System and method for predicting pharmacokinetics

Abstract

A system for predicting pharmacokinetics, comprising: a pump, an airflow control module, a microphysiological module, and tubing, wherein the tubing connects the pump, the airflow control module, and the microphysiological module to form a closed loop. Further provided is a method for predicting pharmacokinetics, comprising: the system for predicting pharmacokinetics; analyzing the state of a drug in a microfluidic apparatus to obtain a first result; and predicting the pharmacokinetics of the drug according to the first result.

Description

Systems and methods for predicting pharmacokinetics TECHNICAL FIELD

[0001] The present disclosure relates to technologies for establishing in vitro dynamic microenvironments, and in particular to in vitro dynamic microfluidic models for predicting pharmacokinetics. BACKGROUND

[0002] Conventionally, drug development mostly relies on animal experiments, using model organisms, such as mice, rats, rabbits, etc., to perform preclinical tests to screen out candidate drugs for investigational new drug application (IND). The preclinical test stage of drugs using model organisms is to simulate the pharmacokinetics (PK) and pharmacodynamics (PD) parameters in the human body. However, although the model organisms have complete drug absorption and metabolism functions, the performance of drugs in the current animal models and in the human body is very different, for example, the physiological structure and gene expression of model organisms and humans are different, resulting in a high drug failure rate of up to 90%, which limits drug development because the animal model cannot accurately correspond to the performance in the human body. In 2022, the US FDA agreed that animal replacement technology can partially replace preclinical test data, among which organ-on-a-chip (OoC) technology is a replacement technology that can successfully reproduce the performance of human organs to meet the current drug development needs.

[0003] Currently, the developing lung drugs can be divided into oral, intravenous injection, or inhalation, etc., among which the inhalation drugs can directly act on the lungs, making them have better therapeutic effect and higher safety compared to other drug administration methods, and at the same time reducing the side effects of drugs on other organs. However, the complex design and high development cost of inhalation drugs reduce the willingness of pharmaceutical companies to develop, and the low success rate in the clinical field makes the development of aerosol drugs using inhalation therapy gradually slow down.

[0004] Therefore, in addition to the above-mentioned animal experiments, there are other tools to assist the development of inhaled drugs, including next generation impactor (NGI), cell culture system or in vitro dissolution model. The in vitro dissolution model can be used as an alternative to animal experiments, reducing the complexity of the experiment and improving the accuracy of predicting in vivo drug absorption. It can be used to study the kinetics of active pharmaceutical ingredients (API) released from different inhaled formulations. However, the mechanisms of drug release in vitro and in vivo are completely different. For example, the in vitro dissolution model lacks drug delivery and lung barrier function, resulting in differences in drug release mechanisms between in vitro and in vivo. It is difficult to accurately reproduce the drug kinetics in the lungs and blood, so it can provide limited information on drug formulation design, drug efficacy or drug safety, greatly limiting the evaluation of changes in drug delivery, release and absorption. In addition, the in vitro dissolution model can only accurately predict oral drugs, and there is a lack of dissolution models suitable for inhaled drug development on the market. The use of nanocarriers such as liposomes and lipid nanoparticles to deliver drugs further increases the difficulty of predicting drug release performance.

[0005] In addition, the current improved inhaled drug development method only studies the dissolution and diffusion characteristics of the drug, and is difficult to simulate drug release with different penetration levels, and is mostly limited to static analysis, making it difficult to observe the differences in drug deposition under different breathing patterns, and unable to reproduce the effects of changes in shear stress on drug release or penetration caused by human respiratory airflow on non-tissue.

[0006] In summary, there is an urgent need in the art for a system and method for predicting drug kinetics that can quickly obtain results similar to the performance of drugs in the human body, including information on the interaction of inhaled drugs with airway surfaces, the clearance of inhaled drugs by lung defense mechanisms, and the time of inhaled drugs on the airway surface, to improve the accuracy of preclinical drug kinetics.

[0007] SUMMARY

[0008] To solve the above problems, the present disclosure provides a system for predicting pharmacokinetics, comprising: a pump; an airflow control module coupled with the pump to generate a biomimetic airflow, and comprising: a speed controller; and a one-way valve coupled with the speed controller; a microphysiological module coupled with the airflow control module to deliver a drug through the biomimetic airflow, and comprising: an atomizer to atomize the drug; and a microfluidic device coupled with the atomizer; and a pipeline connecting the pump, the airflow control module, and the microphysiological module to form a closed loop.

[0009] The present disclosure also provides a method for predicting pharmacokinetics, comprising: providing the system described in the present disclosure; delivering a drug to the microfluidic device; analyzing the state of the drug in the microfluidic device to obtain a first result; and predicting the pharmacokinetics according to the first result. BRIEF DESCRIPTION OF DRAWINGS

[0010] FIG. 1A is a schematic diagram of an air-lifted dynamic loop system for simulating the breathing pattern of a human body according to one embodiment of the present disclosure.

[0011] FIG. 1B is a schematic diagram of an air-lifted dynamic loop system according to one embodiment of the present disclosure.

[0012] FIG. 2A is a schematic diagram of an air-lifted dynamic loop system delivering inhaled particles according to one embodiment of the present disclosure.

[0013] FIG. 2B is a line graph of the relationship between the viscosity and the shear rate of artificial mucus according to one embodiment of the present disclosure.

[0014] FIG. 2C is a fluorescent staining diagram of the deposition amount of fluorescein sodium salt at the inlet, middle section, and outlet of a microfluidic device corresponding to different shear stress sizes according to one embodiment of the present disclosure.

[0015] FIG. 2D is a histogram of the relationship between the weight of deposited fluorescein sodium salt and the shear stress according to one embodiment of the present disclosure.

[0016] FIG. 3A is a schematic diagram of a microfluidic device for simulating a small airway of a human lung according to one embodiment of the present disclosure; and a fluorescent staining diagram of a human small airway tissue cultured in the microfluidic device according to one embodiment of the present disclosure.

[0017] FIG. 3B is a fluorescent staining side view of mucus secreted by a human small airway tissue cultured in a microfluidic device according to one embodiment of the present disclosure.

[0018] FIG. 3C is a fluorescent staining top view of mucus secreted by a human small airway tissue cultured in a microfluidic device according to one embodiment of the present disclosure.

[0019] FIG. 4A is a plot of the clearance trajectory of mucus-ciliary clearance of inhaled particles as a function of time under different breathing patterns for one embodiment of the present disclosure; and FIG. 4A is a plot of the moving distance of mucus-ciliary clearance of inhaled particles as a function of time under different breathing patterns for one embodiment of the present disclosure.

