Application of poloxamer in evaluation of permeability of insoluble compound by in-vitro Caco-2 cell model and evaluation method

Abstract

The invention discloses an application of poloxamer in evaluating the permeability of an indissolvable compound by using an in-vitro Caco-2 cell model and an evaluation method of the poloxamer in evaluating the permeability of the indissolvable compound by using the in-vitro Caco-2 cell model. According to the present invention, the solubility of the indissolvable compound in the transport buffer solution when the permeability is evaluated by using the in vitro Caco-2 cell model can be effectively improved, the non-specific adsorption of the indissolvable compound can be effectively reduced, the toxicity on the Caco-2 cell is not generated, the integrity of the Caco-2 cell membrane is not affected, the influence on the P-gp and BCRP functions is not generated, and the application prospect is broad. A new research strategy is provided for in-vitro permeability evaluation of insoluble compounds through a corresponding permeability division standard after the in-vitro Caco-2 cell model is established and poloxamer is added.

Description

Technical Field

[0001] The present invention relates to the field of pharmacokinetics, and in particular to the application of poloxamer in evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model and a method for evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model. Background Art

[0002] Oral administration is one of the most patient-friendly and convenient administration methods. The permeability of a drug is a key factor affecting its oral bioavailability. During the development of oral drugs, it is crucial to accurately predict the absorption degree of the drug in the gastrointestinal tract. Factors affecting oral drug absorption include solubility, dissolution rate, permeability, formulation, and metabolism. With the rapid development of the pharmaceutical industry, pharmaceutical companies have continuously introduced a large number of candidate compounds. Many new drug molecules show an increasing trend in lipophilicity, with larger molecular weights, smaller polarities, and poor solubility in water. Recent studies have shown that approximately 40% of the marketed drugs and nearly 90% of the in - research candidate drugs have problems with poor solubility.

[0003] In the early stage of drug development, in vitro cell models are usually used to evaluate the permeability of compounds. However, for compounds with poor water solubility or strong non - specific adsorption, traditional methods usually cannot conduct permeability studies at the expected concentration in vitro, and strong non - specific adsorption also affects the accuracy of experimental data. The solubility of a drug is an important physicochemical property during its development. Selecting a suitable method to improve solubility to predict drug absorption in vivo in an in vitro cell model is of great significance for subsequent drug development strategies; non - specific adsorption is another important problem faced in the field of drug development. Non - specific adsorption is the problem of compound adsorption to the solid surface caused by electrostatic or hydrophobic interactions, which runs through the entire experimental process of new drug development and significantly affects the accuracy of experimental results. Therefore, molecules with poor solubility and strong non - specific adsorption not only cause difficulties in in vitro and in vivo analysis but also increase the burden of drug development.

[0004] Selecting a suitable pharmaceutical excipient, while effectively improving the water solubility of poorly soluble compounds and reducing their non - specific adsorption, without affecting the accuracy of the in vitro evaluation system, can further understand the pharmacokinetic properties of the drug and provide support for subsequent drug development strategies. However, when the traditional Caco-2 cell model is used to evaluate the permeability of poorly soluble compounds, there are problems such as low solubility and strong non - specific adsorption. Moreover, the drug is prone to non - specific adsorption in a high - salt - concentration buffer, resulting in the accuracy and consistency of experimental data being affected. In addition, during the process of improving the solubility of compounds by traditional methods, it may have a negative impact on the monolayer integrity of the Caco-2 cell membrane and the normal function of transporters, thereby affecting the reliability of experimental results.

[0005] The International Pharmaceutical Excipients Council (IPEC) defines excipients as components in pharmaceutical preparations that have been reasonably and safely evaluated and do not include active ingredients or precursors. The "Drug Administration Law" also defines excipients as excipients and additives used in the production of drugs and the dispensing of prescriptions, which are important components of pharmaceutical preparations. Using excipients as oral bioavailability enhancers to improve solubility is one of the current research hotspots in the field of pharmacy. Applying this feature to in vitro studies can greatly improve the accuracy of predicting the permeability of poorly soluble and highly non-specifically adsorbed compounds. Selecting appropriate pharmaceutical excipients, while effectively improving the water solubility of poorly soluble compounds and reducing their non-specific adsorption, without affecting the accuracy of the in vitro evaluation system, can further understand the pharmacokinetic properties of drugs and provide support for subsequent drug development strategies.

[0006] Cited Documents

[0007] 1. Document 1: Smith, P.L., et al. "The use of the Caco-2 cell line as a model of the intestinal epithelial barrier." Pharmaceutical Research, 1990. This article details the application and limitations of the Caco-2 cell model in drug permeability research.

[0008] 2. Document 2: Artursson, P., Karlsson, J. "Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells." Biochemical and Biophysical Research Communications, 1991. This article explores the relationship between drug solubility and permeability in the Caco-2 cell model.

[0009] 3. Document 3: Kerns, E.H., Di, L. "Drug-like Properties: Concepts, Structure Design and Methods." Academic Press, 2008. This book outlines various methods for optimizing drug solubility and permeability and their applications.

[0010] Problems and Disadvantages in the Background Art

[0011] 1. Solubility problem: As described in Document 1 and Document 2, compounds with poor water solubility in phosphate buffer have low solubility, which limits their testing within the effective concentration range. This can lead to poor performance of poorly soluble compounds in in vitro permeability assessment.

