Determination of relative FU ratio based on dynamic analysis

Abstract

The present invention aims to provide a method for accurately and quickly determining the unbound fraction (f) of compounds including compounds with a high protein binding rate. u The present invention relates to a method for determining the relative unbound fraction ratio (relative f) of a substance to be measured between different biological samples. u The method comprises: (1) preparing a chamber system (I) in which adjacent chambers are separated from each other by a semipermeable membrane through which a substance to be measured can pass; (2) adding a donor solution containing a first biological sample (A) and a substance to be measured to one chamber (donor side chamber) in the chamber system (I); (3) adding a receptor solution containing a second biological sample (B) to a chamber (receptor side chamber) different from the donor side chamber in the chamber system (I); (4) measuring the concentration of the substance to be measured in the donor solution of step (2) and the receptor solution of step (3) over time; and (5) calculating the relative f using data related to the concentration of the substance to be measured measured in step (4). u Than process.

Description

Technical Field

[0001] The present invention relates to a method for measuring the relative unbound fraction ratio (relative f) of a substance to be measured between different biological samples based on equilibrium dialysis and dynamic analysis. u ratio) method. Background Art

[0002] Drugs absorbed by the body exist in plasma in the form of bound (non-specifically bound to plasma proteins) and unbound (free) forms. It is believed that the bound form cannot act on the target, while the unbound form acts directly on the target (free form hypothesis). According to the free form hypothesis, the factor that affects the expression of drug efficacy / toxicity is the concentration of the unbound form. However, the drug concentration in plasma is usually obtained as the sum of the bound and unbound forms. Therefore, the fraction unbound (f) used to calculate the unbound concentration from the total concentration is used. u value) is positioned as an important parameter. u In non-clinical pharmacokinetic studies (pharmacokinetic studies using mice, rats, dogs, monkeys, cell assays, etc.), the values ​​are obtained from each sample and used for free body concentration information, etc., to improve the prediction accuracy of humans (indicating the disappearance of the drug, the clearance rate of distribution, the prediction of the distribution volume, and the correction of the intrinsic clearance rate of in vitro metabolism) and the risk of drug-drug interaction (DDI).

[0003] As the unbound fraction in plasma (f u A well-known method for measuring the concentration of the target compound (value, absolute value) is equilibrium dialysis. Specifically, in the buffer chamber and the plasma inlet chamber separated by a semipermeable membrane, the concentration ratio when the equilibrium state is reached is used as a premise to reflect the unbound fraction of the target compound (Non-patent Document 1). Equilibrium dialysis is called the gold standard because of its rapidity and simplicity of use, and various methods have been proposed. For example, in Non-patent Document 2, a flux analysis is performed by placing the same plasma into two chambers, and f u It is estimated to be in the range of 0.000013 to 0.22. In addition, in Non-Patent Document 3, a two-way equilibrium dialysis evaluation was performed, and a method was designed to obtain the same f from A to B and from B to A. u On the other hand, as a method of applying equilibrium dialysis, it is proposed to directly calculate the f between the two species. u Attempts to use the ratio of (Non-Patent Document 4, Non-Patent Document 5).

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-Patent Document 1: Eriksson, M. A., et al. (2005). "Studies of drug binding to plasma proteins using a variant of equilibrium dialysis." J Pharm Biomed Anal 38(3): 381 - 389.

[0007] Non-Patent Document 2: Kalvass, J. C., et al. (2018). "Mathematical and Experimental Validation of Flux Dialysis Method: An Improved Approach to Measure Unbound Fraction for Compounds with High Protein.” Drug Metabolism and Disposition, 46: Pages 458 - 469, April.

[0008] Non-Patent Document 3: Chen, Y. C., et al. (2019). "Improving Confidence in the Determination of Free Fraction for Highly Bound Drugs Using Bidirectional Equilibrium Dialysis." J Pharm Sci 108(3): 1296 - 1302.

[0009] Non-Patent Document 4: Deshmukh, S. V. and A. Harsch (2011). "Direct determination of the ratio of unbound fraction in plasma to unbound fraction in microsomal system (fu p / fu mic) for refined prediction of phase I mediated metabolic hepatic clearance." J Pharmacol Toxicol Methods 63(1): 35 - 39.

[0010] Non-patent document 5: Jones, RS, et al. (2020). "Evaluation of a competitiveequilibrium dialysis approach for assessing the impact of protein binding onclearance predictions." J Pharm Sci., Available online 15September 2020.

[0011] Non-patent document 6: Tang, H. and M. Mayersohn (2005). "A novel model for prediction of human drug clearance by allometric scaling." Drug Metab Dispos 33(9): 1297-1303.

[0012] Non-patent document 7: Berry, LM, et al. (2011).Species differences indistribution and prediction of human V(ss) from preclinical data." Drug MetabDispos 39(11):2103-2116.)

[0013] Non-patent document 8: Riccardi K, Cawley S, Yates PD, et al. Plasma protein binding of challenging compounds. J Pharm Sci. 2015; 104: 2627-2636.

[0014] Non-patent literature 9: Kalvass JC, Maurer TS. Influence of nonspecific brain and plasma binding on CNS exposure:implications for rational drug discovery. Biopharm Drug Dispos. 2002; 23:327-338.

[0015] Non-Patent Document 10: Vieira, M. L., et al. (2014). "Evaluation of various static in vitro-in vivo extrapolation models for risk assessment of the CYP3A inhibition potential of an investigational drug." Clin Pharmacol Ther 95(2):189-198.).

[0016] Non-Patent Document 11: Hachad, H., et al. (2010). "A useful tool for drug interaction evaluation: the University of Washington Metabolism and Transport Drug Interaction Database." Hum Genomics 5(1):61-72.

[0017] Non-Patent Document 12: Chu, Q. S., et al. (2008). "A phase I and pharmacokinetic study of lapatinib in combination with letrozole in patients with advanced cancer." Clin Cancer Res 14(14):4484-4490.

[0018] Non-Patent Document 13: Koch, K. M., et al. (2017). "The effects of lapatinib on CYP3A metabolism of midazolam in patients with advanced cancer." Cancer Chemother Pharmacol 80(6):1141-1146.

[0019] Non-patent literature 14: Takano, J., et al. (2016). "The Prediction of the RelativeImportance of CYP3A / P-glycoprotein to the Nonlinear Intestinal Absorption ofDrugs by Advanced Compartmental Absorption and Transit Model." Drug MetabDispos 44(11):1808-1818.

[0020] Non-patent literature 15: FDA DDI guidance (2020)

[0021] Non-patent document 16: Yang, J., et al. (2007). "Prediction of intestinal first-pass drug metabolism." Curr Drug Metab 8(7): 676-684.

[0022] Non-patent document 17: Yang, J., et al. (2007). "Misuse of the well-stirred model of hepatic drug clearance." Drug Metab Dispos 35(3): 501-502.

[0023] Non-patent literature 18: Obach, RS, et al. (2006). "The utility of in vitro cytochrome P450 inhibition data in the prediction of drug-drug interactions." JPharmacol Exp Ther 316(1):336-348. Summary of the invention

[0024] Problems to be solved by the invention

[0025] In these known methods, the processing of f u For compounds with low values ​​(compounds with high protein binding rate in plasma, compounds with high protein binding rate), it is considered difficult to obtain accurate values ​​because it takes a long time to reach equilibrium and the adsorption effect on the buffer chamber wall becomes a problem. Therefore, it is considered that the method of obtaining f by equilibrium dialysis is uThe method with a low value of f is suitable for methods where the compounds used are limited. u Values ​​are prone to errors (for example, f u = 0.00001 and f u =0.00002, etc.), and when these are used for comparison between multiple samples (comparison between mice, rats, dogs, monkeys, and humans), it will lead to an increase in errors and is also considered to be disadvantageous in terms of analytical accuracy. In the method of non-patent document 2, when f is obtained, u In the calculation of the value, the average value of the semipermeable membrane permeation rate (Pmem [m / s]) obtained from the low molecular weight compounds was used to calculate the f of each compound. u However, since Pmem is considered to be a coefficient that is negatively correlated with the molecular weight according to the Stokes-Einstein theory, it is believed that when the molecular weight of a compound such as cyclosporine (molecular weight: 1202.61) is large and Pmem is overestimated, the error of the method of non-patent document 2 will increase. In addition, when the molecular weight of the compound is large, it takes a long time to reach the equilibrium state, so it is believed that for f u For compounds with low f values, it is difficult to obtain accurate f u In the method of non-patent document 3, a buffer chamber is still used, which cannot cope with compounds with strong adsorption to the device such as cyclosporine. In non-patent documents 4 and 5, when calculating f u Therefore, it is difficult to detect the concentration of compounds that have not reached equilibrium. u Compared to, it is not suitable for u Compounds with low protein binding rates.

[0026] In view of the above problems, the present invention provides a method for obtaining high-precision f in a short time for compounds with high protein binding rate. u The method of value ratio is taken as the topic.

[0027] Means for solving problems

[0028] To solve the above problems, the present inventors conducted intensive research and found that, by combining equilibrium dialysis and dynamic analysis, even for compounds with high protein binding rates, the relative unbound fraction ratio (relative f) between different biological samples of the compound can be obtained with high accuracy and in a short time. u ratio). Use this relative u For example, based on the data of drug clearance or distribution volume in a species derived from one biological sample, the data of drug clearance or distribution volume in species derived from other biological samples can be corrected, thereby enabling a high-precision prediction of human pharmacokinetics.

[0029] That is, the present invention relates to the following contents. [1]

[0031] A method for determining the relative unbound fraction ratio (relative f) of a substance to be measured between different biological samples u The method comprises the following steps:

[0032] (1) a step of preparing a chamber system (I) in which adjacent chambers are separated from each other by a semipermeable membrane through which a substance to be measured can pass;

[0033] (2) a step of adding a donor solution containing a first biological sample (A) and the substance to be measured to one chamber (donor side chamber) in the chamber system (I);

[0034] (3) a step of adding an acceptor solution containing a second biological sample (B) to a chamber (acceptor side chamber) different from the donor side chamber in the chamber system (I);

[0035] (4) a step of measuring the concentration of the substance to be measured in the donor solution of the step (2) and the receptor solution of the step (3) over time; and

[0036] (5) Using the data related to the concentration of the substance to be measured measured in the step (4), calculate the relative f u Than process. [2]

[0038] The method according to [1] further comprises the following steps:

[0039] (6) a step of preparing a chamber system (II) different from the chamber system (I), wherein adjacent chambers are separated from each other by a semipermeable membrane that allows the substance to be measured to pass therethrough;

[0040] (7) a step of adding a receptor solution containing the biological sample (A) to one chamber (receptor side chamber) in the chamber system (II);

[0041] (8) a step of adding a donor solution containing the biological sample (B) and the substance to be measured to a chamber (donor side chamber) different from the acceptor side chamber in the chamber system (II);

[0042] (9) a step of measuring the concentration of the substance to be measured in the acceptor solution of the step (7) and the donor solution of the step (8) over time; and

[0043] (10) Using the data related to the concentration of the substance to be measured measured in the step (9), the relative f is calculated. u Than process. [3]

[0045] The method according to [1] or [2], wherein the biological sample (A) and / or (B) is diluted with a buffer. [4]

[0047] The method according to any one of [1] to [3], wherein the molecular weight cutoff of the semipermeable membrane is 50 kDa or less. [5]

[0049] The method according to any one of [1] to [4], wherein the steps (5) and (10) are dynamic analyses. [6]

[0051] The method according to [5], wherein the dynamic analysis is performed using the following equations 1 to 3:

[0052] (Formula 1)

[0053]

[0054] (Formula 2)

[0055]

[0056] (Formula 3)

[0057]

[0058] (Where,

[0059] Formula 1 represents the change in the amount of the analyte in the donor solution per unit time, Formula 2 represents the change in the amount of the analyte in the acceptor solution per unit time, and Formula 3 represents the change in the amount of the analyte in the adsorption fraction in the donor side chamber per unit time;

[0060] V1 represents the volume of the donor solution, V2 represents the volume of the acceptor solution, and fu1 represents the volume of the analyte relative to the biological sample contained in the donor solution. u value, fu2 represents the f of the measured substance relative to the biological sample contained in the receptor solution u value, C1 represents the concentration of the analyte in the donor solution (total concentration: the sum of bound and unbound analyte. However, excluding the analyte adsorbed in the equilibrium dialysis device), C2 represents the concentration of the analyte in the receptor solution (total concentration: the sum of bound and unbound analyte), PS represents the dialysis membrane permeation clearance of the unbound analyte, CLad represents the adsorption clearance of the analyte in the donor side chamber, koff represents the dissociation rate constant of the analyte in the adsorption fraction in the donor side chamber, and Xad represents the adsorbed amount of the analyte in the donor side chamber). [7]

[0062] The method according to any one of [1] to [6], wherein the measurement over time is performed within a time period during which the biological sample and / or the substance to be measured is denatured. [8]

[0064] The method according to any one of [1] to [7], wherein the measurement over time is performed within 24 hours. [9]

[0066] The method according to any one of [1] to [8], wherein the chamber system (I) and / or the chamber system (II) consists of two chambers.

[10]

[0068] The method according to any one of [1] to [9], wherein the protein binding rate of the analyte in the biological sample (A) is 95% or more.

[11]

[0070] The method according to any one of [1] to

[10] , wherein the protein binding rate of the analyte in the biological sample (B) is 95% or more.

[12]

[0072] The method according to any one of [1] to

[11] , wherein the biological sample (A) is one selected from the group consisting of a microsomal fraction, blood, plasma, and serum.

[13]

[0074] The method according to any one of [1] to

[12] , wherein the biological sample (B) is one selected from the group consisting of a microsomal fraction, blood, plasma, and serum.

[14]

[0076] The method according to any one of [1] to

[13] , wherein the biological sample is serum.

[15]

[0078] The method according to any one of [1] to

[14] , wherein the biological sample (A) is derived from a mammal.

[16]

[0080] The method according to any one of [1] to

[15] , wherein the biological sample (B) is derived from a mammal.

[17]

[0082] The method according to any one of [1] to

[16] , wherein the biological sample (B) is derived from an animal species different from the animal species from which the biological sample (A) is derived.

[18]

[0084] The method according to any one of [1] to

[17] , wherein the biological sample (A) is derived from one selected from the group consisting of mouse, rat, rabbit, dog, monkey and human.

[19]

[0086] The method according to any one of [1] to

[18] , wherein the biological sample (B) is derived from one selected from the group consisting of mouse, rat, rabbit, dog, monkey and human.

[20]

[0088] The method according to any one of [1] to

[19] , wherein the CLogP of the substance to be measured is 25 or less. [twenty one]

[0090] The method according to any one of [1] to

[20] , wherein the molecular weight of the substance to be measured is 5000 or less. [twenty two]

[0092] The method according to any one of [1] to

[21] , wherein the substance to be measured is a low molecular weight compound or a peptide compound. [twenty three]

[0094] The method according to any one of [1] to

[22] , wherein the substance to be measured is a peptide compound having a cyclic structure. [twenty four]

[0096] The method according to any one of [1] to

[23] , wherein the substance to be measured is a peptide compound having a non-natural amino acid residue.

[25]

[0098] The method according to

[24] , wherein the non-natural amino acid residue is a non-natural N-substituted amino acid residue.

[26]

[0100] The method according to any one of [1] to

[25] , wherein the substance to be measured is a peptide compound having 7 or more and 30 or less amino acid residues.

[27]

[0102] The relative f obtained using the method described in any one of [1] to

[26] uA method for predicting drug clearance in a species derived from either the biological sample (A) or the biological sample (B) by comparing drug clearance data in the species derived from the other biological sample.

[28]

[0104] The relative f obtained using the method described in any one of [1] to

[26] u A method for predicting the volume of distribution of a drug in a species derived from either the biological sample (A) or the biological sample (B) by comparing the volume of distribution of a drug in a species derived from the other biological sample.

[0105] Effects of the Invention

[0106] According to the method of the present invention, even for compounds with high protein binding rate, the relative f between different biological samples related to the measurement target substance can be obtained with high accuracy and in a short time without necessarily reaching the equilibrium state for equilibrium dialysis. u In addition, by using the relative f u The value of the ratio can accurately predict the data of drug clearance or distribution volume in species derived from other biological samples based on the data of drug clearance or distribution volume in species derived from one biological sample.

[0107] Brief description of the attached figure

[0108] [ Figure 1 ] Figure 1 (1a to 1h) show an example of schematic diagrams of the steps of the method of the present invention.

[0109] [ Figure 2 ] Figure 2 (2a to 2h) are schematic diagrams showing an example of the steps of the method of the present invention. Figure 1 The difference between the two cases is that the biological samples (A) and (B) are placed in the chambers.

[0110] [ Figure 3 ] Figure 3 This is a diagram of a mathematical model when performing analysis using dynamic analysis software (“SimBiology (registered trademark) / MATLAB, version information: 9.7.0.1190202 (R2019b)”, manufactured by The Math Works, Inc.).

[0111] [ Figure 4 ] Figure 4((1) to (4)) show plots of the concentration (vertical axis) of the substance to be measured in each solution at 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction (measured values: circular marks) and fitting curves (predicted values: curves) obtained by dynamic analysis of these plots according to the method of the present invention, using different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber) and nifedipine as the substance to be measured.

[0112] Figure 4 (1) shows the results using human serum and mouse serum. Figure 4 In (1), the fitting curve a represents the time-dependent change of the concentration of (human serum) in the acceptor solution, and the fitting curve b represents the time-dependent change of the concentration of (mouse serum) in the donor solution. The fitting curve c represents the time-dependent change of the concentration of (mouse serum) in the acceptor solution, and the fitting curve d represents the time-dependent change of the concentration of (human serum) in the donor solution.

[0113] Figure 4 (2) shows the results using human serum and rat serum. Figure 4 In (2), the fitting curve a represents the change in concentration of (human serum) in the acceptor solution over time, and the fitting curve b represents the change in concentration of (rat serum) in the donor solution over time. The fitting curve c represents the change in concentration of (rat serum) in the acceptor solution over time, and the fitting curve d represents the change in concentration of (human serum) in the donor solution over time.

[0114] Figure 4 (3) shows the results using human serum and dog serum. Figure 4 In (3), the fitting curve a represents the time-dependent change of the concentration of (human serum) in the acceptor solution, and the fitting curve b represents the time-dependent change of the concentration of (dog serum) in the donor solution. The fitting curve c represents the time-dependent change of the concentration of (dog serum) in the acceptor solution, and the fitting curve d represents the time-dependent change of the concentration of (human serum) in the donor solution.