[0020] FIG. 4B is a histogram of the clearance rate of mucus-ciliary clearance of inhaled particles under different breathing patterns for one embodiment of the present disclosure.

[0021] [Corrected according to Rule 91 on 23.07.2024][deleted]

[0022] FIG. 5A is a schematic diagram of the penetration of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-labeled liposomes into cell layers of a microfluidic device for mimicking human lung small airways under different shear stress for one embodiment of the present disclosure.

[0023] FIG. 5B is a fluorescent staining diagram of DiI-labeled liposomes and carboxylate-polystyrene particles penetrating into cell layers of a microfluidic device for mimicking human lung small airways under different shear stress for one embodiment of the present disclosure.

[0024] FIG. 6A is a histogram of the deposition amount of DiI-labeled liposomes in a first microchannel layer of a microfluidic device for mimicking human lung small airways under different conditions and in a Transwell for one embodiment of the present disclosure.

[0025] FIG. 6B is a histogram of the deposition amount of a model drug calcein in a first microchannel layer of a microfluidic device for mimicking human lung small airways under different conditions and in a Transwell for one embodiment of the present disclosure.

[0026] FIG. 7A is a schematic diagram of the release of a model drug calcein carried by DiI-labeled liposomes in a medium layer of a microfluidic device for mimicking human lung small airways for one embodiment of the present disclosure.

[0027] FIG. 7B is a histogram of the cumulative distribution of a model drug calcein carried by DiI-labeled liposomes in a mucus layer, a cell layer, or a medium layer of a microfluidic device / Transwell for mimicking human lung small airways for one embodiment of the present disclosure.

[0028] Figure 7C is a histogram showing the cumulative distribution of DiI-labeled liposome- loaded model drug calcein in the mucus layer, cell layer, or medium layer of the microfluidic device / Transwell for the in vitro model of the small airway of the human lung according to one embodiment of the present disclosure.

[0029] Figure 7D is a line graph showing the fluorescence signal of DiI-labeled liposome- loaded model drug calcein in the medium layer of the microfluidic device / Transwell for the in vitro model of the small airway of the human lung as a function of time according to one embodiment of the present disclosure.

[0030] Figure 7E is a line graph showing the area under the curve of the release of DiI-labeled liposome-loaded model drug calcein in the second microchannel of the microfluidic device / Transwell for the in vitro model of the small airway of the human lung as a function of time according to one embodiment of the present disclosure.

[0031] Figure 7F is a graph showing the point-to-point comparison of the area under the curve (AUC) of the in vitro model and the area under the curve of the in vivo model according to one embodiment of the present disclosure. DETAILED DESCRIPTION

[0032] The spirit, advantages and effects of the present disclosure can be easily understood by those skilled in the art with reference to the embodiments described herein. However, the embodiments described herein are not intended to limit the present disclosure, and the present disclosure can be implemented or applied in other different embodiments, and each detail described herein can be changed or modified according to different views and applications without departing from the spirit of the present disclosure.

[0033] The proportions, structures, sizes, and other features shown in the drawings accompanying the present disclosure are merely used to facilitate the understanding of the present disclosure by those skilled in the art, and are not intended to limit the scope of the present disclosure. Therefore, any change in proportions, modification of structures, or adjustment of sizes, without affecting the purposes and effects that can be achieved by the present disclosure, should be considered to be within the scope of the technical content disclosed herein.

[0034] When the present disclosure describes "including", "containing", or "having" a specific element, unless otherwise specified, other elements, components, structures, regions, parts, devices, systems, steps, modules, or connection relationships can also be included, and such other elements are not excluded.

[0035] The terms "upper", "lower", "front", "back", and the like, as used herein, are merely for ease of description and are not intended to limit the scope of the disclosure, and the adjustment, interconversion, and change of relative positions and relationships thereof, without materially altering the technical content of the disclosure, shall be deemed to be within the scope of the disclosure.

[0036] The terms "first" and "second" and the like, as used herein, are merely for ease of description or to distinguish one element, component, structure, region, part, device, system, module, and the like, from another, and are not intended to limit the scope of the disclosure, nor are they intended to limit the spatial order of the elements. In addition, unless otherwise explicitly stated herein, the singular forms "a" and "the" as used herein include plural forms, and "or" and "and / or" as used herein can be used interchangeably.

[0037] Any numerical range recited herein, including those numerical ranges that fall within the numerical range recited herein, can be combined with any other numerical range recited herein, including those numerical ranges that fall within the numerical range recited herein, to derive a further numerical range. For example, a numerical range of "200 μm to 2000 μm" should be understood to include any sub-range between the minimum value of 200 μm and the maximum value of 2000 μm, such as 300 μm to 2000 μm, 200 μm to 1500 μm, and 250 μm to 1700 μm. In addition, any numerical endpoint recited herein can be used as a maximum or minimum value to derive a numerical range. For example, 250 μm, 290 μm, and 1800 μm can be used to derive a numerical range of 220 μm to 500 μm, 250 μm to 450 μm, or 500 μm to 2000 μm.

[0038] In some embodiments, "chip device with microfluidic channels", "microfluidic device", "microfluidics", "microfluidic device", "chip", and / or "chip device" as used herein can be used interchangeably, unless otherwise explicitly stated herein.

[0039] In some embodiments, "microphysiology" and "microenvironment" as used herein can be used interchangeably, unless otherwise explicitly stated herein.

[0040] In some embodiments, "pump", "peristaltic pump", "gas pump", and "gas sparger" as used herein can be used interchangeably, unless otherwise explicitly stated herein.

[0041] In some embodiments, "speed controller", "gas flow control valve", "gas flow restriction valve", "flow rate controller", and "adjustment valve" as used herein can be used interchangeably, unless otherwise explicitly stated herein.

[0042] In some embodiments, "gas flow check valve" and "check valve" as used herein can be used interchangeably, unless otherwise explicitly stated herein.

[0043] In some embodiments, “warmer” and “warmer device” as described herein are used interchangeably unless otherwise explicitly stated herein.

[0044] In some embodiments, “central diffuser” and “aerosol diffuser” as described herein are used interchangeably unless otherwise explicitly stated herein.

[0045] In some embodiments, “mode drug,” “inhaled drug,” “inhaled particle,” “aerosol,” and “aerosol suspension” as described herein are used interchangeably unless otherwise explicitly stated herein.