[0012] 2. Non-specific adsorption: Document 3 points out that drugs are prone to non-specific adsorption in high-salt concentration buffers, which affects the accuracy and consistency of experimental data.

[0013] 3. Cell membrane integrity and transporter function: Traditional methods for improving compound solubility may have a negative impact on the monolayer integrity of Caco-2 cell membranes and the normal function of transporters, thereby affecting the reliability of experimental results.

[0014] 4. Lack of corresponding permeability classification criteria after adding solubilizers: In the process of improving compound solubility by traditional methods, no corresponding permeability classification criteria are established, resulting in inaccurate and imprecise permeability assessment results. Summary of the Invention

[0015] To solve at least one of the above technical problems, the present invention provides the use of poloxamer in the assessment of the permeability of poorly soluble compounds in an in vitro Caco-2 cell model. For this purpose, the present invention also provides a method for assessing the permeability of poorly soluble compounds in an in vitro Caco-2 cell model.

[0016] In the first aspect of the present invention, there is provided the use of poloxamer in the assessment of the permeability of poorly soluble compounds in an in vitro Caco-2 cell model. The poorly soluble compound is a compound with a solubility lower than 2 μM in the in vitro Caco-2 cell model permeability assessment system. The in vitro Caco-2 cell model permeability assessment system is the transport buffer used in the permeability evaluation of the in vitro Caco-2 cell model. The transport buffer is Hanks' balanced salt solution (HBSS) of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0017] In some embodiments, the poloxamer is poloxamer 188, and the concentration of the poloxamer is 0.02% - 2% (w / v).

[0018] In some embodiments, the poloxamer is poloxamer 188, and the concentration of the poloxamer is 0.2% - 2% (w / v).

[0019] In some embodiments, the poloxamer is poloxamer 188, and the concentration of the poloxamer is 2% (w / v).

[0020] In some embodiments, the evaluation criterion is the low permeability critical P app value: 0.25×10 -6cm / s, high permeability critical P app Value: 3.85×10 -6 cm / s.

[0021] In some embodiments, the evaluation criterion is the low permeability critical P app Value: (0.25 ± 0.10)×10 -6 cm / s; high permeability critical P app Value: (3.85 ± 0.72)×10 -6 cm / s.

[0022] In a second aspect of the present invention, there is provided a method for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model. Poloxamer is added to the system for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model. The poorly soluble compound is a compound with a solubility lower than 2 μM in the permeability evaluation system of the in vitro Caco-2 cell model. The permeability evaluation system of the in vitro Caco-2 cell model is the transport buffer used in the permeability evaluation of the in vitro Caco-2 cell model.

[0023] In some embodiments, the method for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model includes the following steps:

[0024] S1. Determine whether poloxamer has a solubilizing effect and a reducing non-specific adsorption effect on the poorly soluble compound. If so, continue with the subsequent experiments. The poloxamer is poloxamer 188, and the concentration of poloxamer is 0.02% - 2% (w / v);

[0025] S2. Perform a correlation analysis on the oral absorption percentage of a known model drug in the human body and the apparent permeability coefficient of the known model drug after adding poloxamer to establish a permeability classification criterion for the in vitro Caco-2 cell model;

[0026] S3. Add poloxamer to the system for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model, and evaluate the permeability of the poorly soluble compound in combination with the permeability classification criterion for the in vitro Caco-2 cell model established in step S2.

[0027] In some embodiments, the method for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model includes the following steps:

[0028] S1. Determine whether poloxamer has a solubilizing effect and a reducing non-specific adsorption effect on the poorly soluble compound. If so, continue with the subsequent experiments. The poloxamer is poloxamer 188, and the concentration of poloxamer is 2% (w / v);

[0029] S2. Add poloxamer to the system for evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model, and combine the low permeability critical P app value: (0.25 ± 0.10) × 10 -6 cm / s, and the high permeability critical P app value: (3.85 ± 0.72) × 10 - 6 cm / s to evaluate the permeability of poorly soluble compounds according to the permeability classification criteria of the in vitro Caco-2 cell model.

[0030] In some embodiments, the method for evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model includes the following steps:

[0031] S1. Determine whether poloxamer has a solubilizing effect and a non-specific adsorption reduction effect on the poorly soluble compound. If so, continue with the subsequent experiment. The poloxamer is poloxamer 188, and the concentration of poloxamer is 2% (w / v);

[0032] S2. Add poloxamer to the system for evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model, and combine the low permeability critical P app value: 0.25 × 10 -6 cm / s, and the high permeability critical P app value: 3.85 × 10 -6 cm / s to evaluate the permeability of poorly soluble compounds according to the permeability classification criteria of the in vitro Caco-2 cell model.

[0033] The P app value in the present invention is the apparent permeability coefficient, and Fa is the oral absorption percentage.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) By adding poloxamer in a specific concentration range to the transport buffer in the in vitro Caco-2 cell model, the present invention can effectively improve the solubility of poorly soluble compounds, enabling their permeability evaluation at the expected working concentration and ensuring that the compounds do not precipitate during the experiment. It can also effectively reduce non-specific adsorption, ensure the accuracy of experimental data, and at the same time does not affect the cell integrity, does not affect the functions of P-gp and BCRP, and does not produce toxicity to cells, providing a new research strategy for the in vitro permeability evaluation of poorly soluble compounds.