[0115] Figure 4 (4) shows the results using human serum and monkey serum. Figure 4 In (4), the fitting curve a represents the change in concentration of (human serum) in the acceptor solution over time, and the fitting curve b represents the change in concentration of (monkey serum) in the donor solution over time. The fitting curve c represents the change in concentration of (monkey serum) in the acceptor solution over time, and the fitting curve d represents the change in concentration of (human serum) in the donor solution over time.

[0116] [ Figure 5 ] Figure 5((1) to (4)) show plots (measured values: circular marks) of the concentration of the substance to be measured (vertical axis) in each solution at the time points (horizontal axis) of 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction, when cyclosporine is used as the substance to be measured according to the method of the present invention, and fitting curves (predicted values: curves) obtained by dynamic analysis of these plots.

[0117] Figure 5 (1) shows the results using human serum and mouse serum. Figure 5 In (1), the fitting curve a represents the time-dependent change of the concentration of (human serum) in the acceptor solution, and the fitting curve b represents the time-dependent change of the concentration of (mouse serum) in the donor solution. The fitting curve c represents the time-dependent change of the concentration of (mouse serum) in the acceptor solution, and the fitting curve d represents the time-dependent change of the concentration of (human serum) in the donor solution.

[0118] Figure 5 (2) shows the results using human serum and rat serum. Figure 5 In (2), the fitting curve a represents the change in concentration of (human serum) in the acceptor solution over time, and the fitting curve b represents the change in concentration of (rat serum) in the donor solution over time. The fitting curve c represents the change in concentration of (rat serum) in the acceptor solution over time, and the fitting curve d represents the change in concentration of (human serum) in the donor solution over time.

[0119] Figure 5 (3) shows the results using human serum and dog serum. Figure 5 In (3), the fitting curve a represents the time-dependent change of the concentration of (human serum) in the acceptor solution, and the fitting curve b represents the time-dependent change of the concentration of (dog serum) in the donor solution. The fitting curve c represents the time-dependent change of the concentration of (dog serum) in the acceptor solution, and the fitting curve d represents the time-dependent change of the concentration of (human serum) in the donor solution.

[0120] Figure 5 (4) shows the results using human serum and monkey serum. Figure 5 In (4), the fitting curve a represents the change in concentration of (human serum) in the acceptor solution over time, and the fitting curve b represents the change in concentration of (monkey serum) in the donor solution over time. The fitting curve c represents the change in concentration of (monkey serum) in the acceptor solution over time, and the fitting curve d represents the change in concentration of (human serum) in the donor solution over time.

[0121] [ Figure 6 ] Figure 6((1) to (4)) show plots of the concentration (vertical axis) of the substance to be measured in each solution at 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction (measured values: circular marks) and fitting curves (predicted values: curves) obtained by dynamic analysis of these plots according to the method of the present invention, using different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber) and using ibuprofen as the substance to be measured.

[0122] Figure 6 (1) shows the results using human serum and mouse serum. Figure 6 In (1), the fitting curve a represents the change over time of the concentration of (human serum) in the acceptor solution, and the fitting curve b represents the change over time of the concentration of (mouse serum) in the donor solution.

[0123] Figure 6 (2) shows the results using human serum and rat serum. Figure 6 In (2), the fitting curve a represents the change in concentration of (human serum) in the acceptor solution over time, and the fitting curve b represents the change in concentration of (rat serum) in the donor solution over time. The fitting curve c represents the change in concentration of (rat serum) in the acceptor solution over time, and the fitting curve d represents the change in concentration of (human serum) in the donor solution over time.

[0124] Figure 6 (3) shows the results using human serum and dog serum. Figure 6 In (3), the fitting curve a represents the time-dependent change of the concentration of (human serum) in the acceptor solution, and the fitting curve b represents the time-dependent change of the concentration of (dog serum) in the donor solution. The fitting curve c represents the time-dependent change of the concentration of (dog serum) in the acceptor solution, and the fitting curve d represents the time-dependent change of the concentration of (human serum) in the donor solution.

[0125] Figure 6 (4) shows the results using human serum and monkey serum. Figure 6 In (4), the fitting curve a represents the change in concentration of (human serum) in the acceptor solution over time, and the fitting curve b represents the change in concentration of (monkey serum) in the donor solution over time. The fitting curve c represents the change in concentration of (monkey serum) in the acceptor solution over time, and the fitting curve d represents the change in concentration of (human serum) in the donor solution over time.

[0126] [ Figure 7 ] Figure 7 The relative f obtained by the method of the present invention is u[When different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber) were used and nifedipine was used as the substance to be measured, the f was obtained by dynamic analysis using the data at 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours after the start of the dialysis reaction. u ratio] (x axis) and f obtained by the method based on the reference example u Ratio (using serum from mouse, rat, dog, monkey or human in one chamber and buffer in the other chamber, using data from 24 hours after the start of the dialysis reaction, f was obtained by static analysis u In the figure, the range surrounded by two solid oblique lines represents the f recorded on the vertical axis. u Compared with the f recorded on the horizontal axis u The ratio between the ratios is in the range of 0.5 times or more and 2.0 times or less.

[0127] [ Figure 8 ] Figure 8 The relative f obtained by the method of the present invention is u [When different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber) were used and cyclosporine was used as the substance to be measured, the f was obtained by dynamic analysis using the data at 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours after the start of the dialysis reaction. u ratio] (x axis) and f obtained by the method based on the reference example u Ratio (using serum from mouse, rat, dog, monkey or human in one chamber and buffer in the other chamber, using data from 24 hours after the start of the dialysis reaction, f was obtained by static analysis u In the figure, the range surrounded by two solid oblique lines represents the f recorded on the vertical axis. u Compared with the f recorded on the horizontal axis u The ratio between the ratios is in the range of 0.5 times or more and 2.0 times or less.

[0128] [ Fig. 9 ] Fig. 9 The relative f obtained by the method of the present invention is u Compared with [using different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber), when ibuprofen was used as the measurement object, the f was obtained by dynamic analysis using the data at 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction. u ratio] (x axis) and f obtained by the method based on the reference example uRatio (using serum from mouse, rat, dog, monkey or human in one chamber and buffer in the other chamber, using data from 24 hours after the start of the dialysis reaction, f was obtained by static analysis u In the figure, the range surrounded by two solid oblique lines represents the f recorded on the vertical axis. u Compared with the f recorded on the horizontal axis u The ratio between the ratios is in the range of 0.5 times or more and 2.0 times or less.

[0129] [ Fig.10 ] Fig.10 is to convert f obtained by the method of the comparative example into u Compared with [using different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber), when nifedipine was used as the measurement object, the f obtained by static analysis was obtained using the data at the time of 24 hours from the start of the dialysis reaction. u ratio] (x axis) and f obtained by the method based on the reference example u Ratio (using serum from mouse, rat, dog, monkey or human in one chamber and buffer in the other chamber, using data from 24 hours after the start of the dialysis reaction, f was obtained by static analysis u In the figure, the range surrounded by two solid oblique lines represents the f recorded on the vertical axis. u Compared with the f recorded on the horizontal axis u The ratio between the ratios is in the range of 0.5 times or more and 2.0 times or less.

[0130] [ Fig.11 ] Fig.11 is to convert f obtained by the method based on the comparative example into u Compared with [using different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber), when cyclosporine was used as the measurement object, the f obtained by static analysis was obtained using the data at the time of 24 hours from the start of the dialysis reaction. u ratio] (x axis) and f obtained by the method based on the reference example u Ratio (using serum from mouse, rat, dog, monkey or human in one chamber and buffer in the other chamber, using data from 24 hours after the start of the dialysis reaction, f was obtained by static analysis u In the figure, the range surrounded by two solid oblique lines represents the f recorded on the vertical axis. u Compared with the f recorded on the horizontal axis u The ratio between the ratios is in the range of 0.5 times or more and 2.0 times or less.

[0131] [ Fig.12 ] Fig.12 is to convert f obtained by the method based on the comparative example into u Compared with [using different biological samples (human serum in one chamber and mouse, rat, dog or monkey serum in the other chamber), when ibuprofen was used as the measurement object, the f obtained by static analysis was obtained using the data at 24 hours after the start of the dialysis reaction. u ratio] (x axis) and f obtained by the method based on the reference example u Ratio (using serum from mouse, rat, dog, monkey or human in one chamber and buffer in the other chamber, using data from 24 hours after the start of the dialysis reaction, f was obtained by static analysis u In the figure, the range surrounded by two solid oblique lines represents the f recorded on the vertical axis. u Compared with the f recorded on the horizontal axis u The ratio between the ratios is in the range of 0.5 times or more and 2.0 times or less.

[0132] [ Figure 13-1 ] Figure 13-1 The relative values ​​of the observed steady-state distribution volume of humans (vertical axis: Vss obs (L / kg)) and the relative values ​​of the respective measurement target substances obtained by the method of the present invention (dynamic analysis) in Examples 1, 3, 4, 6, 8 to 12 are shown. u The figure shows a comparison of the predicted values ​​of steady-state distribution volume of humans (horizontal axis: Vss pre (L / kg)) obtained based on a known method for predicting distribution volume by comparing the ratio (rat / human). The solid oblique lines in the figure indicate that the error of the predicted value relative to the observed value is within a range of 0.67 times or more and 1.5 times or less.

[0133] [ Figure 13-2 ] Figure 13-2 The observed value of the steady-state distribution volume in humans (vertical axis: Vss obs (L / kg)) and the f of each of the measurement target substances in Comparative Examples 1, 3, 4, 6, 8 to 12 (static analysis) are shown. u The figure shows a comparison of the predicted values ​​of steady-state distribution volume of humans (horizontal axis: Vss pre (L / kg)) obtained based on a known method for predicting distribution volume by comparing the ratio (rat / human). The solid oblique lines in the figure indicate that the error of the predicted value relative to the observed value is within a range of 0.67 times or more and 1.5 times or less.

[0134] [ Figure 13-3 ] Figure 13-3 The observed value of the steady-state distribution volume in humans (vertical axis: Vss obs (L / kg)) and the f of each of the measurement objects in Reference Examples 1, 3, 4, 6, 8 to 12 (static analysis (vs. buffer)) are shown. uThe figure shows a comparison of the predicted values ​​of steady-state distribution volume of humans (horizontal axis: Vss pre (L / kg)) obtained based on a known method for predicting distribution volume by comparing the ratio (rat / human). The solid oblique lines in the figure indicate that the error of the predicted value relative to the observed value is within a range of 0.67 times or more and 1.5 times or less.

[0135] [ Figure 14-1 ] Figure 14-1 The graph shows the observed value (horizontal axis: AUCR obs) of the area increase ratio (AUCR) under the blood concentration-time curve of midazolam and the relative f obtained in Examples 14 to 20. u The solid oblique line in the figure indicates the line where the ratio of the predicted AUCR value to the observed AUCR value is 1 (the line where the observed AUCR value and the predicted value agree).

[0136] [ Figure 14-2 ] Figure 14-2 The observed value of AUCR of midazolam (horizontal axis: AUCR obs) and the value of f obtained in Reference Examples 14 to 20 are shown. u The solid oblique line in the figure indicates the line where the ratio of the predicted AUCR value to the observed AUCR value is 1 (the line where the observed AUCR value and the predicted AUCR value agree).

[0137] [ Figure 14-3 ] Figure 14-3 The observed value of AUCR of midazolam (horizontal axis: AUCR obs) and the value of f obtained in Reference Examples 21 to 27 are shown. u The solid oblique line in the figure indicates the line where the ratio of the predicted AUCR value to the observed AUCR value is 1 (the line where the observed AUCR value and the predicted value agree).

[0138] Specific embodiments of the invention

[0139] About the terms used in this manual

[0140] The "substance to be measured" in this specification refers to the relative u The compound having a certain ratio is preferably a low molecular weight compound or a peptide compound.

[0141] "Low molecular weight compound" in this specification refers to a compound having a molecular weight of preferably 2000 or less, excluding the peptide compounds described later, including, for example, natural compounds and new compounds (New molecular entity: NME) that are chemically synthesized in the manufacturing process. The molecular weight of the low molecular weight compound is more preferably 1000 or less, further preferably 800 or less, and particularly preferably 500 or less. In addition, the CLogP of the low molecular weight compound is preferably 25 or less, more preferably 16 or less, further preferably 10 or less, further preferably 7 or less, further preferably 6 or less, further preferably 5 or less, and further preferably 4 or less. ClogP is a distribution coefficient calculated by computer by dividing the compound into partial structures (the calculation software is well known. For example, the calculation can be performed using software from Daylight Chemical Information Systems, Inc., etc.).

[0142] The "peptide compound" in this specification is not particularly limited as long as it is a peptide compound in which natural amino acids and / or non-natural amino acids are linked via an amide bond or an ester bond, and is preferably a peptide compound having 5 or more residues, more preferably 7 or more residues, further preferably 8 or more residues, particularly preferably 9 or more residues, and preferably 30 or less residues, more preferably 25 or less residues, further preferably 15 or less residues, and particularly preferably 13 or less residues. For example, the peptide compound may be a peptide compound having preferably 5 or more residues and 30 or less residues, more preferably 7 or more residues and 25 or less residues, further preferably 8 or more residues and 15 or less residues, and particularly preferably 9 or more residues and 13 or less residues.

[0143] The peptide compound that can be used in the present invention preferably contains at least 3 N-substituted amino acids in one peptide, and more preferably contains at least 5 or more N-substituted amino acids. These N-substituted amino acids may be present continuously or discontinuously in the peptide compound. The peptide compound in the present invention may be linear or cyclic, preferably a cyclic peptide compound. In addition, it may also have a branched structure. Among the peptide compounds having non-natural amino acid residues, it is further preferred to have a peptide compound having a non-natural amino acid residue that is N-methylated.

[0144] The molecular weight of the peptide compound is preferably 5000 or less, more preferably 3000 or less, further preferably 2000 or less, preferably 500 or more, more preferably 800 or more, further preferably 1000 or more. For example, the molecular weight of the peptide compound may be preferably 500 or more and 5000 or less, more preferably 800 or more and 3000 or less, further preferably 1000 or more and 2000 or less.

[0145] The CLogP of the peptide compound is preferably 25 or less, more preferably 22 or less, further preferably 20 or less, further preferably 18 or less, further preferably 16 or less, further preferably 15 or less, preferably 5 or more, more preferably 10 or more, further preferably 12 or more. For example, the CLogP of the peptide compound may be preferably 5 or more and 25 or less, more preferably 10 or more and 20 or less, further preferably 12 or more and 18 or less.

[0146] The "cyclic peptide compound" in this specification is not particularly limited as long as it is a peptide compound having a cyclic portion composed of more than 5 amino acids, and its cyclization method is also not limited. It is a cyclic peptide compound that can be obtained by cyclizing the group on the N-terminal side of a linear peptide compound with the group on the C-terminal side. Cyclization can be any form such as cyclization through carbon-nitrogen bonds such as amide bonds, cyclization through carbon-oxygen bonds such as ester bonds and ether bonds, cyclization through carbon-sulfur bonds such as thioether bonds, cyclization through carbon-carbon bonds, or cyclization through heterocyclic construction. Among them, cyclization via covalent bonds such as amide bonds or carbon-carbon bonds is preferred, and cyclization via amide bonds through carboxyl groups on the side chains and amino groups on the main chain of the N-terminal is more preferred. The positions of the carboxyl groups and amino groups used in the cyclization can be on the main chain or on the side chain, and there are no special restrictions as long as they are positions that can be cyclized.

[0147] The "amino acids" in this specification include natural amino acids and non-natural amino acids (sometimes referred to as amino acid derivatives). The "natural amino acids" in this specification refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, Pro. Non-natural amino acids (amino acid derivatives) are not particularly limited, and examples include β-amino acids, D-type amino acids, N-substituted amino acids (except Pro), α,α-disubstituted amino acids, amino acids with side chains different from natural amino acids, hydroxycarboxylic acids, etc. As amino acids in this specification, any spatial configuration is allowed. The choice of the side chain of the amino acid is not particularly limited, and in addition to hydrogen atoms, it is also freely selected from, for example, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and spiro-bound cycloalkyl. Substituents may be given separately, and these substituents are also not limited. For example, one or more substituents may be independently selected from any substituents containing halogen atoms, O atoms, S atoms, N atoms, B atoms, Si atoms, or P atoms. That is, examples include optionally substituted alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, or oxo, aminocarbonyl, halogen atoms, etc. In a non-limiting manner, the amino acid in this specification may be a compound having a carboxyl group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the amino acid).

[0148] Examples of the halogen-derived substituent include fluorine (—F), chlorine (—Cl), bromine (—Br), and iodine (—I).

[0149] As substituents derived from O atoms, there are listed groups such as hydroxyl (-OH), oxy (-OR), carbonyl (-C=OR), carboxyl (-CO2H), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C=O-SR), carbonylthio (-SC=OR), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=OR), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), aminosulfonylamino (-NH-SO2-NHR), thiocarboxy (-C=O-SH), and carboxycarbonyl (-C=O-CO2H).

[0150] Examples of the oxy group (—OR) include an alkoxy group, a cycloalkoxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group, a heteroaryloxy group, and an aralkyloxy group.

[0151] Examples of the carbonyl group (—C═OR) include formyl (—C═OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl and the like.

[0152] Examples of the oxycarbonyl group (—C═O—OR) include an alkyloxycarbonyl group, a cycloalkyloxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, an aralkyloxycarbonyl group and the like.

[0153] Examples of the carbonyloxy group (—OC═OR) include an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group and an aralkylcarbonyloxy group.

[0154] Examples of the thiocarbonyl group (—C═O—SR) include an alkylthiocarbonyl group, a cycloalkylthiocarbonyl group, an alkenylthiocarbonyl group, an alkynylthiocarbonyl group, an arylthiocarbonyl group, a heteroarylthiocarbonyl group, an aralkylthiocarbonyl group and the like.

[0155] Examples of the carbonylthio group (-SC=OR) include an alkylcarbonylthio group, a cycloalkylcarbonylthio group, an alkenylcarbonylthio group, an alkynylcarbonylthio group, an arylcarbonylthio group, a heteroarylcarbonylthio group and an aralkylcarbonylthio group.

