provide results representing the epithelial barrier function state of the subject. The magnitude of the measured impedance can be determined, and a decrease or reduction in this magnitude indicates impaired or reduced epithelial barrier function in the subject. Reference data and / or clinical data can be used in the assessment. In embodiments of the invention, the assessment unit 4 can use a trained assessment program to analyze the measured impedance value dataset, wherein the trained assessment program extracts impedance data reflecting tissue characteristics of epithelial barrier function from the impedance spectrum of the obtained impedance value dataset, and evaluates the obtained impedance dataset to provide results representing the epithelial barrier function state of the subject.

[0052] According to embodiments of the invention, each electrode is equipped with spikes, thereby forming a spiked surface. As discussed above, in a preferred embodiment, the probe may comprise five rectangular regions or rods. In this configuration, each rod contains, for example, an array of 45 (15×3) or 57 (19×3) microspiks. Each rod is approximately 0.75 mm wide and 5 mm long. The distance between adjacent rods is approximately 0.2-0.5 mm. Therefore, the effective portion of the probe is approximately 5×5 mm. Each microspik, measured from the base, is approximately 100 micrometers long and at least 20 micrometers thick. The electrode rods and microspiks can be made from a plastic material by molding. The material may be conductive itself or coated with a conductive layer such as gold. In an alternative embodiment, the electrode rods and microspiks are made, for example, of plastic or silicon and coated with a metal, such as gold, with a thickness of at least 1 micrometer. However, other materials with similar dimensions and conductive surfaces may also be used, but biocompatibility should be chosen. For example, different probe concepts with such microspiky tips are described in the same applicant’s patent applications EP1959828, EP 1600104 and EP 1437091.

[0053] In another embodiment, the electrode rod is non-invasive and generally flat. For example, a probe concept including non-invasive electrodes is described in US 5,353,802, filed by the same applicant.

[0054] In other embodiments of the invention, the probe is spherical, meaning that the surface of the electrode, which is pressed against the skin or tissue during measurement, is spherical. This also means that the electrode may be at least partially spherical.

[0055] For example, each spike may have a length of 0.01 to 1 mm. The spikes may be arranged on the electrodes, sequentially arranged on the probes, wherein each electrode may contain at least two spikes, and in some applications up to 100-200 spikes and any number in between. Examples of preferred spike design embodiments are described in the same applicant's patent US 9,636,035. These spike arrangements, in addition to potentially mitigating the nonlinear effects of the stratum corneum, also allow for increased versatility and adaptability in terms of capacity requirements.

[0056] Control circuit 9 can be configured to control, for example, the switching cycle / sequence of electrode 14 according to a predetermined activation procedure or scheme. Such a predetermined activation scheme may include activating adjacent electrodes in a sequential manner to progressively scan the subject's tissue at a first tissue depth, the scanned tissue being largely dependent on the spacing between the activated electrode pairs, in order to obtain impedance signal matrices from different tissue depths.

[0057] Evaluation unit 4 is configured to preprocess impedance data, such as reducing noise content and / or reducing dimensionality. Noise reduction may include reducing noise in the impedance magnitude and / or phase spectrum. Noise reduction can be achieved, for example, by using a Savitsky-Golay smoothing filter. Diagnostic unit 4 may also utilize data on the subject's physical condition, which can be parameterized and further used in the diagnostic process. Furthermore, preprocessing may include detecting and correcting spikes or other artificial phenomena, allowing the removal of spikes or artificial phenomena from the impedance spectrum (i.e., the magnitude and / or phase spectrum). Spikes can be detected, for example, by a median filter with an appropriate window size. Data points in the filtered data that differ significantly from the original data can be considered spikes or other artificial phenomena and can be corrected, for example, by linear interpolation.

[0058] Evaluation unit 4 may also include a pre-filter to exclude measurements that do not meet one or more specific criteria, such as critical values. The pre-filter can be applied to impedance data that has already been corrected / adjusted, for example, through preprocessing as described above. For example, magnitude and / or phase angle values ​​may be required to be within specified magnitude or phase ranges, respectively, so that measurements are not excluded. If measurements are taken on human / animal skin, criteria, such as ranges, such as excluding non-physiological measurements, can be set. Specific criteria can also be set for a value at a specific frequency.