[0046] In some embodiments, “transport” and “delivery” as described herein are used interchangeably unless otherwise explicitly stated herein.

[0047] In some embodiments, “penetration” and “penetrate” as described herein are used interchangeably unless otherwise explicitly stated herein.

[0048] In some embodiments, “airflow sensor” and “flow meter” as described herein are used interchangeably unless otherwise explicitly stated herein.

[0049] In some embodiments, “suspended particles” and “particles” as described herein are used interchangeably unless otherwise explicitly stated herein.

[0050] In some embodiments, an in vitro model as described herein comprises a microfluidic device of the present disclosure or a Transwell unless otherwise explicitly stated herein.

[0051] In some embodiments, the cells can be human small airway epithelial cells, NuLi-1 cells, CuFi-1 cells, Calu-3 epithelial cells, A549 cells, and / or human intestinal epithelial cells, although the present disclosure is not limited thereto.

[0052] In some embodiments, the culture medium can be PneumaCult Ex Plus medium, airway epithelial cell basal medium, bronchial epithelial cell basal medium, F12K medium, Eagle’s Minimum Essential Medium, and / or Dulbecco’s Modified Medium, although the present disclosure is not limited thereto.

[0053] Materials and Methods

[0054] The present disclosure establishes a human bionic lung-on-a-chip system, which can be used to predict inhaled drug kinetics, comprising an air pump, an airflow controller module, a microphysiological module, and an air filter. The air pump can be a diaphragm pump, but the present disclosure is not limited thereto. The airflow controller module comprises a speed controller, such as but not limited to an airflow control valve (or called a throttle valve or a flow rate controller), an airflow one-way valve (or a check valve), and a fan heater (or called a warmer or a flow rate controller). The microphysiological module comprises an incubator, a nebulizer (or an aerosol generator), a central diffuser matched with the nebulizer, a microfluidic device, and an airflow sensor.

[0055] In some embodiments, the chip device with microfluidic channels can be a lung-on-a-chip and / or a breath-mucocilliary-on-a-chip, but the present disclosure is not limited thereto.

[0056] Preparation of a chip device with microfluidic channels

[0057] In some embodiments, the chip device with microfluidic channels (i.e., the microfluidic device of the present disclosure) is assembled by two polycarbonate (PC) chips, containing a first microfluidic channel and a second microfluidic channel adjacent and parallel to each other, wherein the first microfluidic channel is overlaid on the second microfluidic channel. The height of the first microfluidic channel can be 200 pm to 2000 pm, and the width can be 100 pm to 2000 pm; and the height of the second microfluidic channel can be 200 pm to 2000 pm, and the width can be 100 pm to 2000 pm, but the present disclosure is not limited thereto. The first microfluidic channel and the second microfluidic channel are prepared by injection molding (BIGBRIGHT Precision Machinery, Taiwan), and are separated by a porous polyethylene terephthalate (PET) film between them. The pore size of the porous PET film can be 0.4 pm to 8 pm. The first microfluidic channel and the second microfluidic channel can simulate the air layer and the blood vessel layer of the small airway of the human lung, respectively. First, the PC chips and the PET film are surface treated by oxygen plasma (60 W; 500 mTorr; 2 min) to increase the content of oxygen groups on the surface of the PC chips and the PET film. Then, the PC chips and the PET film are chemically modified, wherein the PC chips are modified with 3% (3-glycidyloxypropyl) trimethoxysilane (Glymo) (Sigma-Aldrich, US); and the PET film is modified with 3% (3-aminopropyl) triethoxysilane (APTES) (Sigma-Aldrich, US). Finally, the PET film is overlaid on the PC chip with the second microfluidic channel, and the PC chip with the first microfluidic channel is overlaid on the PET film, and the PC chip with the first microfluidic channel, the PET film and the PC chip with the second microfluidic channel are clamped with iron sheets (0.9 N / m of torsion) and placed in an oven (80°C) for heating for 40 minutes to dry the excess moisture of the chip device, and finally the chip device is cooled to room temperature after the drying process.

[0058] Establishment of a system for predicting inhaled pharmacokinetics

[0059] The system for predicting inhaled drug kinetics was driven by inExpose (SCIREQ, Canada), which is a pump of the present disclosure, containing a diaphragm gas pump that can output a constant wave or a sinusoid wave simulating human respiration. The airflow output by the diaphragm gas pump and circulating in the system can only flow in one direction. To prevent the airflow from flowing in the opposite direction, a straight speed controller (PSA6D, Airtac) and a one-way valve (CVPU4-4, PISCO) were installed in the system to stabilize the airflow and adjust the gas extracted from the incubator (temperature 37°C; relative humidity 95%; CO2 content 5%) to meet the respiratory airflow frequency, flow size or wave pattern of the human lung microphysiology, which can be used for cell culture or as a delivery power of inhaled drugs. The delivery of inhaled drugs was generated by a commercial nebulizer (ANP-1100, Lab), which can fill the medication reservoir of the nebulizer with at least 200 μL of inhaled drugs, and use FlexiWare8 software (SCIREQ) to set the duration and duty cycle of the nebulizer each time. The aerosol after nebulization of the inhaled drugs is input into the aerosol diffuser through the bionic respiratory airflow to stabilize the flow of the aerosol, so that the aerosol is delivered into the microfluidic device.

[0060] Establishment of a microfluidic device for simulating small airways of human lungs

[0061] First, the primary human small airway epithelial cells (HSAEC) (ATCC, PCS-301-010) were cultured in T-75 cell culture bottles using PneumaCult Ex Plus medium (StemCell, 05040) medium, and when the cell number reached 80-90% of the T-75 cell culture bottle, the cells were separated from the surface of the T-75 cell culture bottle using a separation solution, and after centrifugation, fresh medium was added, and the cell concentration of the cell suspension was adjusted to 6 x 10 6cells / mL. Next, the cells were incubated for 4 hours in the first microchannel of the microfluidic device, after which the cells not adhered to the cell culture flask were flushed and removed. The second microchannel was then connected to a gas pump via a hose line, and the culture medium was continuously supplied (flow rate of 120 μL / hr), and the medium in the first microchannel was replaced every 24 hours. After 72 hours of culturing the cells, the first microchannel was converted to a state with a gas-liquid interface, and the epithelial cells were cultured for a long period (4 to 5 weeks) to differentiate cilia. During the long-term culture, the surface of the small airway epithelial cells was washed 1 to 2 times per week with cell buffer (DPBS) (100-200 μL) to remove excess cell debris and mucus.