[0156] Examples of aminocarbonyl groups (-C=O-NHR) include alkylaminocarbonyl groups, cycloalkylaminocarbonyl groups, alkenylaminocarbonyl groups, alkynylaminocarbonyl groups, arylaminocarbonyl groups, heteroarylaminocarbonyl groups, and aralkylaminocarbonyl groups. In addition to these, compounds in which the H atom bonded to the N atom in -C=O-NHR is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0157] Examples of the carbonylamino group (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C=OR is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0158] Examples of the oxycarbonylamino group (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkyloxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C=O-OR is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0159] Examples of the sulfonylamino group (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, compounds in which the H atom bonded to the N atom in -NH-SO2-R is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0160] Examples of aminosulfonyl groups (-SO2-NHR) include alkylaminosulfonyl groups, cycloalkylaminosulfonyl groups, alkenylaminosulfonyl groups, alkynylaminosulfonyl groups, arylaminosulfonyl groups, heteroarylaminosulfonyl groups, aralkylaminosulfonyl groups, etc. In addition to these, compounds in which the H atom bonded to the N atom in -SO2-NHR is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0161] Examples of sulfamoylamino (-NH-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with a substituent independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, and these two substituents may form a ring.

[0162] Examples of the substituent derived from the S atom include thiol (-SH), sulfenyl (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)2-R), sulfo (-SO3H), and pentafluorosulfanyl (-SF5).

[0163] Examples of the thio group (—SR) include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio and the like.

[0164] Examples of the sulfinyl group (—S═OR) include an alkylsulfinyl group, a cycloalkylsulfinyl group, an alkenylsulfinyl group, an alkynylsulfinyl group, an arylsulfinyl group, a heteroarylsulfinyl group, and an aralkylsulfinyl group.

[0165] Examples of the sulfonyl group (—S(O) 2 —R) include an alkylsulfonyl group, a cycloalkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group, an aralkylsulfonyl group and the like.

[0166] As substituents derived from the N atom, there are listed azide (-N3, also called "azido"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidine (-NH-C(=NH)-NH2), substituted guanidine (-NR-C(=NR"')-NR'R"), and aminocarbonylamino (-NR-CO-NR'R").

[0167] Examples of the secondary amino group (—NH—R) include an alkylamino group, a cycloalkylamino group, an alkenylamino group, an alkynylamino group, an arylamino group, a heteroarylamino group, and an aralkylamino group.

[0168] Examples of tertiary amino groups (-NR(R')) include amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and these arbitrary two substituents may form a ring.

[0169] As examples of substituted amidino groups (-C(=NR)-NR'R"), the three substituents R, R', and R" on the N atom are listed as groups independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, for example alkyl (aralkyl) (aryl) amidino groups, etc.

[0170] Examples of substituted guanidine groups (-NR-C(=NR'")-NR'R") include groups wherein R, R', R", and R'" are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or groups forming a ring thereof.

[0171] Examples of aminocarbonylamino (-NR-CO-NR'R") include groups wherein R, R', and R" are independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or rings formed therefrom.

[0172] As the substituents derived from the B atom, boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')) are listed. These two substituents R and R' are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or they can form a ring. Specifically, cyclic boryl groups are listed, and more specifically, pinacholatoboryl group, neopentanediolatoboryl group, catechol boryl group, etc. are listed.

[0173] As the substituent on the nitrogen atom of the N-substituted amino acid in the present specification, specifically, alkyl, C1-C6 alkyl, C1-C4 alkyl, methyl, C7-C 14 Aralkyl, benzyl, phenethyl, etc.

[0174] The amino group in the main chain of an amino acid may be unsubstituted (-NH2) or substituted (i.e., -NHR. Here, R represents an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group which may optionally have a substituent. In addition, as in proline, the carbon chain bound to the N atom and the carbon atom at the α position may form a ring). Such an amino acid in which the main chain amino group is substituted is sometimes referred to as an "N-substituted amino acid" in this specification. Preferred examples of the "N-substituted amino acid" in this specification include N-alkyl amino acids, N-C1-C6 alkyl amino acids, N-C1-C4 alkyl amino acids, N-methyl amino acids, N-C7-C 14 Aralkyl amino acids, N-phenylethyl amino acids, but not limited to these.

[0175] The "amino acid" in this specification includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom is replaced by an atom with the same atomic number (number of protons) but a different mass number (the sum of the number of protons and neutrons). Examples of isotopes included in the "amino acid" in this specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., respectively. 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 32 P. 35 S. 18 F. 36 Cl et al.

[0176] The "biological sample" used in the present invention refers to a biological sample derived from a living organism, preferably a biological sample derived from a mammal. Preferred mammals include mice, rats, rabbits, dogs, monkeys, or humans. Preferred biological samples include, for example, body fluids, tissues, cells, or parts thereof, and more preferably biological samples selected from the group consisting of microsomal fractions, blood, plasma, and serum.

[0177] In the present invention, "different biological samples" refers to two or more different biological samples. As different biological samples, biological samples from different species (e.g., mice and humans, rats and humans, etc.) or from the same species (e.g., humans and humans) can be used. Furthermore, different biological samples include biological samples from the same biological part or different biological parts of different species, and biological samples from the same biological part or different biological parts of the same species. For example, different biological samples in the present invention include biological sample parts of the same type such as microsome fractions to microsome fractions, blood to blood, plasma to plasma, serum to serum, etc. from the same or different species of mammals, and also include different biological sample parts such as blood to plasma, microsome fractions to plasma, plasma to serum. Even if different biological samples come from the same biological part between the same species, they can be included in the different biological samples of the present invention if they come from different individuals.

[0178] In this specification, "protein binding rate" refers to the ratio (mass ratio or molar ratio or percentage) of the total amount of the substance to be measured that is bound to the protein (e.g., "non-specific protein" such as albumin) in the biological sample. That is, protein binding rate = 1 - unbound fraction (f uIn the present invention, a substance to be measured having a protein binding rate of preferably 95% or more, more preferably 97% or more, further preferably 98% or more, and most preferably 99% or more can be used.

[0179] In this specification, the "unbound fraction (f u "f" refers to the ratio of the substance to be measured that is not bound to the protein in the biological sample (sometimes referred to as "unbound substance to be measured" in this specification) in the total amount of the substance to be measured in the presence of protein (e.g., albumin). That is, the unbound fraction (f u )=1-protein binding rate. "Relative unbound fraction ratio (relative f u The "ratio" refers to the ratio of the unbound fraction of the analyte in each of the different biological samples. For example, as the relative f u The ratio of the measured substance in the biological sample (A) is shown in FIG. u The value (fu1) is different from the f value of the analyte in the biological sample (B) which is different from the biological sample (A). u The ratio of the value (fu2) to the value (fu1 / fu2).

[0180] In the method of the present invention, the "semipermeable membrane" divides the "chamber" into two or more. The "semipermeable membrane" is a membrane through which the selected molecular species can pass by diffusion. As a semipermeable membrane for equilibrium dialysis, a semipermeable membrane that allows the measurement object substance (when present, with a solvent such as a buffer solution) to pass through, but cannot allow the biological sample to pass through, is selected. Preferably, the semipermeable membrane is a size selective membrane, for example, the semipermeable membrane can be characterized by a fractional molecular weight (molecular weight cutoff: MWCO). In this case, the MWCO of the semipermeable membrane is preferably less than 50 kDa, more preferably less than 14 kDa, further preferably less than 8 kDa, preferably more than 3.5 kDa, more preferably more than 4 kDa, and further preferably more than 6 kDa. For example, the MWCO of the semipermeable membrane is preferably more than 3.5 kDa and less than 50 kDa, more preferably more than 4 kDa and less than 14 kDa, and further preferably more than 6 kDa and less than 8 kDa. As the semipermeable membrane that can be used in the method of the present invention, a known semipermeable membrane can be used. For example, a semipermeable membrane composed of regenerated cellulose (Rapid Equilibrium Dialysis (RED) Device Inserts, 8KMWCO, manufactured by Thermo Fisher Scientific) can be used.

[0181] As the chamber, a chamber used in a known equilibrium dialysis method can be used. For example, as the chamber, a chamber made of a material such as Teflon or polypropylene can be used.

[0182] In this specification, "donor side", "donor" or "Donor" refers to the side where a donor solution containing a substance to be measured is added at the initial time of measurement, for example, "donor side chamber" or "donor chamber" refers to the chamber where a donor solution containing a substance to be measured is added at the initial time of measurement. "Acceptor side", "acceptor" or "Acceptor" refers to the side where an acceptor solution not containing a substance to be measured is added at the initial time of measurement, for example, "acceptor side chamber" or "acceptor chamber" refers to the chamber where an acceptor solution not containing a substance to be measured is added at the initial time of measurement.

[0183] In this specification, "donor solution" refers to a solution added to the donor side chamber (or donor chamber) and the solution then present in the donor side chamber (or donor chamber). In addition, "acceptor solution" refers to a solution added to the acceptor side chamber (or acceptor chamber) and the solution then present in the acceptor side chamber (or acceptor chamber).

[0184] In this specification, "initial period" refers to the period when the donor solution and the acceptor solution are added to each chamber and the measurement target substance begins to move between the solutions. For example, the moment when the donor solution and the acceptor solution are first added to the chamber and stirring is immediately started can be regarded as the "initial period". However, when the measurement target substance has the property of being adsorbed on the chamber wall, it is difficult to determine the accurate concentration of the measurement target substance in the donor solution at the initial stage. Therefore, it is preferred to sample the donor solution immediately before adding it to the chamber and measure the concentration, so as to use it as the concentration of the measurement target substance in the donor solution at the initial stage (i.e., time 0) for analysis.

[0185] In this specification, the term "equilibrium state" refers to a state in which the concentration change of the substance to be measured reaches a steady state and the concentration change substantially disappears between the solutions added to the chamber separated by the semipermeable membrane.

[0186] In the present invention, “measurement over time” means measurement over time, and includes the case where measurement is performed continuously in time, and the case where measurement is performed a plurality of times discontinuously at regular time intervals or at regular time intervals.

[0187] In this specification, "dynamic analysis" refers to an analysis method in which f is calculated by performing convergence calculation (fitting) on ​​the measured values ​​using analysis software or the like, by measuring the concentration of the measurement target substance in the solution of the measurement chamber over time. u Ratio (f u In contrast, “static analysis” refers to an analysis method that uses the measured value of the concentration of the substance to be measured in the solution of the chamber only at a specific time or the ratio of the measured values ​​to calculate f u Ratio (f u In addition, the specific time is preferably the time when the operation of the equilibrium dialysis reaction is terminated, and more preferably the time when the equilibrium dialysis reaction is terminated after reaching the equilibrium state.

[0188] Relative f of the analyte between different biological samples u Method for determination of ratio (a)

[0189] The present invention is used to determine the relative unbound fraction ratio (relative f) of the analyte substance between different biological samples. u The method comprises the following steps (1) to (5).

[0190] (1) a step of preparing a chamber system (I) in which adjacent chambers are separated from each other by a semipermeable membrane through which a substance to be measured can pass;

[0191] (2) a step of adding a donor solution containing a first biological sample (A) and the substance to be measured to one chamber (donor side chamber) in the chamber system (I) (for example, Figure 1 a and Figure 1 f);

[0192] (3) a step of adding an acceptor solution containing a second biological sample (B) to a chamber (acceptor side chamber) different from the donor side chamber in the chamber system (I) (e.g., Figure 1 b and Figure 1 e);

[0193] (4) a step of measuring the concentration of the substance to be measured in the donor solution of the step (2) and the receptor solution of the step (3) over time (for example, Figure 1 c to Figure 1 d and Figure 1 g to Figure 1 h); and

[0194] (5) Using the data related to the concentration of the substance to be measured measured in the step (4), calculate the relative f u Than process.

[0195] In the method of the present invention, the order of performing the steps (2) and (3) is not particularly limited. For example, step (3) may be performed after step (2), step (2) may be performed after step (3), or step (2) and step (3) may be performed simultaneously or in parallel (for example, Figure 1 a to Figure 1 b and Figure 1 e to Figure 1 f).

[0196] Below, refer to Figure 1 An example of the present invention is described below. In the figure, the chamber system includes two adjacent chambers (donor side chamber and acceptor side chamber) separated by a membrane M. The size selective membrane M separates the two chambers, allowing the substance to be measured to pass through, but not allowing nonspecific proteins from the first biological sample (A) and nonspecific proteins from the second biological sample (B) to pass through.

[0197] Figure 1 ( Figure 1 a to Figure 1 d, Figure 1 e to Figure 1 h) Represents the dialysis chamber system I during analysis. Figure 1 a represents the state when a donor solution containing a first biological sample (A) containing a non-specific protein 4 and a measurement target substance 3 is added to the donor side chamber 1 (black arrow), wherein the measurement target substance 3 and the non-specific protein 4 are present in the donor solution of the donor side chamber 1. Figure 1 b shows a state when a receptor solution containing a second biological sample (B) containing non-specific protein 5 is added to the receptor side chamber 2 (black arrow), wherein the non-specific protein 5 is present in the receptor solution in the receptor side chamber 2 . Figure 1 c shows the situation at the initial stage when the analyte starts to move from the donor-side chamber 1 to the acceptor-side chamber 2 . Figure 1 d represents the state of the stage of dialysis in which the substance to be measured moves from the donor side chamber 1 to the acceptor side chamber 2, or further moves in the opposite direction from the acceptor side chamber 2 to the donor side chamber 1, and approaches equilibrium. From the initial stage, the concentration of the substance to be measured in the solution added to each chamber is measured multiple times over time.

[0198] Figure 1 e to 1h means Figure 1 a and Figure 1 b is an example of a schematic diagram of the situation after the operation sequence is changed.

[0199] Among the analyte 3 added to the donor side chamber 1, only the non-specific protein 4 or the analyte not bound to the chamber wall passes through the semipermeable membrane. A part of the analyte that passes through the semipermeable membrane binds to the non-specific protein 5 in the acceptor side chamber 2. The concentration of the analyte in the solution tends to be balanced between the two chambers, but since the binding characteristics of the analyte to each non-specific protein are different, the binding characteristics to each non-specific protein affect the distribution of all the analyte.

[0200] In the method of the present invention, the biological sample and the measurement object substance added to the chamber can be diluted by a solvent. As a solvent, there is no particular limitation as long as it has no substantial effect on the characteristics of the biological sample and the measurement object substance, dissolves the biological sample or the measurement object substance, or is a solvent that mixes well with each other. As such a solvent, for example, a buffer solution can be preferably used. As a buffer solution, for example, Good's buffers such as phosphate buffer, Tris buffer solution·HEPES buffer solution, acetate buffer solution, citric acid buffer solution, citric acid phosphate buffer solution, boric acid buffer solution, tartaric acid buffer solution, carbonate buffer solution, phthalic acid buffer solution, oxalic acid buffer solution, etc. are listed.

[0201] The osmotic pressure of the buffer is preferably 0.5 times or more of physiological saline, more preferably 0.9 times or more, preferably 2 times or less of physiological saline, and more preferably 1.1 times or less. For example, the osmotic pressure of the buffer can be set in a range of preferably 0.5 times or more and 2 times or less of physiological saline, and further preferably 0.9 times or more and 1.1 times or less. As the pH of the buffer, it is preferably 6 or more, more preferably 7.0 or more, preferably 8 or less, and further preferably 7.8 or less. For example, the pH of the buffer can be preferably 6 or more and 8 or less, and further preferably 7.0 or more and 7.8 or less.

[0202] Furthermore, the substance to be measured may be dissolved in advance in a solvent such as dimethyl sulfoxide (DMSO) or ethanol in order to improve its solubility, and then mixed with a biological sample in a buffer solution.

[0203] In the method of the present invention, microsomal fractions, blood, plasma, and serum can be preferably used as biological samples, and among these, plasma or serum can be more preferably used, and serum can be further preferably used. When serum is used as a biological sample, adsorption of the measurement object substance to the device etc. can be reduced, and a high recovery rate of the measurement object substance can be achieved, and a decrease in the concentration of the measurement object substance can be suppressed.

[0204] Furthermore, in the method of the present invention, when the biological sample is diluted with a buffer or the like before use, it is more preferable to use diluted serum. By using diluted serum, it is possible to easily capture the initial concentration change in the concentration measurement in the dynamic analysis. Thus, for compounds with high protein binding rates that are difficult to obtain a steady state in dialysis, it is possible to easily capture the initial concentration change in the concentration measurement in the dynamic analysis. u Compared with the previous method, it can achieve further improvement in accuracy and reduction in time.

[0205] In the present invention, the initial concentration of the biological sample in the donor solution or the acceptor solution is not particularly limited, and is, for example, preferably in the range of 1% to 50%, more preferably 1% to 30%, and further preferably 1% to 10% (volume ratio in the solution).

[0206] In the present invention, the initial concentration of the substance to be measured in the donor solution or the acceptor solution is not particularly limited, and is, for example, preferably in the range of 0.1 (μmol / L) to 10 (μmol / L), more preferably in the range of 0.1 (μmol / L) to 1 (μmol / L), and more preferably in the range of 0.1 (μmol / L) to 0.5 (μmol / L).

[0207] After the donor solution and the acceptor solution are added to the chamber, the added sample and substance can be diffused by stirring or freely diffused in the chamber. Preferably, the dialysis can be performed while stirring the solution in the chamber. Stirring can be performed at a rotation speed of preferably 20 rpm or more and 800 rpm or less, more preferably 250 rpm or more and 800 rpm or less.

[0208] The temperature of the solution in the chamber during the dialysis reaction is not particularly limited as long as it does not adversely affect the properties of the biological sample and the substance to be measured, and is, for example, preferably in the range of 0°C to 40°C, more preferably in the range of 20°C to 40°C, and further preferably in the range of 35°C to 40°C.

[0209] In the present invention, the above-mentioned equilibrium dialysis can be performed using a known device, for example, a rapid equilibrium dialysis device or the like.

[0210] In the present invention, the concentration of the substance to be measured in the donor solution and the acceptor solution is measured over time. The concentration measurement can be implemented continuously or at a certain time or at a certain time. In the measurement at each certain time, for example, the solution in each chamber can be dispensed as a sample and measured by a known method. For example, the concentration of the substance to be measured in the sample obtained above can be measured using methods such as liquid chromatography mass spectrometry (LC-MS / MS) and liquid chromatography using a UV detector.