[0059] Evaluation unit 4 may also include a classifier to assess the quality of the measured impedance data. This procedure can be combined with preprocessing and / or pre-filtering to further improve data quality. Examples of such classification include evaluating the variation (e.g., variance or standard deviation) of magnitudes and / or phase angles at one or more frequencies in different permutations. Other examples cover the absolute values ​​of the magnitudes and / or phase angles that can be studied, such as the median or mean of the magnitudes, skewness, derivatives of the magnitudes or phase angles, or phase angles.

[0060] The medical device 10 may also include a communication unit 12 capable of transmitting / receiving data from / to an external unit 15, such as a laptop computer, handheld computer / device, computer embedded in a device, database, cloud configuration, etc., communicating directly with the unit or network itself or via a wireless network 16. In this way, clinical data for evaluation can be provided to the device 10, for example. Furthermore, data acquired by the medical device 10, such as impedance data from measurements, can also be downloaded to the external device 15 via the communication unit 12.

[0061] Furthermore, the medical device 10 includes a pressure application unit 18 configured to apply a predetermined pressure to tissue or skin upon startup and to press the probe 8 against the tissue or skin during measurement. During the measurement phase, the pressure is preferably constant. For example, a pressure in the range of 1-12 N may be applied, or in a preferred embodiment, a pressure in the range of 3-10 N may be applied, or in other preferred embodiments, a pressure in the range of 5-7 N may be applied, or in some embodiments, a pressure in the range of 5.5-6.5 N may be applied. In embodiments of the invention, the applied predetermined pressure may be combined with or replaced by a suction effect, thereby adhering the probe to the tissue or skin during measurement.

[0062] It should be understood that, in the context of this invention, and when referring to electrically connected electrical components, the term "connection" is not limited to meaning a direct connection, but also encompasses a functional connection with intermediate components. For example, on one hand, a direct connection is included if the output of a first component is connected to the input of a second component. On the other hand, the first and second components are also interconnected if a conductor directly or through one or more other components provides a signal from the output of the first component to the input of the second component in a substantially unchanged manner. However, the significance of a functional connection lies in the fact that a gradual or sudden change in the signal from the output of the first component results in a corresponding change or modification of the signal input to the second component.

[0063] Although exemplary embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, or variations can be made to the invention described herein. Therefore, it should be understood that the above description and drawings of the invention should be considered as non-limiting examples and the scope of protection is defined by the appended patent claims.

[0064] Test results

[0065] Therefore, the inventors have discovered that EI spectroscopy can be used to detect epithelial barrier function in vivo. For example... Figure 2 , Figure 2 As shown in (continued), a dose-dependent decrease in EI was detected as early as 1 hour after treatment, reflecting a decline in epithelial barrier function.

[0066] EI spectroscopy measurements showed a significant negative correlation with another biomarker of epithelial barrier damage in the skin, namely, transepidermal water loss (TEWL). Increased TEWL, indicating barrier damage, was synchronous with a decrease in EI spectra, which also indicates barrier damage. Furthermore, to clearly illustrate the effect of papain on EI, we represented it as a vector in a Nyquist plot, plotting the two components (real and imaginary parts) of EI on the X and Y axes, see [link to Nyquist plot]. Figure 8 We clearly observed that the curve obtained 5 hours after papain administration was significantly different from the curve obtained in control mice that received PBS only via epidermal administration.

[0067] Papain-induced barrier disruption was confirmed by histological analysis, showing stratum corneum damage and increased cell infiltration after papain application (Figure 3a). Furthermore, immunofluorescence staining revealed the expression of molecules important for skin barrier function, such as filaggrin or tight junction proteins occludin and occludin-1. The expression of all three barrier molecules was observed to be downregulated in a dose-dependent manner (Figures 4a and 4b).