[0036] (2) The present invention provides a method for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model. By adding a specific concentration of poloxamer, the solubility of poorly soluble compounds is effectively increased and their non-specific adsorption is reduced, without negatively affecting the monolayer integrity and normal function of the Caco-2 cell membrane. A permeability classification standard for the in vitro Caco-2 cell model after adding a specific concentration of poloxamer is established, making this method have good reproducibility and accuracy, and successfully establishing an in vitro permeability evaluation method for poorly soluble compounds.

[0037] (3) The present invention adds a specific concentration range of poloxamer to the transport buffer in the in vitro Caco-2 cell model and provides a method for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model. By increasing the solubility of poorly soluble compounds and reducing non-specific adsorption, it can more accurately predict the absorption and pharmacokinetic properties of poorly soluble compounds in the human body, providing important support for drug screening and development, and improving the efficiency and success rate of new drug research and development.

[0038] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. Description of the Drawings

[0039] Figure 1 is the correlation analysis between the model drug Log P app (A - B) and human Fa in the Caco-2 cell model when containing poloxamer188.

[0040] Figure 2 is the structural formula of zolquidar. Detailed Embodiments

[0041] In order to make the technical means, creative features, achieved purposes and effects of the invention easy to understand, the present invention will be further described below in conjunction with specific illustrations, but the present invention is not limited to the following implemented cases.

[0042] It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they do not have technical essential significance. Any modification of the structure, change of the proportional relationship or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope that the technical content disclosed by the present invention can cover.

[0043] In the current new drug R & D process, the in vitro permeability assessment of compounds is usually carried out in cell models. And to ensure the accuracy and effectiveness of experimental data and avoid the influence of additional additives in the reaction system, buffers such as phosphate buffer are usually used as the incubation system. However, for compounds with poor water solubility, due to the high concentration of salt-type ions in the phosphate buffer, these compounds usually show worse solubility in such a medium. During the in vitro experimental research process, if a compound has poor water solubility, the following problems will be faced: First, the compound cannot be evaluated at the expected working concentration; Second, if the dosing concentration of the compound exceeds its solubility limit, the compound will precipitate during the experimental incubation, and its effective working concentration cannot be evaluated; Third, the non-specific adsorption of the compound on the experimental device and plates is relatively large, seriously affecting the accuracy of experimental data.

[0044] The present invention aims to solve the above technical problems. By selecting a suitable solubilizer, on the basis of effectively improving the solubility of poorly soluble compounds and reducing their non-specific adsorption, while not affecting normal cell functions, a new research strategy is provided for the in vitro permeability assessment of poorly soluble compounds. The present invention screened a series of pharmaceutical excipients, studied the influence on the viability of Caco-2 cells, the solubilization and adsorption reduction effects on poorly soluble drugs, and performed a correlation analysis on the oral absorption percentage of known model drugs in humans and the apparent permeability coefficient of these model drugs in the Caco-2 cell model containing the selected excipients, and successfully established a permeability assessment standard and method for poorly soluble compounds. This standard and method can effectively improve the solubility of poorly soluble compounds and reduce their non-specific adsorption, and has good reproducibility and accuracy, providing a reliable strategy for the in vitro permeability assessment of poorly soluble compounds.

[0045] Starting from the perspective of improving the solubility of compounds, the present invention conducts research on solubilizers, aiming to select a suitable solubilizer. In addition to effectively improving the solubility of poorly soluble compounds and reducing their non-specific adsorption, the solubilizer also needs to meet a series of conditions such as having an impact on Caco-2 cell functions within an allowable range, that is, having no toxicity to Caco-2 cells, not affecting the integrity of the Caco-2 cell membrane, and not affecting the functions of P-gp and BCRP.

[0046] Caco-2 cells are a type of human colon cancer cells. Under specific culture conditions, tight junctions form between Caco-2 cells and they differentiate into cells that are extremely similar in morphology and function to human small intestinal cells, expressing various types of transporters, such as the efflux transporter P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP), etc. Therefore, a monolayer of Caco-2 cells can be used as an in vitro model for studying drug transport in small intestinal epithelial cells. Given the clinical relevance of in vitro models in predicting drug absorption in vivo, the guiding principles of the US Food and Drug Administration and the National Medical Products Administration of China both recommend using the in vitro Caco-2 cell model to predict the oral absorption extent of drugs in humans. The Caco-2 cells in this invention are derived from ATCC (American Type Culture Collection), with the product number HTB-37.

[0047] In this article, the meanings of "Caco-2 cell model" and "Caco-2 cells" are the same, and the meanings of "evaluation criteria", "classification criteria", and "division criteria" are consistent.