[0211] In the present invention, the measurement period in the time measurement depends on the biological sample and the measurement object substance used, and is not particularly limited. For example, appropriate measurement can be performed from the initial stage to preferably 72 hours, more preferably 36 hours, and further preferably 24 hours. When the measurement is performed every certain time or every main time, the number of times is, for example, preferably within 20 times, more preferably within 10 times, and further preferably within 5 times. In such a time range, by performing dynamic analysis by measuring the concentration over time, even if the equilibrium state is not reached, the relative f can be obtained with good accuracy. u Compare.

[0212] In the present invention, the relative f can be calculated using the measured time-dependent data of the concentration of the substance to be measured in the donor solution and the receptor solution. u Ratio. Relative f u The above analysis can be performed using known dynamic analysis software. As such dynamic analysis software, for example, Simbiology / MATLAB R2019b (manufactured by The Math Works, Inc.), Microsoft Excel 2019 (manufactured by Microsoft Corporation), etc. can be used.

[0213] The following is an explanation of the dynamic analysis method of the present invention. The present invention is to directly detect the relative protein-unbound fraction ratio (relative f) of the analyte in different biological samples. u The new analytical technology of the present invention is to establish a relative f for detecting compounds with high protein binding rate by implementing (i) rapid equilibrium dialysis to evaluate the concentration transition of the analyte between different biological samples and (ii) dynamic (kinetic) analysis. u The best conditions than.

[0214] In the dynamic analysis of the present invention, the measured values ​​of the concentration of the substance to be measured in each solution obtained by equilibrium dialysis (for example, 0.5 hour value, 1 hour value, 2 hour value, 4 hour value, 16 hour value, 24 hour value) and the inherent parameters are applied to the following formula 1 (change in the mass of the substance to be measured in the donor solution), the following formula 2 (change in the mass of the substance to be measured in the acceptor solution), and the following formula 3 (change in the amount of the substance to be measured per unit time in the adsorbed fraction in the donor side chamber), and convergence calculation (fitting) is performed to obtain f u 1 and f u 2, the best solution is to obtain f u 1 / f u 2 is set as the relative f u Compare.

[0215] In addition, for the measurement object substance that is adsorbed on the device, in (ii) dynamic analysis, the adsorption clearance rate is set as a parameter in the mass balance equation, and the fitting accuracy is improved by a model based on the mechanism. u The value of the compound) is obtained by analyzing the f u The accuracy of the value is often considered a problem, but in the present invention, if necessary, by implementing (iii) bidirectional evaluation by combining the measurement method (a) with the following measurement method (b), that is, by comparing the relative f obtained by the above method u The relative f was calculated by the same method after replacing the biological sample added to the donor solution and the receptor solution. u Ratio, can be numerically verified, can further improve the relative f u Furthermore, if necessary, by using (iv) diluted serum, the detection conditions of the initial concentration change can be improved, and the relative f can be further improved. u The accuracy of the ratio.

[0216] Specifically, using the above software for dynamic analysis, the concentration transition obtained by the following method can be input as data, and each parameter and f can be calculated using the following conditions and the nonlinear least squares method (lsqnonlin).u Compare.

[0217] (Formula 1):

[0218]

[0219] (Formula 2):

[0220] (Formula 3):

[0221]

[0222] Formula 1 represents the change in the mass of the object to be measured in the donor solution (the change in the amount per unit time). That is, Formula 1 represents the change in the mass of the object to be measured on the donor side, including the reverse movement of the object to be measured from the acceptor side to the donor side (fu2*C2*PS), the movement of the object to be measured from the donor side to the acceptor side (fu1*C1*PS), and the influence of adsorption and desorption on the device wall on the donor side (adsorption of the object to be measured on the device wall (fu1*C1*CLad) and desorption of the adsorbed object to be measured from the device wall (koff*Xad)).

[0223] Formula 2 represents the change in the mass of the object to be measured in the acceptor solution (the change in the amount per unit time). That is, Formula 2 represents the change in the mass of the object to be measured on the acceptor side including the reverse movement of the object to be measured from the acceptor side to the donor side (fu2*C2*PS) and the movement of the object to be measured from the donor side to the acceptor side (fu1*C1*PS) (excluding the influence of adsorption).

[0224] Formula 3 represents the change in the amount of the measurement target substance of the adsorption fraction in the donor side chamber per unit time (adsorption and desorption of the measurement target substance to the device wall per unit time). That is, it represents the change in the adsorption amount of the measurement target substance on the donor side, including the adsorption of the measurement target substance to the donor side device wall (fu1*C1*CLad) and the desorption of the adsorbed measurement target substance from the device wall (koff*Xad)).

[0225] In these equations, the relative f can be obtained by performing convergence calculation (fitting) using the concentration (actual value) of the substance to be measured in the solution measured over time and the inherent parameters (the parameters related to adsorption and desorption to the device wall (CLad, Koff, Xad) are calculated by the computer based on the concentration through convergence calculation and incorporated). u Ratio (f u 1 / f u 2).

[0226] Here, each parameter represents the following.

[0227] V1: Volume of donor solution (fixed value, 3.5×10-4 L)

[0228] V2: Volume of receptor solution (fixed value, 2.0×10 -4 L)

[0229] fu1: The f of the substance to be measured relative to the biological sample contained in the donor solution u Value (calculated value)

[0230] fu2: The f of the substance to be measured relative to the biological sample contained in the receptor solution u Value (calculated value)

[0231] C1: Concentration of the analyte in the donor solution (total concentration: the sum of bound and unbound analytes. However, excluding analytes adsorbed to the equilibrium dialysis device). (measured value, nmol / L)

[0232] C2: Concentration of the analyte in the receptor solution (total concentration: the sum of bound and unbound analyte). (Measured value, nmol / L)

[0233] PS: Dialysis membrane clearance of the substance to be measured (fixed value, 1.0×10 -4 L / h or 1.0×10 -5 L / h)

[0234] CLad: Adsorption clearance rate of the measurement target substance in the donor side chamber (variable value, L / h)

[0235] koff: Dissociation rate constant of the analyte in the donor chamber (variable value, h -1 )

[0236] Xad: adsorption amount of the substance to be measured in the donor side chamber (variable value, nmol)

[0237] The number "1" in each symbol indicates the donor side, and "2" indicates the acceptor side.

[0238] In addition, in the equilibrium dialysis device used in the embodiment of the present application, since the wall surface area on the donor side is very large compared to the acceptor side, the measurement target substance adsorbed in the equilibrium dialysis device only in C1 is considered, and the measurement target substance in the donor side chamber is also considered for CLad, Koff and Xad. However, when using an equilibrium dialysis device in which the wall surface area on the donor side and the wall surface area on the acceptor side are close to each other to a certain extent, the change in the amount of the measurement target substance in the adsorption fraction in the acceptor side chamber per unit time can also be dynamically analyzed as the following formula 3' with the addition of the vertical formula.

[0239] (Formula 3'):

[0240]

[0241] CLad': Adsorption clearance rate of the analyte in the receptor side chamber (variable value, L / h)

[0242] koff': Dissociation rate constant of the analyte in the receptor side chamber (variable value, h -1 )

[0243] Xad': adsorption amount of the analyte in the receptor side chamber (variable value, nmol)

[0244] In this case, Formula 2 is changed to the following Formula 2' for use.

[0245] (Formula 2'):

[0246]

[0247] In addition to the above analysis method, when PS is not fixed to a specific value, (fu1*PS) and (fu2*PS) can be used as variable parameters of a block for convergence calculation (fitting), and the relative f can be obtained by the ratio of (fu1*PS) to (fu2*PS). u Ratio (f u 1 / f u 2).

[0248] In the convergence calculation (fitting), the relative f can be calculated by taking the measurement results of the biological sample replacement contained in the donor solution and the acceptor solution as a group. u The default settings of Fit Data (SimBiology Model Analyzer, Program) can be changed appropriately. For example, the following conditions (Model, Reaction, Rules, Definition, Estimated Parameters) can be recorded and implemented. Figure 3 shown.

[0249] Reaction:

[0250] Eq1.Donor1<->Eq1.Acceptor1:+fu1*PS*Eq1.Donor1 / Vd1-fu2*PS*Eq1.Acceptor1 / Vd2

[0251] Eq1.Donor1->Eq1.Ad:+CL1*fu1*Eq1.Donor1 / Vd1-(Koff1*Eq1.Ad)

[0252] Eq2.Donor2<->Eq2.Acceptor2:+fu2*PS*Eq2.Donor2 / Vd1-fu1*PS*Eq2.Acceptor2 / Vd2

[0253] Eq2.Donor2->Eq2.Ad:+CL2*fu2*Eq2.Donor2 / Vd1-(Koff2*Eq2.Ad)

[0254] Rules:

[0255] rule_1Eq1.Ca1=Eq1.Acceptor1 / Vd2 repeatedAssignment

[0256] rule_2Eq1.Cd1=Eq1.Donor1 / Vd1 repeatedAssignment

[0257] rule_3Eq2.Ca2=Eq2.Acceptor2 / Vd2 repeatedAssignment

[0258] rule_4Eq2.Cd2=Eq2.Donor2 / Vd1 repeatedAssignment

[0259] rule_5Eq1.Donor1=Dose1 initialAssignment

[0260] rule_6Eq2.Donor2=Dose2 initialAssignment

[0261] Definition:

[0262] When the first biological sample is a mouse and the second biological sample is a human, the following four data are used as a set for analysis.

[0263] Data 1 (Eq1.Ca1): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains mouse serum and the receptor solution contains human serum

[0264] Data 2 (Eq1.Cd1): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains mouse serum and the receptor solution contains human serum

[0265] Data 3 (Eq2.Ca2): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains mouse serum

[0266] Data 4 (Eq2.Cd2): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains mouse serum

[0267] Estimated Parameters:

[0268] exp(fu1),InitialValue=1,Bounds=[0.01 100];

[0269] exp(fu2),InitialValue=1,Bounds=[0.01 100];

[0270] exp(Koff1),InitialValue=1,Bounds=[0.01 1000000];

[0271] exp(Koff2),InitialValue=1,Bounds=[0.01 1000000];

[0272] exp(CL1),InitialValue=0.001,Bounds=[1e-07 0.1];

[0273] exp(CL2),InitialValue=0.001,Bounds=[1e-07 0.1];

[0274] exp(Dose1),InitialValue=0.2115,Bounds=[0.1 0.4 or 0.5];

[0275] exp(Dose2),InitialValue=0.2114,Bounds=[0.1 0.4 or 0.5];

[0276] Pooled fit,true

[0277] Relative f of the analyte between different biological samples u Method for determination of ratio (b)

[0278] The present invention is used to determine the relative unbound fraction ratio (relative f) of the analyte substance between different biological samples. u The method for determining the relative unbound fraction ratio (relative f) of the analyte substance between different biological samples may further include replacing the biological sample (A) with the biological sample (B) in the assay method (a) comprising the steps (1) to (5). That is, the method of the present invention is for determining the relative unbound fraction ratio (relative f) of the analyte substance between different biological samples. u The method may further include the following steps (6) to (10).

[0279] (6) a step of preparing a chamber system (II) different from the chamber system (I), wherein adjacent chambers are separated from each other by a semipermeable membrane that allows the substance to be measured to pass therethrough;

[0280] (7) a step of adding a receptor solution containing the biological sample (A) to one chamber (receptor side chamber) in the chamber system (II) (e.g., Figure 2 a and Figure 2 f);

[0281] (8) a step of adding a donor solution containing the biological sample (B) and the analyte to a chamber (donor side chamber) different from the acceptor side chamber in the chamber system (II) (e.g., Figure 2 b and Figure 2 e);

[0282] (9) a step of measuring the concentration of the substance to be measured in the acceptor solution of the step (7) and the donor solution of the step (8) over time (for example, Figure 2 c to Figure 2 d and Figure 2 g to Figure 2 h); and

[0283] (10) Using the data related to the concentration of the substance to be measured measured in the step (9), the relative f is calculated. u Than process.

[0284] In the method, the order of performing step (7) and step (8) is not particularly limited. For example, step (8) may be performed after step (7), step (7) may be performed after step (8), or step (7) and step (8) may be performed simultaneously or in parallel (for example, Figure 2 a to Figure 2 b and Figure 2 e to Figure 2 f).

[0285] Hereinafter, an example of the present invention will be described with reference to the drawings. Figure 2 ( Figure 2 a to Figure 2 d, Figure 2 e to Figure 2 h) Represents dialysis chamber II during analysis. Figure 2 a represents a state where a receptor solution containing the first biological sample (A) containing the non-specific protein 4 is added to the receptor side chamber 20 (black arrow), wherein the non-specific protein 4 is present in the receptor solution in the receptor side chamber 20 . Figure 2b represents the state when a donor solution containing a second biological sample (B) containing a non-specific protein 5 and a measurement object substance 3 is added to the donor side chamber 10 (black arrow), wherein the measurement object substance 3 and the non-specific protein 5 are present in the donor solution of the donor side chamber 10. Figure 2 c shows the situation at the initial stage when the analyte starts to move from the donor-side chamber 10 to the acceptor-side chamber 20 . Figure 2 d represents the state of the stage of dialysis in which the analyte moves from the donor chamber 10 to the acceptor chamber 20, or further moves from the acceptor chamber 20 to the donor chamber 10 in the reverse direction and approaches equilibrium. The concentration of the analyte in the solution added to each chamber is measured over time from the initial stage.

[0286] Figure 2 e to 2h means Figure 2 a and Figure 2 b is an example of a schematic diagram of the situation after the operation sequence is changed.

[0287] Each step (6) to (10) can be implemented in the same manner as the above steps (1) to (5). That is, after the steps (6) to (8), the relative f can be calculated using the time-dependent data of the concentration of the substance to be measured in the donor solution and the receptor solution measured in the step (9). u Ratio (process (10)).

[0288] In the present invention, the concentration changes of the substance to be measured in the solutions added to the two chambers in steps (1) to (4) over time can be introduced into a calculation formula in a dynamic analysis software in which predetermined parameters are incorporated, and the relative f between species differences can be obtained. u In addition, the concentration changes of the substances to be measured in the solutions added to the two chambers in steps (6) to (9) over time can be imported into the calculation formula in the dynamic analysis software with the specified parameters for fitting processing to obtain the relative f between the species differences. u The relative f obtained by such steps (1) to (5) is u ratio (hereinafter sometimes referred to as "relative f u Ratio (a)") and the relative f obtained by steps (6) to (10) u ratio (hereinafter sometimes referred to as "relative f u Since the same biological sample and the substance to be measured are used, the ratio (b)" has the same value in theory.

[0289] Therefore, by comparing the relative f obtained independently by steps (1) to (5) and steps (6) to (10), u ratio (hereinafter, sometimes referred to as "relative f u Ratio (a)", "relative fu Ratio (b)”), it can be verified that each relative f u For example, relative to f u Ratio (a) / relative f u When the ratio (b) is preferably 0.8 or more and 1.2 or less, and more preferably 0.9 or more and 1.1 or less, it can be evaluated as the relative f u Higher accuracy than.

[0290] Furthermore, the concentration changes of the substances to be measured in the solutions added to the four chambers in steps (1) to (4) and steps (6) to (9) over time, i.e., the data related to the concentrations of the substances to be measured measured in steps (4) and (9) can be imported into a calculation formula in a dynamic analysis software incorporating predetermined parameters and fitted to obtain the relative f between the species differences. u By this method, the relative f between species can be further improved. u As described in the examples, the relative f obtained by the method of the present invention is u The value is the same as the f obtained by the reference example method. u The ratio of the values ​​(absolute values) also matches well.

[0291] According to the present invention, for example, when calculating f from a biological sample u Compared with the method of measuring absolute value, it is possible to simultaneously evaluate the relative f between multiple, for example, 2 to 5 biological samples. u ratio, so it is easy to obtain the relative f u For example, to find the relative f between two biological samples u When the relative f between three or more biological samples is evaluated simultaneously, two chambers separated by an equilibrium dialysis membrane are prepared, and solutions containing the biological samples of interest are added to the two chambers for measurement. u When measuring a value ratio, only chambers of the same number as the types of biological samples can be prepared, and they can be separated by an equilibrium dialysis membrane through which the substance to be measured can permeate so that the substance to be measured can diffuse freely. A solution containing the biological sample as the object can be added to each chamber for measurement. Alternatively, two chambers separated by an equilibrium dialysis membrane can be prepared, and solutions containing one or more biological samples can be added to each chamber for measurement.

[0292] In addition, in the present invention, since the relative f relative to two or more biological samples is obtained, u The value of the ratio is thus different from the small value f that is prone to errors. u Compared with the method of value, it can minimize the error and can perform f with higher accuracy. uFurthermore, by setting a biological sample as a reference, such as a reference serum, the validity of each subsequent experiment can be easily verified.

[0293] Furthermore, in the present invention, by adopting dynamic analysis, not only can the relative f be measured in a short time, but also u The relative f ratio can be determined more accurately, and the influence of the time-dependent degradation of the biological sample (the time-dependent change of pH, the change of the solution amount, etc.) can be suppressed to a minimum. u Furthermore, even when the concentration of the substance to be measured in the solution is difficult to reach an equilibrium state, by using dynamic analysis, it is possible to obtain the f measured in the state before reaching an equilibrium state and after reaching an equilibrium state. u The relative f u Therefore, it is particularly effective when the protein binding rate of the analyte is high.

[0294] Provides f from a biological sample by dynamic analysis u When the value is u The average value of Pmem in a known compound group, however, the present invention has a feature that the semipermeable membrane permeation rate (Pmem [m / s]) can be analyzed even without obtaining an experimental numerical value. For example, even in the case of a compound with a large molecular weight such as cyclosporine (molecular weight: 1202.61), by setting Pmem to an arbitrary intrinsic value in the analysis, a relative f can be obtained. u Compare.

[0295] Prediction methods of drug clearance and distribution volume

[0296] The method for predicting drug clearance rate of the present invention comprises using the relative f u A method for predicting drug clearance in species derived from other biological samples by comparing drug clearance data in species derived from one biological sample.

[0297] The “clearance rate” (CL) is defined as the disappearance rate (v) of the drug divided by the concentration (C) at that moment (CL=v / C), and is expressed as the volume of drug that can be removed per unit time (mL / min).