[0068] EI spectroscopy detected a decrease in the mRNA expression of filaggrin, naegrin, and naegrin, indicating that the epithelial barrier function decreased along with the damage to epithelial barrier molecules, suggesting overall impairment of the stratum corneum barrier function. These results demonstrate that skin barrier function is impaired by papain treatment, and this was confirmed by EI spectroscopy.

[0069] Another protease, the serine protease trypsin, was administered to mouse skin via the same protocol as papain administration. As shown in Figures 3a-c, EI significantly decreased and TEWL significantly increased after trypsin treatment, consistent with data from papain exposure. Similarly, EI and TEWL values ​​showed a significant negative correlation.

[0070] To further investigate the accuracy of epithelial barrier detection, we tested the EI and TEWL methods on nude mouse skin with an impaired epithelial barrier achieved through tape peeling, a simple and effective method that uses a continuous adhesive film to remove the cellular layers of the stratum corneum. EI and TEWL measurements were performed before tape peeling and immediately after 5, 10, 15, and 20 tape strips. We detected a significant decrease in EI after tape peeling, reflecting impaired epithelial barrier function. In parallel measurements, we observed an increase in TEWL, demonstrating reduced surface barrier function of the skin (Figure 5a). The same method was used in healthy human volunteers, with the results obtained in mice evidenced by the decrease in EI and increase in TEWL observed after tape peeling in Figure 5b.

[0071] Epithelial barrier damage induced by cholera toxin was detected by EI spectroscopy. Dorsal skin was isolated from C57BL / 6 nude mice and incubated at 37°C for 1 hour in the presence of 2 μg / ml cholera toxin. We observed a significant decrease in EI one hour after cholera toxin treatment compared to the control condition treated with PBS (see [reference needed]). Figure 6 Our observations once again demonstrate the effectiveness of EI spectroscopy as a method for detecting epithelial barrier function.

[0072] Data demonstrate that EI spectroscopy can serve as a direct method for assessing epithelial barrier function of the skin in vivo. Based on our results, EI spectroscopy represents a promising candidate method for studying and characterizing skin inflammations such as Alzheimer's disease (AD). AD affects up to 20% of children and up to 4.9% of adults (References 21, 22). Characterized by pruritus and eczematous skin lesions, it often appears in early childhood and undergoes a process of remission and exacerbation. Epidermal barrier impairment characterized by defective filaggrin protein expression and tight junction protein (TJ) defects has been described (Reference 23). EI spectroscopy aids in the early diagnosis of AD in infants, enabling the determination of disease risk and thus the likelihood of administering preventative measures. Furthermore, EI spectroscopy can be used to track skin lesions to obtain information on the effectiveness of local or systemic treatments and to collect other information to monitor the stage and severity of lesion progression. In addition, it is a useful, non-invasive, and cost-effective tool for overall clinical assessment and patient tracking without the need for complex barrier assessment analyses, such as analysis for filaggrin mutations from DNA.

[0073] Figures 9a-b illustrate test results showing significantly reduced EI values ​​in AD patients compared to healthy subjects, and that their lesions exhibited decreased EI and increased TEWL compared to non-lesion skin. EI (a) and TEWL (b) were measured at the same body sites in healthy controls and AD patients. In patients, measurements were taken at both the lesion site and in non-lesion areas adjacent to the lesion. EI is expressed in kOhm at 1000 Hz. TEWL is expressed in g / m²h. *: p<0.05, **: p<0.01, ****: p<0.0001.

[0074] Figures 10a-b illustrate the test results showing an increase in EI and a decrease in TEWL in the affected skin during a 21-day treatment period. T0 represents the measurement at the first visit, T01 represents the measurement at the mid-treatment visit, and T02 represents the measurement at the last visit. EI(a) and TEWL(b) are measurements of the same body site in AD patients over a 21-day period, corresponding to the duration of treatment at the clinic. EI is expressed in kOhm at 1000 Hz. TEWL is expressed in g / m²h. *: p < 0.05, **: p < 0.01.