[0062] Particle exposure method

[0063] The chip culture platform was set up on a microscope observation stage. Since the state of lung cilia waving is greatly affected by temperature, in order to maintain the state of epithelial cells in the chip close to the physiological conditions of the human body when monitoring outside the incubator, a temperature control system for the in vitro chip was set up, and the temperature in the observation platform was maintained at 35.5°C to 37.5°C during the time of giving the respiratory situation. In terms of humidity control, air from the 95% humidity in the cell culture incubator was used for exposure experiments, but due to the temperature difference between the incubator and room temperature, the humidity in the air was lost a little. In order to maintain the humidity of the air, the nebulizer was set to a working period of 10% during the exposure process. The nebulizer model used in this paper is ANP-1100 The mass median aerodynamic diameter (MMAD) of the generated aerosol was 1.8 μm, and the volume median diameter (VMD) was between 2.5 μm and 4.0 μm.

[0064] Establishment of a microfluidic device for simulating inhaled particle deposition

[0065] To stably and long-term perform gas exposure aerosol on the microfluidic device, the incubator and gas exposure support of the microphysiological module were designed using SolidWorks and 3D printed (C30, QTS) using polylactic acid (PLA). When performing gas exposure, the nebulizer can be placed on the gas exposure support and the microfluidic device can be placed on the incubator. The first microfluidic channel of the microfluidic device is connected to the central diffuser via a hose line, and the working cycle of the nebulizer is controlled by the gas exposure machine (5%; that is, 50 ms of gas exposure behavior in 1000 ms (each working time length)) to perform gas exposure. Before performing gas exposure, the membrane of the microfluidic device can be coated with extracellular matrix (ECM) as a simulated small airway mucus layer to facilitate subsequent analysis of the deposition of inhaled particles in the small airway. The preparation of artificial mucus (AM) that meets the physiological characteristics is configured with 4% (w / v) mucin MUC5B and MUC5AC solution (pH = 7.4), respectively, and 4% PEG-solution (pH = 7.4) is used as a crosslinking agent. MUC5B, MUC5AC and 4% PEG-solution are mixed in a ratio of 3:1:4, and continuously stirred at room temperature for 18 hours to make it into a gel before being injected into the first microfluidic channel of the microfluidic device.

[0066] Method of immunofluorescence staining

[0067] Before staining, the human lung small airway tissue can be washed twice with DPBS to remove the culture medium and fixed with 4% paraformaldehyde (PFA). It is noted that if mucus staining is to be performed, the washing step is not needed and the tissue is directly fixed with 10% acetate. After the tissue is fixed, the tissue is soaked in blocking buffer for 30 minutes to cover the nonspecific binding sites on the cell surface of the small airway tissue. Then, the primary antibody is used to label the biomarkers on the cell surface of the tissue, including tight junction protein (ZO-1), cilia marker (Ac-tub), and mucin (MUC5B), and placed at 4°C (overnight). Before the secondary antibody (Alex 488 and Cy3) is used to label the biomarkers on the cell surface of the tissue, the tissue is again washed twice with DPBS to remove the excess primary antibody. After the tissue is incubated with the secondary antibody solution at room temperature for 2 hours, the excess unbound secondary antibody is washed away with DBPS, and the fluorescent mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) is added to mount the tissue on a glass slide and stain the cell nucleus. For live cell staining of the tissue, the 5 μΜ Calcein-AM live cell fluorescent stain (ABP, A017) is used to treat the tissue for 30 minutes, and then the fluorescent particles with a particle size of 100 nm (used to mark the mucus layer of the present disclosure) are exposed to the tissue for 3 minutes to locate the thickness of the mucus layer of the microfluidic device, and finally the confocal microscope is used for imaging.

[0068] Quantification of the frequency of cilia beating

[0069] To record and quantify the frequency of cilia beating, video recording can be performed. Before recording, the temperature of the microscope stage is maintained at 37°C using a warmer to ensure that the ciliated cells are in the physiological temperature range of the human body, and a 20x microscope objective is used to find the cell area showing the cilia marker. The contrast of the image is then adjusted by the high-speed camera (SP 150) of the Sage Vision bright field imaging system and the SGview software, so that the cilia beating trajectory can be clearly observed. The parameters of the video are then adjusted, including adjusting the image exposure time to 10 ms, the resolution to 1440 x 1080 pixels, the recording length to 10 seconds, and the frame rate (fps) to 100 frames / second. Finally, the recorded video of the cilia beating can be analyzed by the cell area showing the cilia marker and its beating frequency (Hz) using the Matlab software.

[0070] Analysis of mucociliary clearance (MCC) function

[0071] To visualize the rate of mucociliary clearance, first, a 3-minute nebulization of a 0.04% (w / v) 2 pm FluoShperes (F8826, Invitrogen) in DPBS solution was performed to deposit the FluoSpheres on the mucus layer of the microfluidic device with small airway tissue and placed in an incubator for at least 10 minutes. Then, the microfluidic device with small airway tissue was removed and placed on a microscope stage for observation of the mucociliary clearance function and video recording for 3 to 10 minutes with NIS software (Nikon) at a resolution of 536 x 536 pixels. The video of the mucociliary clearance was also analyzed by the software to present the particle (i.e., the nebulized 0.04% (w / v) 2 pm FluoShperes (F8826, Invitrogen) in DPBS solution) clearance trajectory, quantify the clearance rate, and direction.

[0072] Exposure efficiency of aerosol deposition

[0073] To visualize the transport and deposition of aerosol in the microfluidic device, a starting solution (such as, but not limited to, 1 mL of 200 pg / mL fluorescein sodium salt (FSS) solution or 0.04% (w / v) carboxylate- polystyrene (PS) (100 nm) suspension particles (580 / 605) (F8801, Invitrogen)) was added to the reservoir chamber of the nebulizer (Omni Lab, ANP-1100) and the nebulized starting solution was delivered into the first microchannel of the microfluidic device for 15 minutes, wherein the nebulizer was operated at a cycle time of 1000 milliseconds and a duty cycle of 5% (i.e., 50 ms of air exposure for every 1000 ms of operation). After the air exposure of the aerosol, the microfluidic device was removed from the air exposure dynamic circuit system of the present disclosure and 100 pL of deionized water was added to the first microchannel of the microfluidic device (dissolved for 3 to 5 minutes) to extract the deposited aerosol and collect the solution containing the deposited aerosol. The fluorescence intensity of the solution was measured by an enzyme labeler (Awareness Technology, ChroMate 4300) using enzyme-linked immunosorbent assay (ELISA). Finally, a standard curve of the concentration of the fluorescein sodium salt in the solution versus the fluorescence intensity was prepared using Prism 9 (GraphPad) and the above-mentioned standard curve was interpolated to the total amount of the deposited aerosol in the solution.