[0298] As the f obtained by the method of the present invention, uFor the method of predicting the clearance rate, a known method can be used. For example, the f of the drug is applied in the known human clearance estimation method (FCIM: fu-corrected intercept method (Non-patent document 6: Tang, H. and M. Mayersohn (2005). "Anovel model for prediction of human drug clearance by allometric scaling." Drug Metab Dispos 33 (9): 1297-1303.)). u The clearance in humans can be predicted by comparing the clearance data in vivo (e.g., mice, rats, dogs, or monkeys).

[0299] The prediction method of the FCIM method is as follows.

[0300] Apply the clearance rates of various animals to the SA (Simple Allometry) formula shown below to optimize a and b;

[0301] CL=a(weight) b

[0302] (a: scale factor, b: scale index)

[0303] According to the above a and Rf u (Relative f u Ratio (rat / human)), the predicted human clearance was calculated by the following formula;

[0304] CL 人 =33.35x(a / Rf u ) 0.77

[0305] The method for predicting drug distribution volume of the present invention comprises using the relative f u A method for predicting the volume of distribution of a drug in species derived from other biological samples by comparing the volume of distribution data of the drug in a species derived from one biological sample.

[0306] "Volume of distribution" (V) refers to the theoretical amount of fluid required to contain the total amount of administered drug in the body at the same concentration as in blood or plasma. Volume of distribution = amount of drug in the body / blood concentration (eg, L).

[0307] As f obtained by the above method, uThe method for predicting the distribution volume can be changed as needed to use a known method. For example, in the known method for estimating the distribution volume species difference (Non-patent document 7: Berry, LM, et al. (2011). "Species differences in distribution and prediction of human V (ss) from preclinical data." Drug Metab Dispos 39 (11): 2103-2116.), the drug f is used. u By comparing the measured steady-state volume of distribution (e.g., mouse, rat, dog, or monkey, etc.), the expected volume of distribution (e.g., human) can be predicted.

[0308] For example, the following method for predicting the volume of distribution is used: u Method for estimating corrected distribution volume (Non-Patent Document 7).

[0309] Steady-state volume of distribution (Vss, [L])

[0310] Use f u Corrected steady-state volume of distribution (Vss u, [L] = Vss / fu, [L])

[0311] SA (Simple Allometry): Vss u=a (weight) b

[0312] (a: scale factor, b: scale index)

[0313] Here, the f of the animal species used as reference u Let fu,ref, which is relative to the reference animal species f u When the ratio is set to Rfu (=fu / fu,ref), the above SA formula can be transformed into the following formula.

[0314] Vss / Rfu=fu,refxa(weight) b

[0315] By optimizing fu, ref xa and b using measured Vss and Rfu of multiple animal species, Vss / Rfu can be predicted for any species, and Rfu can be used to predict Vss. u The absolute value of does not need to be measured in any species.

[0316] All prior art documents cited in this specification are incorporated into this specification as reference. Example

[0317] Hereinafter, preferred specific embodiments of the present invention will be described as examples, but the present invention is not limited to these.

[0318] [Example 1]

[0319] Obtaining relative f between species through dynamic analysis u Ratio (Measurement object: Nifedipine)

[0320] [Preparation of Donor Solution]

[0321] First, nifedipine was used as the test object, and a 10 mmol / L solution and a 1 mmol / L solution were prepared. Specifically, 0.482 mL of dimethyl sulfoxide (DMSO) was added to 1.67 mg of powdered nifedipine to prepare a 10 mmol / L nifedipine solution. Next, 180 μL of DMSO was added to 20 μL of the 10 mmol / L nifedipine solution to prepare a 1 mmol / L nifedipine solution.

[0322] Next, various serums (mouse serum, rat serum, dog serum, monkey serum, human serum) were dissolved. The various serums dissolved were collected, and 9 times the amount of phosphate buffered saline and 1 / 1000 times the amount of nifedipine 1mmol / L solution were added to prepare donor solutions with a serum concentration of 10% (volume ratio), a nifedipine concentration of 1μmol / L, and a DMSO concentration of 0.1% (volume ratio). The specific amount of each sample used is shown in Table 1 below.

[0323] In addition, each sample used was as follows.

[0324] Nifedipine: “Nifedipine”, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight: 346.335, CLogP: 3.12

[0325] DMSO: "CultureSure (registered trademark) DMSO", manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0326] Mouse serum: "Mouse (CD-1 (ICR) serum", Valley Biomedical

[0327] Rat serum: “Rat (SD (Crl:CD)) serum”, manufactured by Valley Biomedical

[0328] Dog serum: "Beagle mixed serum", made by Japan SLC

[0329] Monkey serum: "Monkey (Cynomolgus) serum", Valley Biomedical

[0330] Human serum: "Human True A serum, pool of donors", manufactured by BIOPREDIC International

[0331] Phosphate buffered saline (PBS): "Dulbecco's phosphate buffered saline (without Ca and Mg)", manufactured by NACALAITESQUE

[0332] [Table 1]

[0333]

[0334] [Preparation of Acceptor Solution]

[0335] A receptor solution having a serum concentration of 10% (volume ratio) and a DMSO concentration of 0.1% (volume ratio) was prepared in the same manner as the donor solution except that a DMSO solution was added instead of the 1 mmol / L nifedipine solution.

[0336] [Dialysis reaction]

[0337] First, a rapid equilibrium dialysis device ("RED (Rapid Equilibrium Dialysis) device", dialysis membrane MWCO: 8 kDa, manufactured by Thermo Fisher SCIENTIFICC) was prepared, and 350 μL of a specific donor solution was dispensed into each white chamber, and 200 μL of a specific receptor solution was dispensed into each red chamber. The donor solution or receptor solution dispensed into each chamber is shown in Table 2 below.

[0338] In Table 2, Table 2-1 is a case where human and mouse serum is used. It shows a total of 6 cases where human serum is set as the donor side and mouse serum is set as the recipient side (tests A to C; same conditions), and where mouse serum is set as the donor side and human serum is set as the recipient side (tests D to F: same conditions), and the measurement is performed at 0.5 hours, 2 hours, 4 hours, 16 hours, and 24 hours. Table 2-2 is a case where human and rat serum is used, Table 2-3 is a case where human and dog serum is used, and Table 2-4 is a case where human and monkey serum is used, and respectively shows the same 6 modes as the above combination of human and mouse, and the measurement is performed at 0.5 hours, 2 hours, 4 hours, 16 hours, and 24 hours.

[0339] Next, the upper part of the RED device was sealed with an aluminum seal ("3M (registered trademark) microplate sealing tape", upper seal of the SURED device, manufactured by 3M Japan Co., Ltd.), and the dialysis reaction was started with an oscillator ("MB100-4AThermo-shaker", manufactured by Hangzhou Allsheng Instruments Co., Ltd.) (set value: stirring speed Rotation 800 rpm, 37°C).

[0340] (Table 2) Chamber types using sera from humans, mice, rats, dogs, and monkeys

[0341] (Table 2-1) Human / Mouse

[0342]

[0343] (Table 2-2) Human / rat

[0344]

[0345] (Table 2-3) Human / Dog

[0346]

[0347] (Table 2-4) Human / Monkey

[0348]

[0349] [Sampling after dialysis reaction]

[0350] At 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction, 20 μL of solution was collected from each chamber into which the donor solution and the acceptor solution were dispensed, mixed with 180 μL of quenching solution 1 (methanol / acetonitrile / DMSO = 85 / 85 / 10 volume ratio) and sealed. The method for preparing quenching solution 1 is as follows: 80 μL of indomethacin solution (concentration 200 μg / mL) was added to 80 mL of methanol / acetonitrile = 1 / 1 solution. 3 mL of DMSO was added to 51 mL of the adjusted mixed solution to prepare quenching solution 1. In addition, the donor solution before the RED device was dispensed was used as the 0 hour value sample, and the same amount as above was collected, mixed with quenching solution 1 in the same way as above and sealed. Each sample was stored at 4°C until it was used for pretreatment.

[0351] [Pretreatment of samples after sampling]

[0352] The total amount of the sample after sampling was transferred to a filter plate ("Multi Screen (registered trademark) Solvinert, filter plate 0.45 μm, low binding hydrophilic PTFE", manufactured by MILLIPORE), and the 96-well PP microplate was stacked downward, and filtered with a filter using a centrifuge ("CF10RXII", manufactured by HITACHI) (3000 rpm, 5 minutes). The filtered sample was sealed with a sealing adhesive and used as a measurement sample for LC-MS / MS.

[0353] [Concentration measurement of target substance by LC-MS / MS]

[0354] For each of the LC-MS / MS measurement samples, the concentration of nifedipine in the solution added to each chamber was measured using the following measurement instrument and measurement conditions. In the Examples, Comparative Examples, or Reference Examples in this specification, when the concentration of a specific target substance is measured in a plurality of wells using the same biological sample type (e.g., serum), the average value of the concentrations measured in each well of the same biological sample type (e.g., serum) group is used for the measurement of the nifedipine concentration in each well. u For example, in this embodiment, the average value of the concentration of the measurement object substance in tests A to C of the same serum group and the average value of the concentration of the measurement object substance in tests D to F of the same serum group are used for f u Calculation of ratio.

[0355] (Measurement Instrument)

[0356] LC: Waters Acquity UPLC

[0357] MS: Xevo TQ-S (Waters Corporation, Milford, Massachusetts)

[0358] Column: YMC-Triart C18 column, column size 2.0x30mm, particle size 1.9μm, pore size 12nm

[0359] (Measurement conditions)

[0360] Column temperature: 50°C (set value)

[0361] Solvent: A) 0.1% formic acid aqueous solution

[0362] B) 0.1% formic acid acetonitrile solution

[0363] Gradient conditions:

[0364] [Table 3]

[0365]

[0366] Flow rate: 0.6mL / min

[0367] Automatic sampler temperature: 10℃ (set value)

[0368] Injection Volume: 1μL

[0369] Preparation of calibration curve samples: To 10 μL of a 1 mmol / L nifedipine solution dissolved in DMSO, add 90 μL of DMSO to prepare a 100 μmol / L nifedipine solution. Based on the 100 μmol / L nifedipine solution, prepare the following dilution series (1)-(8) and a blank solution.

[0370] [(1) To 24 μL of a 100 μmol / L nifedipine solution, add 376 μL of DMSO to prepare a 6000 nmol / L nifedipine solution.

[0371] (2) To 120 μL of 6000 nmol / L nifedipine solution, add 240 μL of DMSO to prepare a 2000 nmol / L nifedipine solution.

[0372] (3) To 120 μL of 2000 μmol / L nifedipine solution, add 280 μL of DMSO to prepare a 600 nmol / L nifedipine solution.

[0373] (4) To 120 μL of the 600 nmol / L nifedipine solution, add 240 μL of DMSO to prepare a 200 nmol / L nifedipine solution.

[0374] (5) To 120 μL of 200 nmol / L nifedipine solution, add 280 μL of DMSO to prepare a 60 nmol / L nifedipine solution.

[0375] (6) To 120 μL of 60 nmol / L nifedipine solution, add 240 μL of DMSO to prepare a 20 nmol / L nifedipine solution.

[0376] (7) To 120 μL of 20 μmol / L nifedipine solution, add 280 μL of DMSO to prepare a 6 nmol / L nifedipine solution.

[0377] (8) Add 240 μL of DMSO to 120 μL of 6 nmol / L nifedipine solution to prepare a 2 nmol / L nifedipine solution. Use 200 μL of DMSO as a blank solution.]

[0378] For 10 μL of the dilution series of (1)-(8) and 10 μL of the blank solution, 20 μL of the matrix solution (animal or human serum / PBS / DMSO = 10 / 90 / 0.1, volume ratio) was added, and further 170 μL of the quenching solution 2 [for 80 mL of the methanol / acetonitrile = 1 / 1 solution, 80 μL of the indomethacin solution (concentration 200 μg / mL) was added] to prepare the calibration curve samples. The calibration curve samples were pretreated in the same way as the samples after sampling. In addition, since the concentration of the dilution series of (1)-(8) was adjusted to 2 times when the actual sample was used as the reference, the concentration adjusted by the dilution series was corrected as half the amount to obtain the concentration of the calibration curve sample.

[0379] Concentrations of calibration curve samples: (8) 1 nmol / L, (7) 3 nmol / L, (6) 10 nmol / L, (5) 30 nmol / L, (4) 100 nmol / L, (3) 300 nmol / L, (2) 1000 nmol / L, (1) 3000 nmol / LMS conditions (settings): ESI positive, Capillary (kV) 2.00, Cone (V) 25.00, Source Offset (V) 50.0, Source Temperature (℃) 150, Desolvation Temperature (℃) 600, Desolvation Gas Flow (L / Hr) 1000, Cone Gas Flow (L / Hr) 150, Collision Gas Flow (mL / Min) 0.20, Nebuliser Gas Flow (Bar) 7.00

[0380] [Table 4]

[0381]

[0382] [obtained through dynamic analysis u Compare]

[0383] The obtained concentration transition data was input using dynamic analysis software ("SimBiology (registered trademark) / MATLAB, version information: 9.7.0.1190202 (R2019b)", manufactured by The MathWorks, Inc.), and each parameter and f were calculated using the following conditions and the nonlinear least squares method (lsqnonlin): u In addition, the set concentration of the substance to be measured in the donor solution when the donor solution is prepared is used as the concentration of the substance to be measured contained in the donor solution at the time of 0 hours from the start of the dialysis reaction.

[0384] (Formula 1):

[0385]

[0386] (Formula 2):

[0387]

[0388] (Formula 3):

[0389]

[0390] Here, each parameter represents the following.

[0391] V1: Volume of donor solution (fixed value, 3.5×10 -4 L)

[0392] V2: Volume of receptor solution (fixed value, 2.0×10 -4 L)

[0393] fu1: fu value of the substance to be measured relative to the biological sample contained in the donor solution (calculated value)

[0394] fu2: fu value of the substance to be measured relative to the biological sample contained in the receptor solution (calculated value)

[0395] C1: Concentration of the analyte in the donor solution (total concentration: the sum of bound and unbound analytes. However, excluding analytes adsorbed to the equilibrium dialysis device).

[0396] (measured value, nmol / L)

[0397] C2: Concentration of the analyte in the receptor solution (total concentration: the sum of bound and unbound analyte). (Measured value, nmol / L)

[0398] PS: Dialysis membrane clearance of unbound analyte (fixed value, 1.0 × 10 -4 L / h)

[0399] CLad: Adsorption clearance rate of the measurement target substance in the donor side chamber (variable value, L / h)

[0400] koff: Dissociation rate constant of the analyte in the donor chamber (variable value, h -1 )

[0401] Xad: adsorption amount of the substance to be measured in the donor side chamber (variable value, nmol)

[0402] The number "1" in each symbol indicates the donor side, and "2" indicates the acceptor side.

[0403] Convergence calculation (fitting):

[0404] In the dynamic analysis, the results of the biological sample replacement in the donor solution and the receptor solution are combined as a group to calculate the relative f u In the default settings of Fit Data (SimBiology Model Analyzer, Program), the following conditions (Model, Reaction, Rules, Definition, Estimated Parameters) are recorded and implemented. Figure 3 shown.

[0405] Reaction:

[0406] Eq1.Donor1<->Eq1.Acceptor1:+fu1*PS*Eq1.Donor1 / Vd1-fu2*PS*Eq1.Acceptor1 / Vd2

[0407] Eq1.Donor1->Eq1.Ad:+CL1*fu1*Eq1.Donor1 / Vd1-(Koff1*Eq1.Ad)

[0408] Eq2.Donor2<->Eq2.Acceptor2:+fu2*PS*Eq2.Donor2 / Vd1-fu1*PS*Eq2.Acceptor2 / Vd2

[0409] Eq2.Donor2->Eq2.Ad:+CL2*fu2*Eq2.Donor2 / Vd1-(Koff2*Eq2.Ad)

[0410] Rules:

[0411] rule_1Eq1.Ca1=Eq1.Acceptor1 / Vd2 repeatedAssignment

[0412] rule_2Eq1.Cd1=Eq1.Donor1 / Vd1 repeatedAssignment

[0413] rule_3Eq2.Ca2=Eq2.Acceptor2 / Vd2 repeatedAssignment

[0414] rule_4Eq2.Cd2=Eq2.Donor2 / Vd1 repeatedAssignment

[0415] rule_5Eq1.Donor1=Dose1 initialAssignment

[0416] rule_6Eq2.Donor2=Dose2 initialAssignment

[0417] Definition:

[0418] When the first biological sample is a mouse and the second biological sample is a human, the following four data are used as a set for analysis.

[0419] Data 1 (Eq1.Ca1): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains mouse serum and the receptor solution contains human serum

[0420] Data 2 (Eq1.Cd1): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains mouse serum and the receptor solution contains human serum

[0421] Data 3 (Eq2.Ca2): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains mouse serum

[0422] Data 4 (Eq2.Cd2): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains mouse serum

[0423] Estimated Parameters:

[0424] exp(fu1), InitialValue=1, Bounds=[0.01 100];

[0425] exp(fu2), InitialValue=1, Bounds=[0.01 100];

[0426] exp(Koff1), InitialValue=1, Bounds=[0.01 1000000];

[0427] exp(Koff2), InitialValue=1, Bounds=[0.01 1000000];

[0428] exp(CL1), InitialValue=0.001, Bounds=[1e-07 0.1];

[0429] exp(CL2), InitialValue=0.001, Bounds=[1e-07 0.1];

[0430] exp(Dose1), InitialValue=0.2115, Bounds=[0.1 0.4];

[0431] exp(Dose2), InitialValue=0.2114, Bounds=[0.1 0.4];

[0432] Pooled fit, true

[0433] The f of the substance to be measured (nifedipine) calculated by the above-mentioned dynamic analysis method relative to each type u The ratio ( / person, serum concentration 1 / 10, person / person 1.00) is shown in Table 5. In addition, the concentration plots of the measurement target substance in the solution added to each chamber at the time of 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction and the fitting curves to these plots by dynamic analysis are shown in Table 5. Figure 4 As shown in (1) to (4).