[0075] Description of test result graph

[0076] Figure 2 , Figure 2 (Continued): Epidermal application of increased concentrations of papain impaired the epithelial barrier, leading to a dose-dependent decrease in EI and an increase in TEWL in the skin. Hair was removed from the backs of WT C57BL / 6 mice using a hair removal cream. Three days after hair removal, 100 μl of a solution containing different doses of the protease papain (0.1 µg / µl, 1 µg / µl, and 10 µg / µl) was applied via skin patch, or PBS was used as a control (unstimulated). EI (a) and TEWL (b) were measured before treatment and at 1, 3, 5, 24, 48, and 72 hours after treatment. (c) shows the Spearman correlation between EI and TEWL at each time point. Electrical impedance is expressed in kOhm at 1000 Hz. TEWL is expressed in g / m²h. Data are presented as mean ± SD (n=8). *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0077] Figures 3a-c: Papain downregulates the expression of molecules involved in epithelial barrier function. (a) Hematoxylin-eosin staining and immunofluorescence staining of filaggrin, occlusin, and occlusin-1 in mouse skin after transdermal application of PBS as a control or after application of 100 μl of solutions containing varying doses of papain (0.1 µg / µl, 1 µg / µl, and 10 µg / µl). (b) mRNA expression of filaggrin, endothelin, occlusin, keratin-10, occlusin-1, and occlusin 5 hours after transdermal treatment with different doses of papain.

[0078] Figures 4a-c: Trypsin showed a similar effect on the skin epithelial barrier, leading to a decrease in EI and an increase in TEWL. Hair was removed from the backs of WTC57BL / 6 mice. Three days after hair removal, 100 μl of a solution containing trypsin (0.5%) or the cysteine ​​protease papain (10 µg / µl), or PBS as a control, was applied via skin patch. EI (a) and TEWL (b) were measured before treatment and at 1, 3, 5, and 24 hours after treatment. (c) shows the correlation between EI and TEWL at each time point. Electrical impedance is expressed in kOhm at 1000 Hz. TEWL is expressed in g / m²h. Data are presented as mean ± SD (n=8). *: p<0.05, **: p<0.01.

[0079] Figures 5a-b: Tape peeling resulted in decreased EI and increased TEWL in the skin. (a) Skin of C57BL / 6 nude mice was damaged by tape peeling, an efficient method for sequentially removing the cellular layers of the stratum corneum using adhesive tape. EI and TEWL were measured before tape peeling and after 5, 10, 15, and 20 tapes. (b) The same protocol was followed in human subjects. Electrical impedance is expressed in kOhm at 1000 Hz. TEWL is expressed in g / m²h. Data are presented as mean ± SD, n = 12 (a), 5 (b). *: p < 0.05, **: p < 0.01, ***: p < 0.001.

[0080] Figure 6 Cholera toxin reduces the electrical impedance (EI) of isolated skin. Dorsal skin from C57BL / 6 nude mice was isolated and incubated at 37°C for 1 h in the presence of 2 μg / ml cholera toxin (B), a microbial product that specifically disrupts epithelial tight junction proteins (TJs). Electrical impedance is expressed in kOhms at 1000 Hz. Data are presented as mean ± SD, n=5 in A and n=7 in B. *: p<0.05.

[0081] Figures 7a-b: After papain treatment and tape peeling, the electrical impedance measured at the surface layer showed a more dramatic decrease compared to deeper layers. Electrical impedance at several different depths was measured. The electrical impedance at depth a (deepest layer) and depth b (deepest layer) is shown before treatment with PBS (control) and papain (10 µg / µl), and at 1, 3, and 5 hours after treatment (a), and before tape peeling and after 5 and 10 tapes (b), expressed in kOhms at 1000 Hz. *p<0.05, **: p<0.01.

[0082] Figure 8 Nyquist plot. Impedance is a complex number consisting of a real part and an imaginary part. A Nyquist plot is obtained by plotting the real part on the X-axis and the imaginary part on the Y-axis. Each point on the curve represents the impedance at a specific frequency. Lower frequency data are on the right side of the plot, while higher frequency data are on the left.