[0048] Combined with another invention published by the inventor of this invention, "A Method for Evaluating the Interaction Relationship between Poorly Soluble Compounds and P-Glycoprotein and Breast Cancer Resistance Protein and the Application of Poloxamer 188" (Application No.: CN202410695834.2), it can be known that adding 0.02 - 2% (w / v) of poloxamer 188 to the transport buffer for evaluating the interaction relationship between compounds and P-glycoprotein and breast cancer resistance protein in the Caco-2 cell model can increase the solubility of poorly soluble compounds, reduce non-specific adsorption, and has no toxicity to Caco-2 cells, does not affect the integrity of the Caco-2 cell membrane, and does not affect the activities of P-glycoprotein and breast cancer resistance protein. Therefore, this invention uses adding 0.02 - 2% (w / v) of poloxamer 188 to this transport buffer system to evaluate the permeability of poorly soluble compounds in the Caco-2 cell model and give the evaluation criteria.

[0049] Poloxamer 188 is a white to slightly yellowish translucent waxy solid, with the trade name Pluronic. It is a new type of high molecular non-ionic surfactant, and its chemical formula is H(C2H4O) a (C3H6O) b (C2H4O) aOH is an α-hydrogen-ω-hydroxy poly(oxyethylene)a-poly(oxypropylene)b-poly(oxyethylene)a block copolymer. It is formed by reacting propylene oxide with propylene glycol to form polyoxypropylene glycol, and then adding ethylene oxide to form a block copolymer. In the copolymer, the oxyethylene unit (a) is 75 - 85, the oxypropylene unit (b) is 25 - 30, the ethylene oxide (EO) content is 79.9% - 83.7%, and the average molecular weight is 7680 - 9510. Poloxamer 188 can improve the solubility of compounds by forming micelles. In this invention, the supplier of poloxamer 188 is BASF, and the product number is F68.

[0050] The poorly soluble compound is a compound with a solubility lower than 2 μM in an in vitro permeability evaluation system. In this invention, the permeability of the poorly soluble compound is evaluated in an in vitro Caco-2 cell model using a transport buffer such as phosphate buffer. At this time, the solubility of the poorly soluble compound in the transport buffer is lower than 2 μM, and the concentration of the transport buffer is also the commonly used concentration in in vitro drug research experiments. This invention selects Hanks balanced salt solution containing 10.0 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as the transport buffer.

[0051] Taking zolquidar as an example, this invention improves the solubility and non-specific adsorption of zolquidar through poloxamer188, and further adds poloxamer188 in an in vitro Caco-2 cell model and establishes corresponding evaluation criteria after adding poloxamer188 to evaluate the permeability of zolquidar.

[0052] Using poloxamer188 for the permeability evaluation of poorly soluble compounds requires the following steps:

[0053] 1. Determine the usage concentration of poloxamer188 in the evaluation system

[0054] Screen out the concentration that does not cause toxicity to Caco-2 cells through a lactate dehydrogenase (LDH) cytotoxicity experiment;

[0055] Screen out the concentration that does not affect the integrity of the Caco-2 cell membrane through a fluorescein detection experiment;

[0056] Combining the critical micelle concentration and the self-solubility of poloxamer188, determine the usage concentration range of poloxamer188 in the evaluation system.

[0057] 2. Judge whether poloxamer188 has a solubilization effect and a non-specific adsorption reduction effect on the poorly soluble compound

[0058] Judge the effects of poloxamer 188 on the solubility and non-specific adsorption of the compound with low solubility to be measured within the above-mentioned concentration range. If poloxamer 188 within this concentration range has a solubilizing effect and can reduce non-specific adsorption on the compound with low solubility to be measured, then poloxamer 188 within this concentration range can be used in subsequent experiments.

[0059] 3. Conduct a correlation analysis on the oral absorption percentage of known model drugs in humans and the apparent permeability coefficients of these model drugs in the Caco-2 cell model containing poloxamer 188, and establish a permeability classification criterion for the in vitro Caco-2 cell model containing poloxamer 188.

[0060] 4. Add poloxamer 188 to the evaluation system for the permeability of the compound with low solubility to be measured in the in vitro Caco-2 cell model, and evaluate the permeability of the compound with low solubility to be measured.

[0061] Therefore, the specific experiments involved in the present invention include the following:

[0062] I. Solubility experiment

[0063] Add a certain concentration of poloxamer 188 to the transport buffer (such as phosphate buffer, etc.), and prepare dosing solutions with final concentrations of 2 μΜ, 10 μΜ, 50 μΜ, and 100 μΜ of the compound to be measured. Take the dosing solutions at each concentration as T0 samples, vortex the dosing solutions at 900 rpm and 37.0 °C for 30 minutes, and then centrifuge at 3220 × g and 37.0 °C for 30 minutes. After centrifugation, collect the upper layer samples (S) and the lower layer samples (R) in equal volumes respectively, and analyze all samples by LC-MS / MS.

[0064] The following formula is used to calculate the theoretical concentration dissolution percentage (% Nominal Conc):

[0065] Peak Area Ratio (PAR) = compound peak area / internal standard peak area × dilution factor

[0066] % Nominal Conc = PAR (s) / PAR (T0) × 100%

[0067] where PAR (s) represents the peak area ratio of the upper layer sample, and PAR (T0) represents the peak area ratio of the T0 sample. If % Nominal Conc is greater than 70%, it is considered that the compound is soluble in the matrix (i.e., the transport buffer).