[0434] [Table 5]

[0435] <![CDATA[Relative to f u Ratio ( / person)]]> Mouse 1.37 Rat 0.06 dog 1.40 monkey 1.10 people 1.00

[0436] [Comparative Example 1]

[0437] Obtain the relative f between species through static analysis u Ratio (Measurement object: Nifedipine)

[0438] A donor solution (10% mouse, rat, dog or monkey serum) and a receptor solution (10% human serum) were prepared in the same manner as in Example 1, and the dialysis reaction was started. The concentration of the substance to be measured in the solution added to each chamber at the time when 24 hours had passed since the start of the dialysis reaction was measured. The obtained data were applied to the following formula 4 to obtain the f of nifedipine in various sera (mouse, rat, dog or monkey): u When the concentration of a specific target substance is measured in multiple wells using the same serum, the average value of the concentration measured in each well of the same serum group is used for the ratio (relative to human, human / human is 1.00). u Calculation of ratio.

[0439] (Formula 4):

[0440]

[0441] The results are shown below.

[0442] [Table 6]

[0443] <![CDATA[f u Ratio( / person)]]> Mouse 1.25 Rat 0.12 dog 1.29 monkey 1.01 people 1.00

[0444] [Reference Example 1]

[0445] The f between species was obtained by static analysis (relative to buffer) u Ratio (Measurement object: Nifedipine)

[0446] [Preparation of donor solution]

[0447] A donor solution having a serum concentration of 10% (volume ratio), a nifedipine concentration of 1 μmol / L, and a DMSO concentration of 0.1% (volume ratio) was prepared in the same manner as in Example 1, except that the amounts of various sera (mouse, rat, dog, monkey, and human (A to E)), phosphate-buffered saline, and 1 mmol / L nifedipine solution were set to the amounts shown in Table 7.

[0448] [Table 7]

[0449]

[0450] [Preparation of receptor solution]

[0451] A 1 / 1000-fold amount of DMSO solution (20 μL) was added to phosphate buffered saline (PBS) (20 mL) to prepare a receptor solution (buffer) having a DMSO concentration of 0.1% (volume ratio).

[0452] [Dialysis reaction]

[0453] The dialysis reaction was carried out in the same manner as in Example 1 except that the solutions dispensed into each chamber were as shown in Table 8.

[0454] [Table 8]

[0455]

[0456] [Sampling after dialysis reaction]

[0457] Sampling was performed in the same manner as in Example 1, except that the sampling timing was set only to the time when 24 hours had passed from the start of the dialysis reaction.

[0458] [Pretreatment of samples after sampling and concentration measurement of target substances by LC-MS / MS]

[0459] The pretreatment of the sample after collection and the measurement of the concentration of the substance to be measured in the sample were performed in the same manner as in Example 1. When the concentration of a specific substance to be measured in a plurality of wells using the same serum, the average value of the concentrations measured in each well of the same serum group was used for the calculation of the concentration of the substance to be measured.u Calculation of ratio.

[0460] [Use various types of f u The relative f between species u Compare]

[0461] First, the concentration data obtained were applied to the following formula 5 to obtain the f of nifedipine in various serums (mouse, rat, dog, monkey or human): u value.

[0462] (Formula 5):

[0463]

[0464] Next, the above f u The value is applied to the following formula 6 to calculate the relative f for various people. u Compare.

[0465] (Formula 6):

[0466] f u Ratio = α / β

[0467] (where α represents the f of each species (mouse, rat, dog or monkey) u value, β represents the f u The result obtained by ) is shown below.

[0468] [Table 9]

[0469] <![CDATA[f u Ratio( / person)<!-- 31 --> ]]> Mouse 1.48 Rat 0.12 dog 1.37 monkey 1.17 people 1.00

[0470] [Example 2]

[0471] Obtaining relative f between species through dynamic analysis u Ratio (Measurement object: Cyclosporine )

[0472] [Preparation of donor solution]

[0473] First, cyclosporine was used as the test object, and a 10 mmol / L solution and a 1 mmol / L solution were prepared. Specifically, 0.566 mL of dimethyl sulfoxide (DMSO) was added to 6.81 mg of powdered cyclosporine ("cyclosporine", manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight 1202.61, CLogP: 14.36, number of amino acid residues 11) to prepare a 10 mmol / L solution of cyclosporine. Next, 180 uL of DMSO was added to 20 μL of the 10 mmol / L solution of cyclosporine to prepare a 1 mmol / L solution of cyclosporine.

[0474] Next, various sera (mouse serum, rat serum, dog serum, monkey serum, human serum) were dissolved. The various dissolved sera were collected, and 9 times the amount of phosphate buffered saline and 1 / 1000 times the amount of cyclosporine 1mmol / L solution were added to prepare donor solutions with a serum concentration of 10% (volume ratio), a cyclosporine concentration of 1μmol / L, and a DMSO concentration of 0.1% (volume ratio). The specific amount of each sample used is shown in Table 10 below.

[0475] The samples used were the same as those in Example 1 except for cyclosporine.

[0476] [Table 10]

[0477]

[0478] [Preparation of receptor solution]

[0479] The same method as described in Example 1 was carried out.

[0480] [Dialysis reaction]

[0481] The same method as described in Example 1 was carried out.

[0482] [Sampling after dialysis reaction]

[0483] The same method as described in Example 1 was carried out.

[0484] [Pretreatment of samples after sampling]

[0485] The same method as described in Example 1 was carried out.

[0486] [Concentration measurement of target substance by LC-MS / MS]

[0487] For each of the LC-MS / MS measurement samples, the cyclosporine concentration in the solution added to each chamber was measured using the following measurement instrument and measurement conditions. When the concentration of a specific target substance was measured in a plurality of wells using the same serum, the average value of the concentrations measured in each well of the same serum group was used for the calculation of the concentration of the specific target substance. u Calculation of ratio.

[0488] (Measurement Instrument)

[0489] LC: Waters Acquity UPLC

[0490] MS: Xevo TQ-S (Waters Corporation, Milford, Massachusetts)

[0491] Column: YMC-Triart Bio C4, column size 2.1x30mm, particle size 1.9μm, pore size 30nm

[0492] (Measurement conditions)

[0493] Column temperature: 40°C (set value)

[0494] Solvent: A) 0.1% formic acid aqueous solution

[0495] B) 0.1% formic acid acetonitrile solution

[0496] Gradient conditions:

[0497] [Table 11]

[0498]

[0499] Flow rate: 0.6mL / min

[0500] Automatic sampler temperature: 10℃ (set value)

[0501] Injection Volume: 1μL

[0502] Preparation of calibration curve samples: The same method as described in Example 1 was carried out except that cyclosporine was used instead of nifedipine.

[0503] Calibration curve 1nmol / L, 3nmol / L, 10nmol / L, 30nmol / L, 100nmol / L, 300nmol / L, 1000nmol / L, 3000nmol / L

[0504] MS conditions (set values): ESI positive, Capillary (kV) 2.00, Cone (V) 25.00, SourceOffset (V) 50.0, Source Temperature (℃) 150, Desolvation Temperature (℃) 600, Desolvation Gas Flow (L / Hr) 1000, Cone Gas Flow (L / Hr) 150, Collision Gas Flow(mL / Min)0.20、Nebuliser Gas Flow(Bar)7.00

[0505] [Table 12]

[0506]

[0507] [obtained through dynamic analysis u Compare]

[0508] The obtained concentration transition was inputted using the software for dynamic analysis ("SimBiology (registered trademark) / MATLAB, version information: 9.7.0.1190202 (R2019b)", manufactured by The Math Works, Inc.) in the same manner as in Example 1, and each parameter and f were calculated under the following conditions: u Compare.

[0509] The relational expressions involved in the dynamic analysis are the same as the equations 1 to 3 described in Example 1. The parameters in the equations are respectively represented as follows.

[0510] V1: Volume of donor solution (fixed value, 3.5×10 -4 L)

[0511] V2: Volume of receptor solution (fixed value, 2.0×10 -4 L)

[0512] fu1: fu value of the substance to be measured relative to the biological sample contained in the donor solution (calculated value)

[0513] fu2: fu value of the substance to be measured relative to the biological sample contained in the receptor solution (calculated value)

[0514] C1: Concentration of the analyte in the donor solution (total concentration: the sum of bound and unbound analytes, excluding analytes adsorbed to the equilibrium dialysis device) (measured value, nmol / L)

[0515] C2: Concentration of the analyte in the receptor solution (total concentration: the sum of bound and unbound analyte) (measured value, nmol / L)

[0516] PS: Dialysis membrane clearance of the substance to be measured (fixed value, 1.0×10 -5 L / h)

[0517] CLad: Adsorption clearance rate of the measurement target substance in the donor side chamber (variable value, L / h)

[0518] Koff: Dissociation rate constant of the analyte in the donor chamber (variable value, h -1 )

[0519] Xad: adsorption amount of the substance to be measured in the donor side chamber (variable value, nmol)

[0520] Fitting: The same parameters as in Example 1 were used except that the following parameters were used.

[0521] Estimated Parameters:

[0522] exp(fu1),InitialValue=1,Bounds=[0.01 100];

[0523] exp(fu2),InitialValue=1,Bounds=[0.01 100];

[0524] exp(Koff1),InitialValue=1,Bounds=[0.01 1000000];

[0525] exp(Koff2),InitialValue=1,Bounds=[0.01 1000000];

[0526] exp(CL1),InitialValue=0.001,Bounds=[1e-07 0.1];

[0527] exp(CL2),InitialValue=0.001,Bounds=[1e-07 0.1];

[0528] exp(Dose1),InitialValue=0.2115,Bounds=[0.1 0.5];

[0529] exp(Dose2),InitialValue=0.2114,Bounds=[0.1 0.5];

[0530] The f of the substance to be measured (cyclosporine) relative to various types calculated by the above-mentioned dynamic analysis method u The ratio ( / person, serum concentration 1 / 10, person / person 1.00) is shown in Table 13. In addition, the concentration plots of the measurement target substance in the solution added to each chamber at the time of 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction and the fitting curves to these plots by dynamic analysis are shown in Table 13. Figure 5 As shown in (1) to (4).

[0531] [Table 13]

[0532] <![CDATA[Relative to f u Ratio (relative person)]]> Mouse 0.63 Rat 0.99 dog 0.82 monkey 2.01 people 1.00

[0533] [Comparative Example 2]

[0534] Relative fu ratio between species obtained by static analysis (substance to be measured: cyclosporine)

[0535] A donor solution (10% mouse, rat, dog or monkey serum) and a receptor solution (10% human serum) were prepared in the same manner as in Example 2, and the dialysis reaction was started. The concentration of the substance to be measured in the solution added to each chamber at the time when 24 hours had passed since the start of the dialysis reaction was measured. When the concentration of a specific substance to be measured in a plurality of wells using the same serum, the average value of the concentrations measured in each well of the same serum group was used as the average value. u The obtained data were applied to the following formula 7 to obtain the f of cyclosporine in various serums (mouse, rat, dog or monkey): u Ratio (relative to person, person / person is 1.00).

[0536] (Formula 7):

[0537]

[0538] The results are shown below.

[0539] [Table 14]

[0540] <![CDATA[f u Ratio( / person)]]> Mouse 0.23 Rat 0.45 dog 0.27 monkey 0.78 people 1.00

[0541] [Reference Example 2]

[0542] The f between species was obtained by static analysis (relative to buffer) u Ratio (Measurement object: cyclosporine)

[0543] [Preparation of donor solution]

[0544] A donor solution having a serum concentration of 10% (volume ratio), a cyclosporine concentration of 1 μmol / L, and a DMSO concentration of 0.1% (volume ratio) was prepared in the same manner as in Example 2, except that the amounts of various sera (mouse, rat, dog, monkey, and human), phosphate-buffered saline, and 1 mmol / L cyclosporine solution were set to the amounts shown in Table 15.

[0545] [Table 15]

[0546]

[0547] [Preparation of receptor solution]

[0548] The same method as described in Reference Example 1 was carried out.

[0549] [Dialysis reaction]

[0550] The same method as described in Reference Example 1 was carried out.

[0551] [Sampling after dialysis reaction]

[0552] The same method as described in Reference Example 1 was carried out.

[0553] [Pretreatment of samples after sampling and concentration measurement of target substances by LC-MS / MS]

[0554] The same method as described in Example 2 was carried out.

[0555] [Use various types of f u The relative f between species u Compare]

[0556] The same method as described in Reference Example 1 was carried out.

[0557] The results are shown below.

[0558] [Table 16]

[0559] <![CDATA[f u Ratio (relative person)]]> Mouse 1.14 Rat 1.79 dog 1.31 monkey 2.55 people 1.00

[0560] [Example 3]

[0561] Obtaining relative f between species through dynamic analysis u Ratio (Measurement object: Ibuprofen)

[0562] [Preparation of donor solution]

[0563] First, ibuprofen was used as the test object, and a 10 mmol / L solution and a 1 mmol / L solution were prepared. Specifically, 0.926 mL of dimethyl sulfoxide (DMSO) was added to 1.91 mg of powdered ibuprofen ("ibuprofen", manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight 206.28, CLogP: 3.68) to prepare a 10 mmol / L ibuprofen solution. Next, 180 uL of DMSO was added to 20 uL of the 10 mmol / L ibuprofen solution to prepare a 1 mmol / L ibuprofen solution.

[0564] Next, various serums (mouse serum, rat serum, dog serum, monkey serum, human serum) were dissolved. The various serums dissolved were collected, and 9 times the amount of phosphate buffered saline and 1 / 1000 times the amount of ibuprofen 1mmol / L solution were added to prepare donor solutions with a serum concentration of 10% (volume ratio), an ibuprofen concentration of 1μmol / L, and a DMSO concentration of 0.1% (volume ratio). The specific amount of each sample used is shown in Table 17 below.

[0565] The samples used were the same as those in Example 1 except for ibuprofen.

[0566] [Table 17]

[0567]

[0568] [Preparation of receptor solution]

[0569] The same method as described in Example 1 was carried out.

[0570] [Dialysis reaction]

[0571] The same method as described in Example 1 was carried out.

[0572] [Sampling after dialysis reaction]

[0573] The same method as described in Example 1 was carried out.

[0574] [Pretreatment of samples after sampling]

[0575] The same method as described in Example 1 was carried out.

[0576] [Concentration measurement of target substance by LC-MS / MS]

[0577] For each of the LC-MS / MS measurement samples, the ibuprofen concentration in the solution added to each chamber was measured using the following measurement instrument and measurement conditions. When the concentration of a specific target substance was measured in a plurality of wells using the same serum as in Example 1, the average value of the concentrations measured in each well of the same serum group was used for the calculation of the concentration of the specific target substance. u Calculation of ratio. (Measurement instrument)

[0578] LC: Waters Acquity UPLC

[0579] MS: Xevo TQ-S (Waters Corporation, Milford, Massachusetts)

[0580] Column: YMC-Triart C18 column, column size 2.0x30mm, particle size 1.9μm, pore size 12nm

[0581] (Measurement conditions)

[0582] Column temperature: 50°C (set value)

[0583] Solvent:

[0584] A) 10mmol / L ammonium formate aqueous solution (water: ammonium formate solution 1mol / L, 1000ml:10ml)

[0585] B) 10mmol / L ammonium formate MeOH solution (MeOH: ammonium formate solution 1mol / L, 1000ml:10ml)

[0586] Gradient conditions:

[0587] [Table 18]

[0588]

[0589] Flow rate: 0.6mL / min

[0590] Automatic sampler temperature: 10℃ (set value)

[0591] Injection Volume: 10μL

[0592] Preparation of calibration curve samples: To 10 μL of a 1 mmol / L solution of ibuprofen dissolved in DMSO, 90 μL of DMSO was added to prepare a 100 μmol / L solution of ibuprofen. Based on the 100 μmol / L solution of ibuprofen, the following dilution series (1) to (9) and a blank solution were prepared.

[0593] [(1) To 24 μL of a 100 μmol / L ibuprofen solution, 376 μL of DMSO was added to prepare a 6000 nmol / L ibuprofen solution.

[0594] (2) To 240 μL of the ibuprofen 6000 nmol / L solution, add 240 μL of DMSO to prepare an ibuprofen 3000 nmol / L solution.

[0595] (3) To 240 μL of the 3000 nmol / L ibuprofen solution, 120 μL of DMSO was added to prepare a 2000 nmol / L ibuprofen solution.

[0596] (4) To 120 μL of the 2000 nmol / L ibuprofen solution, add 280 μL of DMSO to prepare a 600 nmol / L ibuprofen solution.

[0597] (5) To 240 μL of the 600 nmol / L ibuprofen solution, add 240 μL of DMSO to prepare a 300 nmol / L ibuprofen solution.

[0598] (6) To 240 μL of the 300 nmol / L ibuprofen solution, 120 μL of DMSO was added to prepare a 200 nmol / L ibuprofen solution.

[0599] (7) To 120 μL of the 200 nmol / L ibuprofen solution, add 280 μL of DMSO to prepare a 60 nmol / L ibuprofen solution.

[0600] (8) To 240 μL of 60 nmol / L ibuprofen solution, add 240 μL of DMSO to prepare a 30 nmol / L ibuprofen solution.

[0601] (9) Add 120 μL of DMSO to 240 μL of 30 nmol / L ibuprofen solution to prepare 20 nmol / L ibuprofen solution. Use 200 μL of DMSO as a blank solution.

[0602] For 10 μL of the dilution series of (1)-(9) and 10 μL of the blank solution, 20 μL of the matrix solution (animal or human serum / PBS / DMSO = 10 / 90 / 0.1, volume ratio) was added, and further 170 μL of the quenching solution 2 [for 80 mL of the methanol / acetonitrile = 1 / 1 solution, 80 μL of the indomethacin solution (concentration 200 μg / mL) was added] to prepare the calibration curve samples. The calibration curve samples were pretreated in the same way as the samples after sampling. In addition, since the concentration of the dilution series of (1)-(9) was adjusted to 2 times when the actual sample was used as the reference, the concentration adjusted by the dilution series was corrected as half the amount to obtain the concentration of the calibration curve sample.