[0083] Figures 9a-b show a significant decrease in EI values ​​in AD patients compared to healthy subjects, and the lesion characteristics compared to non-lesion skin are decreased EI and increased TEWL. EI (a) and TEWL (b) were measured at the same body sites in healthy controls and AD patients. In patients, measurements were taken at lesion sites and in non-lesion areas adjacent to the lesions. EI is expressed in kOhm at 1000 Hz. TEWL is expressed in g / m²h. *: p<0.05, **: p<0.01, ****: p<0.0001.

[0084] Figures 10a-b show an increase in EI and a decrease in TEWL in the affected skin during a 21-day treatment period. EI (a) and TEWL (b) are measurements of the same body sites in AD patients over a 21-day period, corresponding to the duration of treatment at the clinic. EI is expressed in kOhm at 1000 Hz. TEWL is expressed in g / m²h. *: p < 0.05, **: p < 0.01.

[0085] References

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[0091] 6. Wawrzyniak P, Wawrzyniak M, Wanke K, Sokolowska M, Bendelja K, Rückert B, et al. Regulation of bronchial epithelial barrier integrity in asthmatic patients via type 2 cytokines and histone deacetylases. *Journal of Allergy and Clinical Immunology*, 2016.

[0092] 7. Soyka MB, Wawrzyniak P, Eiwegger T, Holzmann D, Treis A, Wanke K, et al. Defective epithelial barrier in chronic sinusitis: regulation of tight junctions via IFN-γ and IL-4. Journal of Allergy and Clinical Immunology 2012;130(5):1087-96.e10.

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[0094] 9. Mitamura Y, Nunomura S, Nanri Y, Ogawa M, Yoshihara T, Masuoka M, et al. The IL-13 / periostein / IL-24 pathway contributes to epithelial barrier dysfunction in allergic dermatitis. Allergy, 2018.

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Claims

1. A medical device for assessing and monitoring in vivo epithelial barrier function in a subject using electrical impedance measurement, the device comprising: An impedance measurement unit configured to obtain skin impedance values ​​of a target tissue region by passing an electric current through the subject's skin, the impedance measurement unit comprising a plurality of electrodes adapted to be placed in contact with the tissue, wherein each electrode is equipped with a spike, thereby forming a spiked surface, and the data comprising at least one impedance value measured in a target tissue region at different tissue layers. A switching circuit is adapted to sequentially scan subject tissue at a first tissue depth by activating adjacent electrodes in a continuous manner, thereby obtaining a series of impedance signals from the selected tissue depth; and An assessment unit is configured to apply an assessment procedure to analyze the epithelial barrier function in a target tissue region based on a dataset of impedance values ​​measured in different tissue layers, and to evaluate the obtained impedance value dataset to provide results representing the epithelial barrier function status of the subject; and The evaluation unit is configured to analyze a dataset of measured impedance values ​​using a trained evaluation program, wherein the trained evaluation program performs the following functions: Impedance data reflecting tissue characteristics of epithelial barrier function are extracted from the impedance spectrum of the obtained impedance value dataset. The extracted impedance data includes numerical information, which includes absolute values, magnitude gradients, and / or phase information including phase angles; and The obtained impedance value dataset is evaluated to provide results representing the epithelial barrier function status of the subject.

2. The medical device of claim 1, wherein the evaluation unit is configured to use an evaluation of the acquired data to determine atopic dermatitis (AD) in a patient, wherein a decrease in impedance compared to non-lesioned skin indicates AD.

3. The medical device of claim 1, wherein the evaluation unit is configured to use an evaluation of the acquired data to determine atopic dermatitis (AD) in a patient, wherein a decrease in impedance over time indicates AD.

4. The medical device of claim 1, wherein the obtained impedance value dataset is evaluated to provide results representing the epithelial barrier function status of the subject, wherein the evaluation unit is further configured to determine the patient response to epithelial barrier treatment based on the results representing the epithelial barrier function status and clinical data including treatment prescriptions.