[0074] Visualization of aerosol deposition

[0075] To visualize the deposition of aerosol, after aerosol deposition on the first microfluidic channel of the microfluidic device, the microfluidic device was placed in a custom holder inside a high content system (HCS) (Molecular Devices) and the whole first microfluidic channel of the microfluidic device was imaged using a Molecular Devices ImageXpress Micro4. Finally, the MetaXpress software (Molecular Devices) was used to stitch the captured images of the first microfluidic channel of the microfluidic device together to evaluate the aerosol distribution in the first microfluidic channel.

[0076] Analysis of liposome penetration

[0077] To investigate the effect of shear stress on the penetration of inhaled particles into the mucus layer, after delivering DiI-labeled liposomes (Taiwan Liposome Company) to the surface of the cell layer (e.g., but not limited to HSAEC) using the above-mentioned method of gas-exposed aerosol, the cells were fixed and immunofluorescently stained. Then, three-dimensional images of the cells were taken by a confocal microscope (40x magnification), in which the side view of the three-dimensional images could observe the penetration depth (Z-axis) and location of the liposomes, and the three-dimensional cell images were analyzed using MATLAB program code, the upper and lower boundaries of the cell layer could be identified by the fluorescent signal of DAPI, so that the cell layer could be equally divided into 5 sections (subsections) along the Z-axis, and the distribution of the fluorescent signal of the liposomes at different depths could be calculated. Finally, the proportion of the fluorescent signal of each subsection to the total fluorescent signal was quantified.

[0078] Establishment of a model for in vitro release (IVR) of inhaled particles

[0079] Release analysis of inhaled particles can be performed by establishing an in vitro model of release and comparing the release of inhaled particles (e.g., aerosol) in the in vitro model of the present disclosure (i.e., microfluidic device for mimicking human lung small airway) and a conventional Transwell. Prior to aerosolization, the microfluidic device for mimicking human lung small airway / Transwell is removed from the tubing line connected to the peristaltic pump to maintain the second microfluidic channel of the microfluidic device or the downstream channel of the Transwell at static, and 200 pL and 700 pL of fresh medium is added to the second microfluidic channel and the downstream channel of the Transwell, respectively, to ensure cell survival. During aerosolization, a flow of air (100 mL / min) is provided to the first microfluidic channel of the microfluidic device for 3 min to facilitate deposition of the aerosol (e.g., but not limited to, lipid nanoparticle) in the first microfluidic channel of the microfluidic device; at the same time, the Transwell is placed under the nebulizer to allow the aerosol (e.g., but not limited to, liposome) to be deposited on the mucus layer of the Transwell by sedimentation. After deposition of the aerosol (e.g., but not limited to, liposome), the microfluidic device is immediately (e.g., but not limited to, within 1 min) exposed to a mimicking breathing airflow (50 mL / min) (i.e., dynamic chip set of the present disclosure) or not exposed to a mimicking breathing airflow (50 mL / min) (i.e., static chip set of the present disclosure), and both chip sets are placed in an incubator (37 °C; 5% CO2). The Transwell is not exposed to a mimicking breathing airflow (50 mL / min) (i.e., Transwell set of the present disclosure), and it is placed in an incubator (37 °C; 5% CO2). At six time points (i.e., 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h) after deposition of the aerosol (e.g., but not limited to, liposome), the dynamic chip set, the static chip set, and the Transwell set are removed from the incubator, and 200 pL of medium from the second microfluidic channel of the dynamic chip set / static chip set or the downstream channel of the Transwell set is collected, and an equal volume of fresh medium is added to the second microfluidic channel of the dynamic chip set / static chip set or the downstream channel of the Transwell set. At the 24 h time point after deposition, the mucus layer and cell layer of the dynamic chip set, the static chip set, and the Transwell set are removed, the mucus layer of each set is extracted using 6.5 mM dithiothreitol (DTT) solution, and after 30 min (37 °C) of DTT solution addition, the DTT solution is removed, and the cell layer of each set is washed twice with DPBS; then, 1% Triton X-100 is added to extract the cell layer (for 10 min), and it is washed twice with DPBS to complete the extraction of the cell layer (i.e., the cells are washed down from the chip or Transwell, and the cell layer and its liquid are aspirated).Before conducting the analysis of the release of the inhalable particles in the in vitro model, all the extracted mucus and cell layers were stored at -20°C and were thawed at the time of analysis. When conducting the analysis of the release of the inhalable particles in the in vitro model, the concentration of the aerosol (i.e., comprising the DiI-labeled liposomes and the calcein loaded therein) in the extracted mucus or cell layers was quantified using a microplate reader (CLARIOstar, BMG Labtech). The extracted mucus or cell layers were added to a 96-well plate (100 μL / well) (165305, Nunc), and a reader was placed in each well and the corresponding fluorescence excitation and emission filters were configured (DiI: λex: 548 / 8 nm; DiI: λem: 567 / 8 nm; calcein: λex: 493 / 8 nm; calcein: λem: 527 / 8 nm) to measure the fluorescence intensity of the extracted mucus or cell layers in each well by the reader. Finally, the measured fluorescence intensity was converted to the relative concentration (%) using a standard curve.

[0080] Correlation of the release fraction of the inhalable particles in the in vitro model and the in vivo model

[0081] The results of the release of the inhalable particles in the in vitro model according to the present disclosure were compared with the results of the in vivo drug absorption. The results of the in vivo drug absorption were obtained from the HCQ liposome absorption experiment in mice. The results of the release of the inhalable particles in the in vitro model were compared with the results of the in vivo drug absorption using noncompartmental analysis (NCA). The area under curve (AUC) of the cumulative 0.25 hours, 1 hour, 4 hours, and 24 hours after the release / absorption of the inhalable particles or the drug was used to calculate the release concentration in the in vitro model and the in vivo model. Finally, Pearson correlation analysis was performed to obtain the correlation (R 2 ) of the release fraction of the in vitro model and the in vivo model results.