[0068] II. Non-specific adsorption experiment

[0069] Add a certain concentration of poloxamer 188 to the transport buffer (such as phosphate buffer), and prepare a working solution of the compound to be tested with a final concentration of 2.00 μM. The working solution after centrifugation at 3220×g for 20 minutes is used as the dosing solution. Take 250 μL of the dosing solution and transfer it to a 96-well receiving plate, and incubate it for 120 minutes at 37.0 °C, 5.0% CO2 and a relative saturation humidity of 95%, with three parallels for each concentration. Take 1200 μL of the termination solution (acetonitrile solution containing 250 ng / mL tolbutamide) and add it to 400 μL of the transport buffer as a blank sample; take 100 μL of the dosing solution and add it to 300 μL of the termination solution as the T0 sample. After incubating for 120 minutes, all samples are mixed 3 times, then 100 μL of the sample is taken out and transferred to a new 96-well plate containing 300 μL of the termination solution as the dosing-end sample. Remove the remaining samples in the receiving plate and pat them dry, then add 250 μL of the termination solution, mix 3 times, take 100 μL of the sample from the receiving plate and transfer it to a 96-well plate containing 200 μL of the termination solution, and then add 100 μL of the transport buffer as the adsorbed sample. After vortexing all samples, centrifuge them at 3220×g for 20 minutes. Then take out 200 μL of the supernatant and reconstitute it with 200 μL of pure water, vortex for 10 minutes, and analyze the samples using LC-MS / MS.

[0070] The following formula is used to calculate the non-specific adsorption rate (% Binding):

[0071] Peak Area Ratio (PAR) = compound peak area / internal standard peak area

[0072] % Binding = PAR (adsorbed sample) / PAR (T0 sample) × 100%

[0073] If % Binding is greater than 30%, it is considered that the compound has strong non-specific adsorption.

[0074] III. LDH cytotoxicity study of poloxamer 188 on Caco-2 cells

[0075] Seed Caco-2 cells at 1.0×10 5Cells were inoculated into a 96-well cell culture plate at a concentration of

[0076] IV. Study on the integrity of the Caco-2 cell membrane by poloxamer188

[0077] Caco-2 cells were seeded at a density of 1.0×10 5 cells / cm 2 into a 96-well Corning transwell cell plate and cultured for 21 - 28 days to investigate the effect of poloxamer188 on the integrity of the Caco-2 monolayer cell membrane. Hanks balanced salt solution (HBSS) containing 10.0 mM 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES) (pH 7.40 ± 0.05) was used as the transport buffer in this study. Poloxamer188 was dissolved in the transport buffer to prepare the incubation solution with final concentrations of 0.02%, 0.2%, and 2% (w / v). The incubation solution was added to the corresponding wells of the cell plate (75.0 μL and 250 μL were added to each apical and basolateral well, respectively). After adding the samples, the Caco-2 cell plate was incubated at 37.0 °C, 5.0% CO2, and 95% humidity for 120 minutes.

[0078] After incubation, the integrity of the Caco-2 cell layer was detected by Lucifer Yellow Rejection Assay. The remaining solutions in the apical and basolateral wells were removed. 75.0 μL of the transport buffer containing 100 μM Lucifer Yellow was added to the apical well, and 250 μL of the transport buffer was added to the basolateral well. The cell plate was incubated in a cell culture incubator at 37.0 °C, 5.0% CO2, and 95% humidity for 30 minutes. Then, 20.0 μL of the sample was taken from the apical side and mixed with 60.0 μL of the transport buffer, and 80 μL of the sample was taken from the basolateral side. The RFU value was measured using a microplate reader at a wavelength of 425 / 528 nm (excitation / emission).

[0079] The transmittance of Lucifer Yellow (%) is calculated using the following formula

[0080]

[0081] where RFU Apical and RFU Basolateral are the relative fluorescence intensities of Lucifer Yellow at the apical and basolateral ends respectively, and V Apical and V Basolateral are the sample volumes at the apical and basolateral ends respectively (0.075 mL at the AP end and 0.25 mL at the BL end).

[0082] If the % Lucifer Yellow value of Caco-2 cells is less than 1.0, it indicates that poloxamer 188 does not affect the integrity of the Caco-2 cell membrane.

[0083] V. Study on the effect of poloxamer 188 on the function of transporters in Caco-2 cells

[0084] Using digoxin as the P-gp substrate at a test concentration of 10 μM and estrone 3-sulfate as the BCRP substrate at a test concentration of 5 μM, a bidirectional transport experiment was carried out in the absence or presence of a certain concentration of poloxamer 188 to investigate the effect of poloxamer 188 on the percentage of transport activity of P-gp and BCRP in Caco-2 cells (%of transport activity).

[0085] The formula for calculating the percentage of transport activity (%of transport activity) is as follows

[0086]

[0087] %of transport activity=(ERT / ERV)×100

[0088] where CR is the average concentration of digoxin or estrone 3-sulfate at the receiver end, VR is the volume of the solution at the receiver end (the volumes at the apical side and basolateral side are 0.0750 and 0.250 mL respectively). ER is the abbreviation of Efflux Ratio, ERT represents the efflux rate of digoxin or estrone 3-sulfate in the presence of the test compound, and ERv represents the efflux rate of digoxin or estrone 3-sulfate in the absence of the test compound.