[0603] Concentrations of calibration curve samples: (9) 10 nmol / L, (8) 15 nmol / L, (7) 30 nmol / L, (6) 100 nmol / L, (5) 150 nmol / L, (4) 300 nmol / L, (3) 1000 nmol / L, (2) 1500 nmol / L, (1) 3000 nmol / L

[0604] Calibration curve 10nmol / L, 15nmol / L, 30nmol / L, 100nmol / L, 150nmol / L, 300nmol / L, 1000nmol / L, 1500nmol / L, 3000nmol / L

[0605] MS conditions (set values): (ESI negative), Capillary (kV) 2.00, Cone (V) 25.00, SourceOffset (V) 50.0, Source Temperature (℃) 150, Desolvation Temperature (℃) 600, Desolvation Gas Flow (L / Hr) 1000, Cone Gas Flow (L / Hr) 150, Collision Gas Flow(mL / Min)0.20、Nebuliser Gas Flow(Bar)7.00

[0606] [Table 19]

[0607]

[0608] [obtained through dynamic analysis u Compare]

[0609] Using the method described in Example 1, the f of ibuprofen was determined. uRegarding mouse / human, since the concentration of mouse receptors was below LLOQ and could not be detected, the results of the human donor → mouse receptor assay were not used for analysis, and f was obtained only from the results of the mouse donor → human receptor assay, i.e., a one-way analysis. u Compare.

[0610] Fitting (Human / Mouse):

[0611] In the case of human / mouse, the following conditions were described in the default settings of Fit Data (SimBiology Model Analyzer, Program) and implemented.

[0612] Definition:

[0613] When the first biological sample is a mouse and the second biological sample is a human, the following two data are used as a set for analysis.

[0614] Data 1 (Eq1.Ca1): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains mouse serum and the receptor solution contains human serum

[0615] Data 2 (Eq1.Cd1): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains mouse serum and the receptor solution contains human serum

[0616] Estimated Parameters:

[0617] exp(fu1),InitialValue=1,Bounds=[0.01 100];

[0618] exp(fu2),InitialValue=1,Bounds=[0.01 100];

[0619] exp(Koff1),InitialValue=1,Bounds=[0.01 1000000];

[0620] exp(CL1),InitialValue=0.001,Bounds=[1e-07 0.1];

[0621] exp(Dose1),InitialValue=0.2115,Bounds=[0.1 0.5];

[0622] Pooled fit,true

[0623] Other setting values: Same as Example 1.

[0624] Fit (human / rat, human / dog, human / monkey):

[0625] In the case of human / rat, human / dog, and human / monkey, the same parameters as in Example 1 were used except that the following parameters were used.

[0626] Estimated Parameters:

[0627] exp(fu1),InitialValue=1,Bounds=[0.01 100];

[0628] exp(fu2),InitialValue=1,Bounds=[0.01 100];

[0629] exp(Koff1),InitialValue=1,Bounds=[0.01 1000000];

[0630] exp(Koff2),InitialValue=1,Bounds=[0.01 1000000];

[0631] exp(CL1),InitialValue=0.001,Bounds=[1e-07 0.1];

[0632] exp(CL2),InitialValue=0.001,Bounds=[1e-07 0.1];

[0633] exp(Dose1),InitialValue=0.2115,Bounds=[0.1 0.5];

[0634] exp(Dose2),InitialValue=0.2114,Bounds=[0.1 0.5];

[0635] The f of the substance to be measured (ibuprofen) calculated by the above dynamic analysis method relative to each type u The ratio ( / person, serum concentration 1 / 10, person / person 1.00) is shown in Table 20. In addition, the concentration plots of the measurement target substance in the solution added to each chamber at the time of 0.5 hours, 2 hours, 4 hours, 16 hours and 24 hours from the start of the dialysis reaction and the fitting curves to these plots by dynamic analysis are shown in Table 20. Figure 6 As shown in (1) to (4).

[0636] [Table 20]

[0637] <![CDATA[Relative to f u Ratio (relative person)]]> Mouse 17.02 Rat 4.58 dog 1.06 monkey 2.41 people 1.00

[0638] [Comparative Example 3]

[0639] Relative fu ratio between species obtained by static analysis (substance to be measured: ibuprofen)

[0640] A donor solution (10% mouse, rat, dog or monkey serum) and a receptor solution (10% human serum) were prepared in the same manner as in Example 3, and the dialysis reaction was started. The concentration of the substance to be measured in the solution added to each chamber at the time when 24 hours had passed since the start of the dialysis reaction was measured. When the concentration of a specific substance to be measured in a plurality of wells using the same serum, the average value of the concentrations measured in each well of the same serum group was used as the average value. u The obtained data were applied to the following formula 8 to obtain the f of ibuprofen in various serums (mouse, rat, dog or monkey): u Ratio (relative to person, person / person is 1.00).

[0641] (Formula 8):

[0642]

[0643] The results are shown below.

[0644] [Table 21]

[0645] <![CDATA[f u Ratio( / person)]]> Mouse 7.58 Rat 3.37 dog 0.59 monkey 1.26 people 1.00

[0646] [Reference Example 3]

[0647] The f between species was obtained by static analysis (relative to buffer) u Ratio (Measurement object: Ibuprofen)

[0648] [Preparation of donor solution]

[0649] Except that the amounts of various sera (mouse, rat, dog, monkey, human), phosphate-buffered saline and ibuprofen 1 mmol / L solution were set to the amounts shown in Table 22, a donor solution with a serum concentration of 10% (volume ratio), an ibuprofen concentration of 1 μmol / L, and a DMSO concentration of 0.1% (volume ratio) was prepared in the same manner as in Example 2.

[0650] [Table 22]

[0651]

[0652] [Preparation of receptor solution]

[0653] The same method as described in Reference Example 1 was carried out.

[0654] [Dialysis reaction]

[0655] The same method as described in Reference Example 1 was carried out.

[0656] [Sampling after dialysis reaction]

[0657] The same method as described in Reference Example 1 was carried out.

[0658] [Pretreatment of samples after sampling and concentration measurement of target substances by LC-MS / MS]

[0659] The same method as described in Example 3 was carried out.

[0660] [Use various types of f u The relative f between species u Compare]

[0661] The same method as described in Reference Example 1 was carried out.

[0662] The results are shown below.

[0663] [Table 23]

[0664] <![CDATA[f u Ratio (relative person)]]> Mouse 16.08 Rat 5.01 dog 1.76 monkey 3.34 people 1.00

[0665] [Relative differences between species obtained in Examples 1 to 3, Comparative Examples 1 to 3 and Reference Examples 1 to 3 f u Comparison ]

[0666] The relative f between the species differences obtained in Example 1, Comparative Example 1 and Reference Example 1, Example 2, Comparative Example 2 and Reference Example 2, and Example 3, Comparative Example 3 and Reference Example 3 are u The results are as follows: Figures 7 to 12 shown.

[0667] Thus, the relative f of the method of the present invention is u Ratio (serum from any species is added to the donor solution and the acceptor solution, and the dialysis reaction is performed for 24 hours from the start of the dialysis reaction. The interspecies difference f is obtained by dynamic analysis based on the concentration change until this time. u ratio) and f obtained by the conventionally known method shown in the reference example u Ratio (serum from any species is added to the donor solution, phosphate buffered saline is used as the acceptor solution, and the dialysis reaction is performed until 24 hours from the start of the dialysis reaction, and the f obtained from the concentration ratio at that time is used u Value, calculate the f of animals and humans u The absolute average fold errors (AAFE) were 1.16 times for nifedipine, 1.46 times for cyclosporine, and 1.22 times for ibuprofen. Figure 7 , Figure 8 , Fig. 9 )

[0668] On the other hand, the f u Ratio (serum from any species is added to the donor solution and the receptor solution, and the dialysis reaction is performed for 24 hours from the start of the dialysis reaction. The f of animals and humans is obtained based on the concentration ratio at that time. u The ratio) is compared with the f obtained by the conventionally known method shown in the reference example. u The correlation of the ratios was within the error range of 2 times, falling within 5 plots in nifedipine, 1 plot in 5 plots in cyclosporine, and 3 plots in 5 plots in ibuprofen. It was confirmed that the absolute mean error was 1.08 times in nifedipine, 3.16 times in cyclosporine, and 1.90 times in ibuprofen. ( Fig.10 , Fig.11 , Fig.12 ) That is, in nifedipine, which takes a short time to reach equilibrium, no relative difference between the conventional method and the present invention was found. u There are advantages and disadvantages, but for cyclosporine and ibuprofen, which are difficult to reach equilibrium, the f obtained by the existing method (static analysis) is u The accuracy of the ratio is relatively good compared with the relative accuracy of the method (dynamic analysis) of the present invention. u Compared with f obtained by the conventionally known method shown in the reference example u The accuracy is worse than that.

[0669] Therefore, it can be seen that by using the method of the present invention, even for a substance to be measured that is difficult to reach an equilibrium state, the f can be obtained without waiting for the equilibrium state to be reached (that is, in a shorter time) as compared with the conventionally known method. u The relative f u Compare.

[0670] [Examples 4 to 13]

[0671] Obtaining relative f between species through dynamic analysis u Ratio (Substances to be measured: Anapravir, Apalutamide, E Basacevir·Dutasteride·Deferasirox·Aripiprazole·Belabuvir·Lomitapaide·Ibrutinib·Montelukast )

[0672] [Preparation of Donor Solution]

[0673] Anaprevir, apalutamide, ibasvir, dutasteride, deferasirox, aripiprazole, belabuvir, lomitapide, ibrutinib, and montelukast were used as the test substances, and 10 mmol / L solutions were prepared for each of them. The specific samples used and their amounts are shown in Table 24 below.

[0674] [Table 24]

[0675]

[0676] First, a 1 mmol / L solution of each measurement target substance was prepared. Specifically, 90 μL of DMSO was added to 10 μL of a solution containing 10 mmol / L of each measurement target substance.

[0677] Next, various serums (rat serum, human serum) were dissolved. The various serums dissolved were collected, and 9 times of phosphate buffered saline were added respectively. To the solution, 1 / 5000 times of anaprevir 1mmol / L solution, 1 / 5000 times of apalutamide 1mmol / L solution, 1 / 5000 times of ibasivir 1mmol / L solution, 1 / 5000 times of dutasteride 1mmol / L solution and 1 / 5000 times of deferasirox 1mmol / L solution were added. In addition, other various serums were collected, and 9 times of phosphate buffered saline were added respectively. To the solution, 1 / 5000 times of aripiprazole 1mmol / L solution, 1 / 5000 times of belabuvir 1mmol / L solution, 1 / 5000 times of lomitapide 1mmol / L solution, 1 / 5000 times of ibrutinib 1mmol / L solution and 1 / 5000 times of montelukast 1mmol / L solution were added respectively. In this way, donor solutions were prepared with a serum concentration of 10% (volume ratio), a DMSO concentration of 0.1% (volume ratio), and a concentration of 0.2 μmol / L for anaprevir, apalutamide, ibasvir, dutasteride, and deferasirox, or a concentration of 0.2 μmol / L for aripiprazole, belabuvir, lomitapide, ibrutinib, and montelukast. The specific amount of each sample used is shown in Tables 25 and 26 below.

[0678] The samples used were the same as those in Example 1 except for nifedipine.

[0679] [Table 25]

[0680]

[0681] [Table 26]

[0682]

[0683] [Preparation of Acceptor Solution]

[0684] A receptor solution having a serum concentration of 10% (volume ratio) and a DMSO concentration of 0.1% (volume ratio) was prepared in the same manner as the donor solution except that a DMSO solution was added instead of the 1 mmol / L solution containing each measurement target substance.

[0685] [Dialysis reaction]

[0686] The same method as in Example 1 was used except that the substance to be measured was changed from nifedipine to each substance to be measured in Examples 4 to 13. The donor solution or the acceptor solution dispensed into each chamber is shown in Table 27 below.

[0687] Table 27 shows a total of 6 situations, including the case where rat serum was set as the donor side and human serum was set as the recipient side (tests A to C; same conditions), and the case where human serum was set as the donor side and rat serum was set as the recipient side (tests D to F: same conditions), in which measurements were performed at 0.5 hours, 2 hours, 4 hours, 20 hours and 24 hours, respectively.

[0688] (Table 27) Chamber model using rat / human serum

[0689]

[0690] [Sampling after dialysis reaction]

[0691] The same method as in Example 1 was carried out except that the collected donor solution and acceptor solution were sealed without using a quenching solution and each sample was stored at -80°C until the pretreatment.

[0692] [Pretreatment of samples after sampling]

[0693] 20 μL of the sample after sampling was added with 5 μL of DMSO and 100 μL of IS solution, and the mixture was transferred to filter plates ("MultiScreen filter plate 0.45 um, low binding", manufactured by MILLIPORE) and filtered. 50 μL of water was added to the filtered sample to prepare a sample for LC-MS / MS measurement. The composition of the IS solution is shown in Table 28 below.

[0694] [Table 28]

[0695]

[0696] [Concentration measurement of target substance by LC-MS / MS]

[0697] For each of the LC-MS / MS measurement samples, the concentration of each measurement target substance in each chamber was measured using the following measurement instrument and measurement conditions. When the concentration of a specific target substance was measured in a plurality of wells using the same serum, the average value of the concentrations measured in each well of the same serum group was used for the measurement. u Calculation of ratio.

[0698] (Measurement Instrument)

[0699] LC:Nexera(SHIMADZU)

[0700] MS: QTRAP 6500 (Sciex)

[0701] Column: InertSustain C18 HP 3um, 2.1x30mm (UP)

[0702] (Measurement conditions)

[0703] Column temperature: 40°C (set value)

[0704] Solvent: A) 0.1% formic acid aqueous solution

[0705] B) 0.1% formic acid acetonitrile solution

[0706] Gradient conditions:

[0707] [Table 29]

[0708] Time (minutes) A％ B％ 0.0 95 5 0.5 70 30 0.9 5 95 1.2 5 95 1.25 95 5 1.8 95 5

[0709] Flow rate: 0.6mL / min

[0710] Automatic sampler temperature: 4°C (set value)

[0711] Injection Volume: 2 μL in Examples 4 to 8, 1 μL in Examples 9 to 13

[0712] [Preparation and pretreatment of calibration curve samples]

[0713] For 50 μL of rat serum or human serum, add 450 μL of PBS as 10% serum (blank matrix), and for 20 μL of blank matrix, add 5 μL of solution of calibration curve sample dissolved in DMSO and 100 μL of IS solution, respectively, transfer to filter plates ("Multi Screen filter plate 0.45 um, low binding", made by MILLIPORE), and filter. Add 50 μL of water to the filtered sample as a measurement sample for LC-MS / MS. The working solution for the calibration curve is prepared as follows. A sample in which 5 μL of DMSO is added instead of 5 μL of the standard curve sample solution is used as a blank sample.

[0714] Preparation of calibration curve sample solution: For 190 μL of DMSO, add 2 μL each of 10 mmol / L solutions of anaprevir, apalutamide, ibasvir, dutasteride, and deferasirox dissolved in DMSO, for a total of 200 μL, to prepare a solution with a concentration of 100 μmol / L for each analyte, and prepare the following dilution series (1)-(8).

[0715] [(1) To 60 μL of a 100 μmol / L solution of each substance to be measured, 90 μL of DMSO was added to prepare a 40,000 nmol / L solution of the compound.

[0716] (2) To 60 μL of a 40,000 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 12,000 nmol / L solution of the compound.

[0717] (3) To 60 μL of a 12000 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 4000 nmol / L solution of the compound.

[0718] (4) To 60 μL of a 4000 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 1200 nmol / L solution of the compound.

[0719] (5) To 60 μL of a 1200 nmol / L solution of each analyte, 120 μL of DMSO was added to prepare a 400 nmol / L solution of the compound.

[0720] (6) To 60 μL of a 400 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 120 nmol / L solution of the compound.

[0721] (7) To 60 μL of a 120 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 40 nmol / L solution of the compound.

[0722] (8) To 60 μL of a 40 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 12 nmol / L solution of the compound.]

[0723] Since the dilution series of (1) to (8) were adjusted to 4 times the concentration when the actual sample was used as the reference, the concentration adjusted by the dilution series was corrected to 1 / 4 times the concentration to be used as the concentration of the calibration curve sample.

[0724] Concentrations of calibration curve samples: (8) 3 nM, (7) 10 nM, (6) 30 nM, (5) 100 nM, (4) 300 nM, (3) 1000 nM, (2) 3000 nM

[0725] To 190 μL of DMSO, 2 μL of each 10 mmol / L solution of aripiprazole, berabuvir, lomipatapide, ibrutinib, and montelukast dissolved in DMSO was added to a total of 200 μL to prepare a solution with a concentration of 100 μmol / L for each analyte. The following dilution series (1) to (8) were prepared.

[0726] [(1) To 6 μL of a 100 μmol / L solution of each substance to be measured, 144 μL of DMSO was added to prepare a 4000 nmol / L solution of the compound.

[0727] (2) To 60 μL of a 4000 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 1200 nmol / L solution of the compound.

[0728] (3) To 60 μL of a 1200 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 400 nmol / L solution of the compound.

[0729] (4) To 60 μL of a 400 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 120 nmol / L solution of the compound.

[0730] (5) To 60 μL of a 120 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 40 nmol / L solution of the compound.

[0731] (6) To 60 μL of a 40 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 12 nmol / L solution of the compound.

[0732] (7) To 60 μL of a 12 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 4 nmol / L solution of the compound.

[0733] (8) To 60 μL of the 4 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 1.2 nmol / L solution of the compound.]

[0734] Since the dilution series of (1) to (8) were adjusted to 4 times the concentration when the actual sample was used as the reference, the concentration adjusted by the dilution series was corrected to 1 / 4 times the concentration to be used as the concentration of the calibration curve sample.

[0735] Concentrations of calibration curve samples: (8) 0.3 nM, (7) 1 nM, (6) 3 nM, (5) 10 nM, (4) 30 nM, (3) 100 nM, (2) 300 nM

[0736] [Table 30]

[0737]

[0738] In Table 30, DP is declustering potential, CE is collision energy, and CEP is collision entrance potential.

[0739] MS conditions: (ESI positive) Curtain Gas TM flow(CUR)30,IonSpray TM Voltage (IS)5500,Temperature(TEM)650,Ion SourceGas1(GS1)20,Ion Source Gas2(GS2)20

[0740] [obtained through dynamic analysis u Compare]

[0741] The rat-human f was calculated in the same manner as in Example 1 except that the following conditions were changed. u Compare.

[0742] Definition:

[0743] When the first biological sample is a rat and the second biological sample is a human, the following four data are used as a set for analysis.