5. The medical device of claim 4, wherein a value of the measured impedance is determined, and a decrease or reduction in the value indicates impaired or reduced epithelial barrier function of the subject, wherein the change in impedance value is correlated with increased transepidermal water loss in a subject with impaired epithelial barrier function, and wherein patient response predicts the patient's disease.

6. The medical device according to any one of claims 1-5, wherein the evaluation unit is configured to determine a decrease in electrical impedance (EI) and an increase in transepidermal water loss (TEWL) measured at the body site of the AD patient compared to a non-lesioned skin site of the AD patient, to indicate that the body site is diseased.

7. The medical device according to any one of claims 1-5, wherein the assessment unit is configured to determine the electrical impedance (EI) difference between the non-lesioned skin of an AD patient and the healthy skin of a subject to predict AD in the subject.

8. The medical device according to any one of claims 1-5, wherein reference data and / or clinical data are used in the evaluation.

9. A medical device that uses electrical impedance measurement to determine a patient's response to a drug, comprising: An impedance measurement unit is configured to pass an electric current through the subject's skin to obtain the skin impedance value of a target tissue area. The impedance measurement unit includes a plurality of electrodes adapted to be placed in contact with the tissue, wherein each electrode is equipped with a spike, thereby forming a spiked surface. The data includes at least one impedance value measured in a target tissue area at different tissue layers. A switching circuit is adapted to sequentially scan subject tissue at a first tissue depth by activating adjacent electrodes in a continuous manner, thereby obtaining a series of impedance signals from the selected tissue depth; as well as An assessment unit is configured to apply an assessment procedure to analyze the epithelial barrier function in a target tissue region based on a dataset of impedance values ​​measured in different tissue layers, and to evaluate the obtained impedance value dataset to provide results representing the epithelial barrier function status of the subject; and The evaluation unit is configured to analyze a dataset of measured impedance values ​​using a trained evaluation program, wherein the trained evaluation program performs the following functions: Impedance data reflecting tissue characteristics of epithelial barrier function are extracted from the impedance spectrum of the obtained impedance value dataset. The extracted impedance data contains quantitative information, including absolute values, quantitative gradients, and / or phase information including phase angles. as well as The obtained impedance value dataset is evaluated to provide results representing the epithelial barrier function status of the subject; The assessment unit is configured to determine patient response to a drug based on the state of epithelial barrier function and clinical data, including drug prescriptions.

10. The medical device of claim 9, wherein reference data and / or clinical data are used in the evaluation.

11. A medical device for determining a patient's response to a drug using electrical impedance measurement, comprising: An impedance measurement unit configured to obtain skin impedance values ​​of a target tissue region by passing an electric current through the skin of a subject, the impedance measurement unit comprising a plurality of electrodes adapted for placement in contact with the tissue, each electrode being equipped with a spike to form a spiked surface, the data comprising impedance values ​​measured in at least one target tissue region at different tissue layers, the impedance measurement unit being configured to initiate an impedance measurement phase including using the plurality of electrodes to pass an electric current through the skin of a subject to obtain skin impedance values ​​of the target tissue region, selectively activating electrode pairs to progressively scan the subject's tissue to obtain a series of impedance signals from a selected tissue depth; and An assessment unit is configured to apply an assessment procedure to analyze epithelial barrier function in a target tissue region based on a dataset of impedance values ​​measured in the target tissue region at different tissue layers; assess the obtained impedance value dataset to provide results representing the epithelial barrier function status of the subject, wherein the assessment of the obtained impedance value dataset provides results representing the epithelial barrier function status of the subject; and determine patient response to a drug for epithelial barrier therapy based on the results representing the epithelial barrier function status and clinical data including drug prescriptions, wherein the measured impedance values ​​are determined, and a decrease or reduction in the values ​​indicates impaired or reduced epithelial barrier function of the subject, wherein changes in impedance values ​​are correlated with increased transepidermal water loss in subjects with impaired epithelial barrier function, and wherein patient response predicts the patient's disease.

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