[0082] Results

[0083] Functional validation of the system for predicting inhalable pharmacokinetics

[0084] The present disclosure provides a system for mimicking human small airway, which integrates micro-engineering technology with aerosol exposure platform and lung-on-a-chip to simulate and analyze the behavior of inhaled drug delivery to the mimicked human small airway environment. As shown in the left part of FIG. 1A, human respiratory patterns include from slow and gentle breathing pattern at rest to rapid and fast breathing pattern during exercise, etc. As shown in the right part of FIG. 1A, the side view of the microfluidic device 122 shows its structure and composition, including dynamic mimicked breathing airflow into the first microfluidic channel 1221A, the first substrate 1221 with the first microfluidic channel 1221A, mucus layer, cell layer (e.g. including but not limited to lung epithelial cells), thin film layer 1220, the second substrate 1222 with the second microfluidic channel 1222A, and culture medium 124. As shown in FIG. 1B, the system 1 for predicting pharmacokinetics includes pipeline 13, incubator 126, nebulizer 120, liquid storage chamber 1200, central diffuser 121, microfluidic device 122, cells 123 (not shown in FIG. 1B), airflow sensor 125, air filter 14, pump 10, speed controller 110, one-way valve 111, and warmer 112. After the airflow passes through the airflow control module 11 (i.e. airflow speed controller 110, one-way valve 111, and warmer 112), it can be stably outputted to the microphysiological module 12 (i.e. incubator 126, nebulizer 120, liquid storage chamber 1200, central diffuser 121, microfluidic device 122, cells 123 (not shown in FIG. 1B), and airflow sensor 125) as a one-way airflow with half-sine waveform. Referring to the shear stress in each branch of human small airway (all less than or equal to 1 Pa), a sine-wave airflow with a frequency of 12 breaths / min is introduced into the microfluidic device 122; and the airflow flow rate is two modes, 0 mL / min to 2.5 mL / min (airflow flow rate mode one) and 0 mL / min to 16 mL / min (airflow flow rate mode two), to ensure that the airflow can stably pass through the first microfluidic channel 1221A of the microfluidic device 122 in different modes. When the introduced airflow flow rate mode reaches the peak value (i.e. airflow flow rate is 2.5 mL / min and 16 mL / min), the shear stress corresponding to the first microfluidic channel 1221A of the microfluidic device 122 is 0.11625 Pa and 0.744 Pa, respectively. It should be noted that since the airflow flow rate of 2.5 mL / min is close to the limit that can be stably controlled by the speed controller 110, in order to stably introduce the target airflow waveform (e.g. but not limited to sine waveform) into the first microfluidic channel 1221A of the microfluidic device 122, the airflow flow rate of 0 mL / min to 2.5 mL / min is set as the parameter of the smaller airflow flow rate mode (i.e. airflow flow rate mode one).In addition, in order to solve the stability problem of the aerosol inhalation drug, the present disclosure provides an aerosol support in the system, which can be used to support the atomizer 120 and the central diffuser 121, so that the atomizer 120 and the central diffuser 121 can be stably placed in the biosafety cabinet (BSC) to carry out the aerosol inhalation of the drug, so as to keep the environment of the operation of the aerosol sterile and the operation of the experimental personnel safe (the leaked inhalation drug is intercepted by the filtering equipment of the biosafety cabinet).

[0085] Test of microfluidic device for simulating inhalation particle deposition

[0086] Since the mucus layer on the surface of the cell layer (for example, but not limited to, the small airway of the human lung) of the microfluidic device is the first biological barrier encountered by the inhalation particles entering the first microchannel of the microfluidic device, and the viscosity of the mucus layer affects the deposition (for example, the inhalation particles are deposited in the mucus layer) and release (for example, the release of the model drug calcein from the inhalation particles in the present disclosure) of the inhalation particles, in order to test the credibility of the microfluidic device for simulating inhalation particle deposition, artificial mucus (AM) is coated in the first microchannel of the microfluidic device by using a synthetic material, and the composition of mucin in the artificial mucus is similar to that of healthy human tracheal mucus (the ratio of MUC5B and MUC5AC is 3:1). Figure 2A shows a schematic diagram of delivering inhalation particles into the microfluidic device 122. As shown in Figure 2B, the viscosity of the artificial mucus can decrease with the increase of the applied shear rate, showing the phenomenon of shear thinning, which is consistent with the characteristics of human mucus, wherein the viscosity measurement uses a logarithm-frequency sweep mode, and there are 10 data points between each 10-fold difference on the logarithmic scale, to obtain the change of the viscosity of the artificial mucus under the action of shear stress at low shear rate (LSS; 1 to 10 1 / s) and high shear rate (HSS; 10 to 100 1 / s). Then, the atomized sodium fluorescein saline solution is aerosolized in the first microchannel 1221A of the microfluidic device 122 to analyze the distribution and deposition of the actual aerosol in the microfluidic device 122. As shown in Figure 2C, the fluorescence performance of the aerosolized sodium fluorescein salt deposited in the first microchannel 1221A of the microfluidic device 122 under the shear stress of HSS is higher than that under the shear stress of LSS; and as shown in Figure 2D, the quantified results can further determine the results of Figure 2C.

[0087] Establishment of microfluidic device for mimicking human small airway and its application for clearance of inhaled drugs

[0088] The left part of FIG. 3A shows a schematic diagram of the microfluidic device for mimicking human small airway, including a first microfluidic channel and a second microfluidic channel. The first microfluidic channel includes an air layer, a mucus layer, and a cell layer. The second microfluidic channel includes a culture medium layer, which can be used to simulate human microvessels. As shown in FIG. 3B, after culturing human small airway cells in an environment with an air-liquid interface (ALI) for 28 days, the cells can be well differentiated, the tight junction protein (ZO-1) presents a complete fishnet-like pattern, and cilia structures (Ac-Tub) are successfully differentiated. The conventional immunostaining method requires separating the bonded upper and lower PC chips, i.e., separating the PC chip with the first microfluidic channel and the PC chip with the second microfluidic channel of the present disclosure, removing the PET film, and then fixing and staining the cell layer and the mucus layer. Since the structure of the cell layer and the mucus layer can be easily damaged during the process, and the mucus layer has fluidity, a live cell stain (calcein-AM) can be used to stain the cell layer. The live cells can absorb colorless calcein-AM, and through the enzyme in the live cells, the AM group is removed to produce green fluorescent calcein, which is used to present the thickness of the cell layer. The mucus layer can be exposed to negatively charged 100 nm PS suspended particles (red fluorescent), the size of the PS suspended particles (or PS particles) can pass through the pore structure of the mucus layer, but is repelled by the electrostatic force between the negatively charged mucus layer and the cell membrane, so that the PS suspended particles (or PS particles) are deposited in the mucus layer and are not easy to penetrate into the cell layer, and thus can be used to present the thickness of the mucus layer. As shown in the right part of FIG. 3A, the thickness of the fixed cell mucus layer observed by the conventional immunostaining method is about 5 to 10 μm; and the thickness of the live cell mucus layer observed by the live cell stain staining and PS suspended particle (or PS particle) exposure is about 10 to 50 μm. As shown in FIG. 3C, the shape of the mucus layer does not differ between the conventional immunostaining method or the live cell stain and PS suspended particle (or PS particle) exposure method, and both methods can observe filamentous mucus (white arrow) structures and mucus (yellow arrow) lumps.