[0089] VI. Establishment of permeability classification criteria for Caco-2 cell model in the presence of poloxamer 188

[0090] Twenty-seven model drugs with known human oral absorption percentage (Fa) were selected and subjected to bidirectional transport experiments in the Caco-2 cell model in the presence of poloxamer 188. Using Log P app (A - B) and Fa values were subjected to Boltzmann nonlinear fitting. The 50% and 85% oral absorption percentages were used as the critical values for low permeability and high permeability respectively, to establish a new permeability classification criterion for the Caco-2 cell model, that is, the evaluation criterion.

[0091] VII. Evaluation of the permeability of zolaquindar using the Caco-2 cell model in the presence of poloxamer 188

[0092] Using the transport buffer containing poloxamer 188, bidirectional permeability experiments of zolaquindar were carried out in the Caco-2 cell model. The permeability of zolaquindar was evaluated in combination with the permeability classification criterion of the Caco-2 cell model in the presence of poloxamer 188. The evaluation results were compared with the literature results to verify the feasibility and accuracy of this method. At the same time, bidirectional permeability experiments of zolaquindar were carried out in the Caco-2 cell model without poloxamer 188 as a control group, and the obtained experimental results were compared to verify the beneficial effects of this method.

[0093] Taking zolaquindar as an example in the present invention, its structural formula is shown in Figure 2 , due to the combination with the published invention "A method for evaluating the interaction between poorly soluble compounds and P-glycoprotein and breast cancer resistance protein and the application of poloxamer 188", it is known that poloxamer 188 at a concentration of 0.02 - 2% (w / v) can improve the solubility and non-specific adsorption of zolaquindar, and has no toxicity to Caco-2 cells, does not affect the integrity of the Caco-2 cell membrane, and does not affect the activities of P-glycoprotein and breast cancer resistance protein. Considering that the higher the concentration of poloxamer 188, the greater the impact on the solubility and non-specific adsorption of poorly soluble compounds, and the good solubility of poloxamer 188 itself, 2% (w / v) is preferably selected in the present invention. Based on the addition of poloxamer 188 at a concentration of 2% (w / v), the permeability classification criterion of the in vitro Caco-2 cell model at this concentration was established to further evaluate the permeability of zolaquindar in the in vitro Caco-2 cell model. The establishment of the permeability classification criterion of the Caco-2 cell model after adding poloxamer 188 and the evaluation of the permeability of zolaquindar in the Caco-2 cell model in the presence of poloxamer 188 are elaborated in detail below.

[0094] Example 1 Establishment of Permeability Classification Criteria for Caco-2 Cell Model after Adding Poloxamer 188

[0095] 1) Caco-2 cells were inoculated into the wells of a Corning 96-well Transwell cell culture insert at a seeding density of 1.0×10 5 cells / cm 2 . After 21 - 28 days of culture, TEER was measured. If the TEER value > 200 Ω·cm 2 , it indicated that a monolayer-intact cell membrane structure had been formed and could be used for the next bidirectional transport experiment.

[0096] 2) In this technical solution, Hank's balanced salt buffer (HBSS, pH 7.40 ± 0.05) containing 2% (w / v) poloxamer 188 and 10.0 mM HEPES was used as the transport buffer. Nadolol was used as a low-permeability control compound at a test concentration of 2 μM, metoprolol was used as a high-permeability control compound at a test concentration of 2 μM, digoxin was used as a P-gp substrate at a test concentration of 10 μM, and estrone 3-sulfate was used as a BCRP substrate at a test concentration of 5 μM. The test compounds were dissolved in the transport buffer to prepare the dosing solution with a final concentration of the compound of 2.00 μM and a final concentration of DMSO of 0.6%. The transport buffer containing 0.6% DMSO was used as the receiving solution. The dosing solution and the receiving solution were respectively added to the corresponding wells of the cell culture insert (75.0 and 250 μL were added to each apical and basolateral well), and the bidirectional transport experiment was started. After adding the samples, the Caco-2 cell culture insert was placed at 37.0 °C, 5.0% CO2 and 95% humidity and incubated for 120 minutes.

[0097] 3) The initial dosing solution was the T0 sample, which was mixed with the transport buffer and the termination solution in a certain proportion. After 120 minutes of incubation, the final samples were collected from the dosing side and the receiving side, and used as the dosing-side sample and the receiving-side sample respectively, which were also mixed with the transport buffer and the termination solution in a certain proportion. After the dosing-side and receiving-side samples were collected, the cell culture insert was gently tapped, the remaining solution in the cell culture insert was discarded, the termination solution was added to lyse the cells, and the cell lysate was collected after pipetting up and down 5 times and mixed with the transport buffer and the termination solution in a certain proportion.

[0098] 4) After vortexing all the samples, they were centrifuged at 3220×g for 20 minutes at 20.0 °C. An appropriate volume of the supernatant was transferred to the sample analysis plate and analyzed by LC-MS / MS.

[0099] 5) The integrity of the Caco-2 monolayer cell membrane after the transport experiment was detected by Lucifer Yellow Rejection Assay. A fluorescence yellow transmittance of less than 1% indicates good cell membrane integrity. The following formula was used to calculate P app , Efflux ratio, % Solution Recovery, % Total Recovery, and % Lucifer Yellow.