[0089] Results of mucus-cilia clearance under different breathing modes

[0090] [Corrected according to Rule 91 on 23.07.2024] Previous studies have shown that mucociliary clearance can correspond to different breathing patterns, resulting in periodic clearance trajectories of inhaled particles, and that the rate of mucociliary clearance under more vigorous breathing patterns such as coughing or breathing is 27.2% higher than under static breathing patterns. In some embodiments, the shear stress of the HSS can simulate more vigorous breathing patterns such as coughing or breathing, while the shear stress of the LSS can simulate the breathing patterns of the human body under most circumstances. FIGS. 4A-4B show the clearance trajectories of 2 pm PS suspended particles (or PS particles) in the mucus layer, and the differences in mucociliary clearance rates of the microfluidic device under the no air flow mode (w / o AF), the simulated static breathing mode (LSS), and the simulated vigorous breathing mode (HSS). FIG. 4A shows the clearance trajectories of fluorescent PS suspended particles (or PS particles) under the three breathing modes described above (i.e., w / o AF, LSS, and HSS) within 100 seconds, where the color of the clearance trajectory represents the change in time, and the color change corresponding to 0-100 seconds is red, green, yellow, and white in order; and FIG. 4B shows the relationship between the movement distance of fluorescent PS suspended particles and the corresponding change in time, according to the clearance trajectories of PS suspended particles, the PS suspended particles under the shear stress of the HSS can have periodic clearance trajectories corresponding to the change in shear stress, whereas no significant periodic clearance trajectories were observed under the no air flow mode (w / o AF) or the simulated static breathing mode (LSS). As shown in FIG. 4B, the rate of mucociliary clearance of the microfluidic device under the simulated static breathing mode (LSS) and the simulated vigorous breathing mode (HSS), i.e., under the action of shear stress, is significantly higher than that of the microfluidic device under the no air flow mode (w / o AF). However, there is no significant difference in the frequency (Hz) of cilia oscillation of the microfluidic device under the no air flow mode (w / o AF) or the air flow mode (w / AF) and the simulated vigorous breathing mode (HSS).

[0091] Penetration of inhaled particles

[0092] To analyze the penetration behavior of inhaled particles (such as, but not limited to, PS suspension particles) and drugs (such as, but not limited to, DiI-labeled liposomes) in the human lung small airway tissue (such as, but not limited to, small airway epithelial tissue) of the microfluidic device under the action of horizontal shear stress, two types of nanoscale particles with different surface properties were used, including hydrophilic 100 nm PS suspension particles (i.e., carboxylate-modified PS microspheres) and hydrophobic DiI-labeled liposomes, and the PS suspension particles and DiI-labeled liposomes were exposed to the first microfluidic device of the microfluidic device. After the PS suspension particles and DiI-labeled liposomes were deposited on the surface of the small airway tissue (such as, but not limited to, small airway epithelial tissue), the breathing mode (shear stress of the HSS or LSS described in the present disclosure) was continuously maintained for 10 to 20 minutes, and then the small airway tissue (such as, but not limited to, small airway epithelial tissue) was fixed, and the penetration behavior of the above two types of nanoscale particles in the small airway tissue (such as, but not limited to, small airway epithelial tissue) under the action of horizontal shear stress was observed by confocal microscopy. FIG. 5A shows a schematic diagram of the penetration behavior of nanoscale particles (such as, but not limited to, liposomes) in the small airway tissue under the action of different shear stresses. As shown in FIG. 5B, the PS suspension particles (or PS particles) (red fluorescence) and the DiI-labeled liposomes (red fluorescence) did not have obvious penetration behavior under the action of the shear stress of the LSS, i.e., the two types of nanoscale particles were all retained in the mucus layer (green fluorescence), however, under the action of the shear stress of the HSS, the DiI-labeled liposomes (red fluorescence) had obvious penetration behavior, i.e., the depth was significantly increased, and the red fluorescence signal of the DiI-labeled liposomes could be observed in the cell layer (blue fluorescence), while the PS suspension particles did not have obvious penetration behavior, which was speculated to be because the PS particles themselves were negatively charged, and the negative repulsion with the mucus layer caused them to be unable to penetrate smoothly through the airflow, while the neutral DiI-labeled liposomes accelerated the penetration rate of the mucus under the action of shear stress, and then reached the cell layer, which indicated that shear stress helps to accelerate the penetration effect of neutral labeled liposomes.