[0100]

[0101]

[0102] V R is the volume of the receiving solution (0.0750 mL for side A and 0.250 mL for side B); Area is the relative surface area of the cell monolayer (0.0804 cm 2 ); Time is the incubation time (the incubation time is 5,400 s); C0 is the peak area ratio of the sample at the start of the compound administration end; V D is the volume of the administration end (0.0750 mL for side A and 0.250 mL for side B); C D and C R are the peak area ratios of the samples at the compound administration end and the receiving end, respectively; C Lysate is the peak area ratio of the sample in the compound cell lysate; V A is the volume of the cell lysate (0.0750 mL). RFU Apical and RFU Basolateral are the relative fluorescence intensities of lucifer yellow at the apical and basolateral ends, respectively. V Apical and V Basolateral are the sample volumes at the apical and basolateral ends, respectively (0.0750 mL and 0.250 mL, respectively).

[0103] Table 1 Model drug information

[0104]

[0105]

[0106] Twenty-seven model drugs with known human oral absorption percentages (Fa) (Table 1) were selected, and a bidirectional transport experiment was carried out in a transport buffer containing 2% (w / v) poloxamer 188. The experiment was carried out three times, and the average Log P app (A - B) was nonlinearly fitted with the Fa value by Boltzmann. The results are as Figure 1As shown, in the presence of 2% (w / v) poloxamer 188, the Log P of the model drug obtained in the Caco-2 cell model app (A - B) has a good correlation with Human Fa (%), and its R 2 value is 0.86, further proving that this method has good reproducibility. Taking Fa values of 50.0% and 85.0% as the critical values for low permeability and high permeability respectively, the P app values in the Caco-2 cell model are respectively (0.25 ± 0.10)×10 - 6 cm / s and (3.85 ± 0.72)×10 -6 cm / s, and their standard deviations are extremely low, further proving that this method has high accuracy.

[0107] Example 2 Evaluation of the permeability of zolquidar using the Caco-2 cell model under conditions containing poloxamer 188

[0108] Using a transport buffer containing 2% (w / v) poloxamer 188 (the same transport buffer as in Example 1), zolquidar was formulated into a working solution with a final concentration of 2.00 μM, and a bidirectional permeability experiment was carried out in the Caco-2 cell model. The experimental results are as follows:

[0109]

[0110] Under the condition of containing 2% (w / v) poloxamer 188, the apparent permeability coefficient of zolquidar in the A - B direction in the Caco-2 cell model is 1.46×10 -6 cm / s. The solution recovery rate of zolquidar in the A - B direction is relatively low, which may be due to the high lipophilicity of zolquidar, resulting in accumulation in Caco-2 cells (Int J Pharm X. 2021;3:100089. Published 2021 Jul 7.). According to the judgment criteria of Example 1, zolquidar can be judged as moderately permeable.

[0111] The results of the bidirectional permeability experiment of zolquidar in a transport buffer without 2% (w / v) poloxamer 188 are as follows:

[0112]

[0113] After adding 2% (w / v) poloxamer 188, the apparent permeability coefficient and recovery rate of zuoquindar in both directions were improved, which might be due to the fact that poloxamer 188 promoted the solubility of zuoquindar and reduced its non-specific adsorption. Combining with the references (Clin Cancer Res. 2004; 10(21): 7220-7228., Clin Cancer Res.

[0114] 2004; 10(10): 3265-3272. and Cancer Chemother Pharmacol. 2005; 56(2): 154-160), the first-pass elimination in the intestine and liver is the main reason for the low bioavailability of zuoquindar. Oral administration of zuoquindar can reach a relatively high plasma drug concentration, that is, the oral absorption of zuoquindar is good. Without 2% (w / v) poloxamer 188, zuoquindar shows low permeability, which might be due to its own solubility limitation and strong non-specific adsorption. The permeability evaluation results are more accurate in the presence of 2% (w / v) poloxamer 188.

[0115] A method for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model specifically includes the following steps:

[0116] 1) Evaluate the solubilization effect and non-specific adsorption reduction effect on the poorly soluble compound by adding poloxamer 188 at a specific concentration (0.02%-2% (w / v)) in the transport buffer.

[0117] 2) If the addition of poloxamer 188 at the specific concentration in step 1) can effectively increase the solubility of the poorly soluble compound (% Nominal Conc > 70%) and reduce its non-specific adsorption (% Binding < 30%), then based on the above concentration, perform a correlation analysis on the oral absorption percentage of the known model drug in humans and the apparent permeability coefficient of the known model drug after adding poloxamer, and establish a permeability classification standard for the in vitro Caco-2 cell model.

[0118] 3) Add the above concentration of poloxamer to the system for evaluating the permeability of the poorly soluble compound using the in vitro Caco-2 cell model, and evaluate the permeability of the poorly soluble compound in combination with the permeability classification standard of the in vitro Caco-2 cell model established in step 3).

[0119] It can also be carried out through the following steps:

[0120] 1) Evaluate the solubilization effect and non-specific adsorption reduction effect on the poorly soluble compound by adding poloxamer 188 at a concentration of 2% (w / v) in the transport buffer.