[0093] Release / absorption of inhaled particles

[0094] To analyze whether the release of inhaled particles (e.g., but not limited to, liposomes) in the microfluidic device is affected by the horizontal shear stress (i.e., in the presence of air flow), calcein was used as a model drug encapsulated in DiI-labeled liposomes (DiI-calcein liposomes), and the release of calcein from DiI-labeled liposomes was tracked by quantifying the change in fluorescence intensity of calcein (see FIG. 7A). FIG. 7B compares the final cumulative fraction of calcein in the mucus layer, cell layer, and medium layer 24 hours after air exposure of DiI-calcein liposomes in the traditional static drug release method (Transwell), in the model without air flow (static chip), and in the model with air flow (dynamic chip). FIG. 6A and FIG. 6B show the amount of DiI-labeled liposomes and calcein deposited in the Transwell, static chip, and dynamic chip, respectively. As shown in FIG. 6A, there was no significant difference in the amount of DiI-labeled liposomes (containing calcein) deposited in the first microfluidic channel of the Transwell and / or static chip. After 24 hours of air exposure of DiI-calcein liposomes, the results of the cumulative distribution of calcein showed that the penetration of calcein was higher in the Transwell and static chip models than in the dynamic chip model; the penetration of calcein in the dynamic chip model was only 1.97%, which was significantly different from the results in the Transwell and static chip models (p = 0.0004 for the Transwell; p = 0.006 for the static chip), and about 87.69% of calcein remained in the mucus layer of the dynamic chip (see FIG. 7B), which was similar to the results of the in vivo model described above. FIG. 7C shows the Calcein / DiI (C / D) ratio in the Transwell, static chip, and dynamic chip 24 hours after air exposure of DiI-calcein liposomes, in which the C / D ratio in the mucus layer of the dynamic chip was much higher than that of fresh DiI-calcein liposomes (i.e., DiI-calcein liposomes mixed in fresh medium) (see FIG. 7C), indicating that dynamic shear stress (i.e., in the model with air flow described in the present disclosure) can increase the release rate of drugs from carriers, while the calcein C / D ratio in the Transwell and static chip was not different from that of fresh DiI-calcein liposomes (i.e., DiI-calcein liposomes mixed in fresh medium).As shown in FIG. 7D, the calcein concentration in the medium layer over time can compare the difference in the release rate of calcein in the Transwell, static chip and / or dynamic chip mode, the release rate of calcein in the Transwell and static chip mode is obviously higher than that in the dynamic chip mode, wherein the highest concentration of calcein in the Transwell mode (C. max ) is 71.9 times the highest concentration of calcein in the dynamic chip mode, however, calcein in the dynamic chip mode has a more obvious release rate change rate, it is worth noting that after 2 hours of air exposure of DiI-calcein microspheres, the cumulative release rate of calcein in the group of air exposure in the dynamic chip mode is obviously slower than that in the Transwell and static chip mode.

[0095] Establishment of in vitro / in vivo correlation (IVIVC)

[0096] In order to evaluate whether the microfluidic device for simulating human lung small airway of the present disclosure (i.e. lung chip with dynamic airflow) has the ability to predict IVIVC, the results of DiI-calcein microspheres released in vitro models (including Transwell and microfluidic device of the present disclosure (dynamic chip / static chip mode)) are converted into the change of area-under-curve (AUC) over time using non-compartmental model analysis (see FIG. 7E), the AUC curve of in vitro model can reflect the difference in the release rate of calcein from DiI-calcein microspheres in dynamic (dynamic chip) or static (Transwell or static chip) mode, the AUC of Transwell or static chip in static mode changes linearly over time, dynamic chip has a higher AUC change rate from 0 to 4 hours, which makes DiI-calcein microspheres have a higher penetration rate.

[0097] Then, according to the method of level A IVIVC, the AUC curves of in vitro models and the AUC curves of HCQ microspheres absorption results of animal experiments described herein are compared point-to-point, the results show that both present a high correlation (R 2 above 0.9) (see FIG. 7F), therefore, the dynamic chip of the present disclosure can provide a dynamic chip with high in vitro / in vivo correlation, which can be used as a tool to predict the pharmacokinetics of inhaled drugs in vivo.

[0098] The above merely describes preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the present application should be included in the scope of the present application.

[0099] wherein the reference signs are as follows: 1: system for predicting pharmacokinetics 10: pump 11: air flow control module 110: speed controller 111: one-way valve 112: warmer 12: microphysiological module 120: nebulizer 1200: liquid storage chamber 121: central diffuser 122: microfluidic device 1220: thin film layer 1221: first substrate 1221A: first microfluidic channel 1222: second substrate 1222A: second microfluidic channel 123: cell 124: culture medium 125: air flow sensor 126: incubator 13: tube 14: air filter

[0100] Biological material deposit

[0101] None

Claims

1. A system for predicting pharmacokinetics, characterized in that, include: Pump; An airflow control module, coupled to the pump to generate an artificial airflow, includes: Speed ​​controller; and A one-way valve coupled to the speed controller; A microphysiological module, coupled to the airflow control module, for drug delivery via the biomimetic gas flow, and comprising: A nebulizer, used to atomize the drug; and A microfluidic device coupled to the atomizer; and The pipeline connects the pump, the airflow control module, and the microphysiology module to form a closed loop.

2. The system as described in claim 1, characterized in that, The airflow control module also includes a heater.

3. The system as described in claim 1, characterized in that, The microphysiological module also includes: Incubator; A central diffuser, connected to the nebulizer, receives the nebulized drug from the nebulizer and delivers the drug; Cells, which are cultured within the microfluidic device; Culture medium, which is located within the microfluidic device; and An airflow sensor, coupled to the microfluidic device, senses the biomimetic gas flow, wherein the nebulizer, the central diffuser, the microfluidic device, the cells, the culture medium, and the airflow sensor are located in the incubator.

4. The system as described in claim 3, characterized in that, It also includes an air filter, which is coupled to the incubator and the pump.

5. The system as described in claim 1, characterized in that, The microfluidic device includes a first substrate having a first microchannel, a thin film layer, and a second substrate having a second microchannel, wherein the thin film layer is disposed between the first substrate and the second substrate, one side of the thin film layer is in contact with the first substrate, and the other side of the thin film layer is in contact with the second substrate.

6. The system as described in claim 1, characterized in that, The nebulizer includes a reservoir for storing the drug.

7. The system as described in claim 1, characterized in that, The drugs include inhaled drugs and / or nanoscale drugs.

8. The system as described in claim 1, characterized in that, The microphysiological module further includes an aeration support and / or a culture rack, wherein the aeration support supports the nebulizer and the central diffuser, and the culture rack supports the microchannel device.

9. The system as described in claim 1, characterized in that, The first substrate and the second substrate comprise polycarbonate, and the thin film layer comprises porous polyethylene terephthalate.

10. A method for predicting pharmacokinetics, characterized in that, include: Provide the system as described in claim 1; Delivery of drugs to the microfluidic device; The state of the drug in the microfluidic device is analyzed to obtain a first result; as well as The pharmacokinetics are predicted based on the first result.

11. The method as described in claim 10, characterized in that, The drugs include inhaled drugs and / or nanoscale drugs.

12. The method as described in claim 10, characterized in that, The first results include the amount of drug deposited in the mucus layer of the microfluidic device, the cell layer of the cells and / or the culture layer of the culture medium, penetration rate, absorption rate, permeability, release rate, clearance rate, migration rate and / or migration trajectory.

13. The method as described in claim 10, characterized in that, It also includes point-to-point analysis of the first result and the second result obtained from the in vivo drug release model to establish in vitro-in vivo correlation.

14. The method as described in claim 13, characterized in that, The second result includes the deposition amount, penetration rate, absorption rate, permeability, release rate, clearance rate, migration rate, and / or migration trajectory of the drug in the in vivo drug release model.

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