[0121] 2) If the addition of 2% (w / v) poloxamer 188 can effectively increase the solubility of the poorly soluble compound (% Nominal Conc > 70%) and reduce its non-specific adsorption (% Binding < 30%), then combined with the low permeability critical P app value: (0.25 ± 0.10) × 10 -6 cm / s, high permeability critical P app value (3.85 ± 0.72) × 10 -6 cm / s, the permeability of the poorly soluble compound is evaluated according to the permeability classification criteria of the in vitro Caco-2 cell model.

[0122] It can also be carried out through the following steps:

[0123] 1) Evaluate the solubilization effect and non-specific adsorption reduction effect on the poorly soluble compound by adding 2% (w / v) poloxamer 188 to the transport buffer.

[0124] 2) If the addition of 2% (w / v) poloxamer 188 can effectively increase the solubility of the poorly soluble compound (% Nominal Conc > 70%) and reduce its non-specific adsorption (% Binding < 30%), then combined with the low permeability critical P app value: 0.25 × 10 -6 cm / s, high permeability critical P app value 3.85 × 10 -6 cm / s, the permeability of the poorly soluble compound is evaluated according to the permeability classification criteria of the in vitro Caco-2 cell model.

[0125] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations according to the concept of the present invention without creative labor. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention based on the concept of the present invention through logical analysis, reasoning or limited experiments should be within the protection scope determined by the claims.

Claims

1. Use of poloxamer in evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model, characterized in that, The poorly soluble compound is a compound with a solubility lower than 2 μM in the in vitro Caco-2 cell model permeability evaluation system, and the in vitro Caco-2 cell model permeability evaluation system is the transport buffer used in the permeability evaluation of the in vitro Caco-2 cell model.

2. The application according to claim 1, characterized in that, The poloxamer is poloxamer 188; the concentration of the poloxamer is 0.02%-2% (w / v).

3. The application according to claim 1, wherein The poloxamer is poloxamer 188; the concentration of the poloxamer is 0.2%-2% (w / v).

4. The application according to claim 1, characterized in that, The poloxamer is poloxamer 188; the concentration of the poloxamer is 2% (w / v).

5. The application according to claim 4, characterized in that, The evaluation criteria are as follows: low permeability critical P app value: (0.25 ± 0.10) × 10 -6 cm / s; high permeability critical P app value: (3.85 ± 0.72) × 10 -6 cm / s.

6. The application according to claim 4, characterized in that, The evaluation criteria are the low-permeability critical P app value: 0.25×10 -6 cm / s; the high-permeability critical P app value: 3.85×10 -6 cm / s.

7. A method for evaluating the permeability of poorly soluble compounds using an in vitro Caco-2 cell model, characterized in that, Poloxamer is added to the system for evaluating the permeability of a poorly soluble compound in an in vitro Caco-2 cell model. The poorly soluble compound is a compound with a solubility lower than 2 μM in the in vitro Caco-2 cell model permeability evaluation system, and the in vitro Caco-2 cell model permeability evaluation system is the transport buffer used in the permeability evaluation of the in vitro Caco-2 cell model.

8. The method according to claim 7, characterized in that It includes the following steps: S1. Determine whether the poloxamer has a solubilizing effect and a non-specific adsorption reducing effect on the poorly soluble compound. If so, continue with the subsequent experiments. The poloxamer is poloxamer 188, and the concentration of the poloxamer is 0.02%-2% (w / v); S2. Analyze the correlation between the oral absorption percentage of a known model drug in humans and the apparent permeability coefficient of the known model drug with the addition of the poloxamer, and establish a permeability classification standard for the in vitro Caco-2 cell model; S3. Add the poloxamer to the system for evaluating the permeability of the poorly soluble compound in the in vitro Caco-2 cell model, and evaluate the permeability of the poorly soluble compound in combination with the permeability classification standard of the in vitro Caco-2 cell model established in step S2.

9. The method according to claim 7, wherein It includes the following steps: S1. Determine whether the poloxamer has a solubilizing effect and a non-specific adsorption reducing effect on the poorly soluble compound. If so, continue with the subsequent experiments. The poloxamer is poloxamer 188, and the concentration of the poloxamer is 2% (w / v); S2. Add the poloxamer to the system for evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model, and combine with the low permeability critical P app value: (0.25 ± 0.10) × 10 -6 cm / s, high permeability critical P app value: (3.85 ± 0.72) × 10 - 6 cm / s, and evaluate the permeability of the poorly soluble compound according to the permeability classification criteria of the in vitro Caco-2 cell model.

10. The method according to claim 7, wherein It includes the following steps: S1. Determine whether the poloxamer has a solubilizing effect and a non-specific adsorption reducing effect on the poorly soluble compound. If so, continue with the subsequent experiments. The poloxamer is poloxamer 188, and the concentration of the poloxamer is 2% (w / v); S2. Add the poloxamer to the system for evaluating the permeability of poorly soluble compounds in an in vitro Caco-2 cell model, and combine the low permeability critical P app value: 0.25×10 -6 cm / s, high permeability critical P app value: 3.85×10 -6 cm / s to conduct the permeability evaluation of the poorly soluble compound according to the permeability classification criteria of the in vitro Caco-2 cell model.

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