[0744] Data 1 (Eq1.Cd1): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains rat serum and the receptor solution contains human serum

[0745] Data 2 (Eq1.Ca1): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains rat serum and the receptor solution contains human serum

[0746] Data 3 (Eq2.Cd2): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains rat serum

[0747] Data 4 (Eq2.Ca2): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains rat serum

[0748] Estimated Parameters:

[0749] exp(fu1).InitialValue=0.01; Bounds=[1e-05 1];

[0750] exp(fu2).InitialValue=0.01; Bounds=[1e-05 1];

[0751] exp(Koff1).InitialValue=1; Bounds=[1e-05 10000];

[0752] exp(Koff2).InitialValue=1; Bounds=[1e-05 10000];

[0753] exp(CL1).InitialValue=0.001; Bounds=[1e-07 100];

[0754] exp(CL2).InitialValue=0.001; Bounds=[1e-07 100];

[0755] exp(Dose1).InitialValue=0.2115; Bounds=[0.01 0.3];

[0756] exp(Dose2).InitialValue=0.2114; Bounds=[0.01 0.3];

[0757] The results are shown below.

[0758] [Table 31]

[0759] <![CDATA[f u Ratio (rat / human)]]> Anapravir 3.34 Apalutamide 2.50 Ibasvir 2.90 Dutasteride 1.94 Deferasirox 3.12 Aripiprazole 1.74 belabwe 1.34 Lomitapide 6.52 Ibrutinib 0.26 Montelukast -

[0760] [Comparative Examples 4 to 13]

[0761] Relative fu ratio between species obtained by static analysis (substances to be measured: the same as in Examples 4 to 13)

[0762] The donor solution and the acceptor solution were prepared in the same manner as in Examples 4 to 13, the dialysis reaction was started, and the concentration of the substance to be measured in the solution added to each chamber at the time when 24 hours had passed since the start of the dialysis reaction was measured. When the concentration of a specific substance to be measured in a plurality of wells using the same serum, the average value of the concentrations measured in each well of the same serum group was used for the calculation of the concentration of the substance to be measured. u The obtained data is applied to Formula 4 of Comparative Example 1 to obtain the f of the measured substance relative to various types. u Ratio (rat / human, serum concentration 1 / 10).

[0763] The results are shown below.

[0764] [Table 32]

[0765] <![CDATA[f u Ratio (rat / human)]]> Anapravir 0.69 Apalutamide 3.36 Ibasvir 0.18 Dutasteride 1.70 Deferasirox 1.39 Aripiprazole 0.98 belabwe 0.84 Lomitapide 0.57 Ibrutinib 0.23 Montelukast -

[0766] [Reference Examples 4 to 13]

[0767] Static analysis (relative to buffer) The difference between the species f u Obtaining the ratio (substance to be measured: compared with Examples 4 to 13 same)

[0768] [Preparation of Donor Solution]

[0769] Anaprevir, apalutamide, ibasvir, dutasteride, deferasirox, aripiprazole, berabuvir, lomitapide, ibrutinib, and montelukast were used as the test substances, and 10 mmol / L solutions were prepared for each. The specific samples used and their amounts are shown in Table 33 below.

[0770] [Table 33]

[0771]

[0772] First, prepare a mixed solution (Cocktail solution) of the measured object substance. Specifically, 2 μL of anaprevir 10 mmol / L solution, 2 μL of apalutamide 10 mmol / L solution, 2 μL of ibasivir 10 mmol / L solution, 2 μL of dutasteride 10 mmol / L solution, and 2 μL of deferasirox 10 mmol / L solution were mixed with 90 μL of DMSO to prepare a total of 100 μL of mixed solution. In addition, 2 μL of aripiprazole 10 mmol / L solution, 2 μL of belabuvir 10 mmol / L solution, 2 μL of lomitapide 10 mmol / L solution, 2 μL of ibrutinib 10 mmol / L solution, and 2 μL of montelukast 10 mmol / L solution were mixed with 90 μL of DMSO to prepare a total of 100 μL of mixed solution.

[0773] Then, various serums (rat serum, human serum) were dissolved. The various serums dissolved were collected, and 9 times of phosphate buffered saline were added respectively, and a mixed solution of anaprevir, apalutamide, ibasvir, dutasteride, and deferasirox containing each concentration of 0.2 μmol / L was added to the solution 1 / 1000 times or a mixed solution of aripiprazole, belabuvir, lomitapide, ibrutinib, and montelukast containing each concentration of 0.2 μmol / L was added 1 / 1000 times to prepare a donor solution. The amount of each sample used is shown in Table 34 and Table 35 below. In addition, each sample used except nifedipine is the same as that in Example 1.

[0774] [Table 34]

[0775]

[0776] [Table 35]

[0777]

[0778] [Preparation of Acceptor Solution]

[0779] A receptor solution having a DMSO concentration of 0.1% (volume ratio) was prepared in the same manner as in Examples 4 to 13.

[0780] [Dialysis reaction]

[0781] The rat / human f in each of the assay substances was obtained by the same method as in Reference Example 1 except that the donor solution was changed to the donor solution prepared in Reference Examples 4 to 13 and the conditions were as follows. u value.

[0782] Table 36 shows the cases where rat serum or human serum was used as the donor side and phosphate-buffered saline was used as the recipient side (Tests A to C; same conditions), in total 6 cases, in which measurements were performed at 24 hours.

[0783] [Table 36]

[0784]

[0785] [Sampling after dialysis reaction]

[0786] The same steps as in Examples 4 to 13 were used.

[0787] [Pretreatment of samples after sampling]

[0788] 20 μL of the sample after sampling was added with 10 μL of DMSO and 100 μL of IS solution, and the mixture was transferred to filter plates ("MultiScreen filter plate 0.45 um, low binding", manufactured by MILLIPORE) and filtered. 70 μL of water was added to the filtered sample to prepare a sample for LC-MS / MS measurement. The composition of the IS solution is shown in Table 37 below.

[0789] [Table 37]

[0790]

[0791] Preparation of calibration curve samples: For 1 μL of a mixed solution of anaprevir, apalutamide, ibasvir, dutasteride, and deferasirox (2 μL each) dissolved in DMSO, add 99 μL of DMSO to prepare a 2 μmol / L mixed solution of the compound. For 1 μL of a mixed solution of aripiprazole, belabuvir, lomitriparib, ibrutinib, and montelukast (2 μL each) dissolved in DMSO, add 99 μL of DMSO to prepare a 2 μmol / L mixed solution of the compound. Prepare the following (1)-(8) dilution series and blank solutions.

[0792] [(1) To 60 μL of a 2 μmol / L solution of each substance to be measured, 40 μL of DMSO was added to prepare a 1200 nmol / L solution of the compound.

[0793] (2) To 60 μL of a 1200 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 400 nmol / L solution of the compound.

[0794] (3) To 60 μL of a 400 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 120 nmol / L solution of the compound.

[0795] (4) To 60 μL of a 120 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 40 nmol / L solution of the compound.

[0796] (5) To 60 μL of a 40 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 12 nmol / L solution of the compound.

[0797] (6) To 60 μL of a 12 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 4 nmol / L solution of the compound.

[0798] (7) To 60 μL of a 4 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 1.2 nmol / L solution of the compound.

[0799] (8) To 60 μL of a 1.2 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 0.4 nmol / L solution of the compound.]

[0800] Since the dilution series of (1) to (8) were adjusted to 2 times the concentration when the actual sample was used as the reference, the concentration adjusted by the dilution series was corrected to 1 / 2 times to obtain the concentration of the calibration curve sample.

[0801] Concentrations of calibration curve samples: (8) 0.2 nM (8) 0.6 nM, (7) 2 nM, (6) 6 nM, (5) 20 nM, (4) 60 nM, (3) 200 nM, (2) 600 nM

[0802] [Concentration measurement of target substance by LC-MS / MS]

[0803] For each of the LC-MS / MS measurement samples, the concentrations of anaprevir, apalutamide, ibasvir, dutasteride, and deferasirox, and the concentrations of aripiprazole, belabuvir, lomitriparib, ibrutinib, and montelukast in each chamber were measured using the following measuring instruments and measuring conditions. When the concentration of a specific target substance was measured in a plurality of wells using the same serum, the average value of the concentrations measured in each well of the same serum group was used for the analysis. u Calculation of ratio.

[0804] (Measurement Instrument)

[0805] LC: Waters Acquity UPLC

[0806] MS: XEVO-TQS#WAA696 (Waters Corporation, Milford, Massachusetts)

[0807] Column: YMC-Triart C18 column, 30x 2.0mm ID, S-1.9um, 12nm

[0808] (Measurement conditions)

[0809] Column temperature: 40°C (set value)

[0810] Solvent: A) 0.1% formic acid aqueous solution

[0811] B) 0.1% formic acid acetonitrile solution

[0812] Gradient conditions:

[0813] [Table 38]

[0814] Time (minutes) A％ B％ 0.0 80 20 0.2 80 20 0.4 35 65 0.6 15 85 0.8 5 95 1.2 5 95 1.3 80 20 1.6 80 20

[0815] Flow rate: 0.6mL / min

[0816] Automatic sampler temperature: 10℃ (set value)

[0817] Injection Volume: 1 μL (Reference Examples 5, 7 to 13), 1.5 μL (Reference Example 6)

[0818] [Concentration measurement of target substance by LC-MS / MS]

[0819] For each of the LC-MS / MS measurement samples, the concentration of Anaprevir in each chamber was measured using the following measurement instrument and measurement conditions.

[0820] (Measurement Instrument)

[0821] LC: Waters Acquity UPLC

[0822] MS: XEVO-TQS#WAA696 (Waters Corporation, Milford, Massachusetts)

[0823] Column: YMC-Triart Bio C4, 30x 2.1mm ID, S-1.9um, 30nm

[0824] (Measurement conditions)

[0825] Column temperature: 40°C (set value)

[0826] Solvent: A) 0.1% formic acid aqueous solution

[0827] B) 0.1% formic acid acetonitrile solution

[0828] Gradient conditions:

[0829] [Table 39]

[0830] Time (minutes) A％ B％ 0.0 60 40 0.2 60 40 0.4 15 85 0.6 5 95 0.8 1 99 1.2 1 99 1.4 1 99 1.41 60 40 2 60 40

[0831] Flow rate: 0.6mL / min

[0832] Automatic sampler temperature: 10°C (set value)

[0833] Injection Volume: 1μL

[0834] [Table 40]

[0835]

[0836] MS conditions:

[0837] ESI positive

[0838] Capillary 2kV, Cone 25V, Source Offset 50V, Source Temperature150℃, Desolvation Temperature 600℃, Cone Gas Flow 150L / Hr, Collision Gas Flow 0.2mL / min, Nebuliser Gas Flow 7Bar

[0839] The obtained f of each measurement object substance u The results of the ratio (rat / human) are shown below.

[0840] [Table 41]

[0841] <![CDATA[f u Ratio (rat / human)]]> Anapravir 1.52 Apalutamide 2.30 Ibasvir 1.46 Dutasteride 1.47 Deferasirox 3.53 Aripiprazole 1.45 belabwe 1.21 Lomitapide 2.77 Ibrutinib 0.40 Montelukast -

[0842] [Evaluation Example 1]

[0843] Method for predicting human volume of distribution of drugs

[0844] The f obtained in Examples 1 to 13, Comparative Examples 1 to 13, and Reference Examples 1 to 13 was used. u Ratio, predicting the volume of distribution in humans.

[0845] "Volume of distribution" (V) refers to the theoretical amount of fluid required to contain the total amount of administered drug in the body at the same concentration as in blood or plasma. Volume of distribution = amount of drug in the body / blood concentration (eg, L).

[0846] The method for predicting human distribution volume (Non-patent document 7: Berry, LM, et al. (2011). "Species differences in distribution and prediction of human V (ss) from preclinical data." Drug Metab Dispos 39 (11): 2103-2116) was used to calculate the steady-state distribution volume per body weight (rat) by removing the drug f obtained by the above method. u The predicted value of human distribution volume per body weight was calculated by comparing the obtained predicted value with the observed value of human distribution volume to verify the prediction accuracy.

[0847] Prediction of volume of distribution

[0848] Vss 人 (Predicted value) = Vss 大鼠 / Rfu

[0849] Vss 大鼠 : Steady-state volume of distribution per body weight of rats (Vss [L / kg])

[0850] Rfu: relative f u Ratio (rat / human)

[0851] The diluted f obtained in Reference Examples 4 to 13 was diluted using the following dilution formula: u Value (rat, serum concentration 1 / 10), f u The values ​​(human, serum concentration 1 / 10) were corrected to serum concentration 1 / 1. u Compounds with values ​​of 0.01 or less (i.e., compounds that are difficult to obtain an equilibrium state) are used for distribution volume prediction.

[0852] Dilution

[0853] In the formula, D represents the dilution ratio. This time, 10-fold diluted serum was used, so D=10.

[0854] The above dilution formula refers to the following document.

[0855] Non-patent document 8: Riccardi K, Cawley S, Yates PD, et al. Plasma protein binding of challenging compounds. J Pharm Sci. 2015; 104: 2627-2636.

[0856] Non-patent literature 9: Kalvass JC, Maurer TS. Influence of nonspecific brain and plasma binding on CNS exposure:implications for rational drug discovery. Biopharm Drug Dispos. 2002; 23:327-338.

[0857] The corrected f u The resulting values ​​are shown below.

[0858] [Table 42]

[0859]

[0860] According to the results in Table 42, the effects of anaprevir, ibasvir, dutasteride, deferasirox, aripiprazole, belabuvir, lomitapide, and ibrutinib on the performance of rats or humans were not significantly different from those of humans. u The value is 0.01 or less. For compounds in which the observed distribution volume in rats and humans is obtained, the relative f is used. u In addition, the distribution volume prediction was performed using the same method as that used in rats or humans. u The relative f of nifedipine and ibuprofen used in Examples 1 and 3, Comparative Examples 1 and 3, and Reference Examples 1 and 3 for compounds having a value of 0.01 or less u The results of the predicted steady-state distribution volumes (Vdss, Vss) are shown in Tables 43, 44 and Figure 13-1 to Figure 13-3 shown.

[0861] [Table 43]

[0862]

[0863] [Table 44]

[0864]

[0865] In the relative f of the method of the present invention u Ratio (serum from any species is added to the donor side chamber and the acceptor side chamber, and the dialysis reaction is carried out for 24 hours from the start of the dialysis reaction. The interspecies difference f is obtained by dynamic analysis based on the concentration change until this time u In the prediction of the distribution volume, 67% of the predictions fell within the error range of 1.25 times, and 67% of the predictions fell within the error range of 1.5 times. The absolute average error (AAFE) was 1.61 times the prediction of the distribution volume. Figure 13-1 ).

[0866] In the comparative example method, u Ratio (serum from any species is added to the donor side chamber and the acceptor side chamber, respectively, and the dialysis reaction is performed for 24 hours from the start of the dialysis reaction, and the concentration ratio at the time of 24 hours is used to obtain f u Value, calculate the f of animals and humans u The distribution volume predicted 22% within the error range of 1.25 times, and the distribution volume predicted 33% within the error range of 1.5 times. The AAFE was 2.38 times. Figure 13-2 ).

[0867] In the case of f obtained by the conventionally known method shown in the reference example u In the comparison, 11% of the predictions based on the distribution volume fell within the error range of 1.25 times, 56% of the predictions based on the distribution volume fell within the error range of 1.5 times, and the AAFE was 1.68 times. Figure 13-3 )

[0868] Using the relative f obtained by the method of the present invention (dynamic analysis) u The accuracy of the distribution volume prediction of the ratio is within the range of 1.25 times and 1.5 times the accuracy of the distribution volume prediction shown in the comparative example, and the prediction accuracy is excellent for each AAFE. That is, by using the method of the present invention, it is known that the relative f can be obtained with good accuracy even for the measurement object substance that is difficult to reach an equilibrium state. u In addition, the method of the reference example (relative buffer) requires the exploration of the time when the compound concentration in the serum and buffer chamber approaches the equilibrium state for each compound through prior research, but this is not necessary in the method of the present invention (dynamic analysis), which is considered to be a more practical method.

[0869] [Examples 14 to 20]

[0870] Dynamic analysis was used to obtain the difference between samples of the same type (human) and different organisms (serum, liver microsomes ) Relative f u Comparison (Substances to be measured: aripiprazole, deferasirox, lapatinib, ticagrelor, ketoconazole, itraconazole, RG12525 )

[0871] The method of the present invention can not only compare different sera, but also obtain the same kind of different biological samples of the same f u The ratio of the unbound fraction of serum to liver microsomes (f u Serum vs SF u mic The ratio of f u Ratio (f u Serum vs SF u mic The ratio of the measured and analyzed substances was set as aripiprazole, deferasirox, lapatinib, ticagrelor, ketoconazole, itraconazole, and RG12525. These suggest that they may competitively inhibit CYP3A4, a hepatic drug metabolizing enzyme, and have a high protein binding rate in human serum (f u The serum value is below 0.01).

[0872] Non-patent literature 10: (Vieira, ML, et al. (2014). "Evaluation of various static in vitro-in vivo extrapolation models for risk assessment of the CYP3A inhibition potential of an investigational drug." Clin Pharmacol Ther 95(2): 189-198.), Non-patent literature 11: Hachad, H., et al. (2010). "A useful tool for drug interaction evaluation: the University of Washington Metabolism and Transport Drug Interaction Database." Hum Genomics 5(1): 61-72., Non-patent literature 12: Chu, QS, et al. (2008). "A phase I and pharmacokinetic study of lapatinib in combination with letrozole in patients with advanced cancer." Clin Cancer Res 14(14): 4484-4490., Non-patent literature 13: Koch, KM, et al. (2017). "The effects of lapatinib onCYP3Ametabolism of midazolam in patients with advanced cancer." Cancer Chemother Pharmacol 80(6):1141-1146).

[0873] These compounds should only be used according to current FDA guidelines. u If the value of human serum is 0.01, it is considered that the compound is difficult to predict DDI. However, if the method of the present invention (dynamic analysis) can be used to obtain a high-precision f u Ratio (f u Serum vs SF u mic The accuracy of DDI prediction can be expected to improve if the ratio of

[0874] [Table 45

[0875]

[0876] For each of the measurement target substances, 1 mmol / L solutions were prepared, specifically, 2 μL of a 5 mmol / L solution containing itraconazole was adjusted by adding 8 μL of DMSO, or 2 μL of a 10 mmol / L solution containing aripiprazole, deferasirox, lapatinib, ticagrelor, ketoconazole, or RG12525 was adjusted by adding 18 μL of DMSO.

[0877] Furthermore, for RG12525, 5 μL of DMSO was added to 5 μL of the 1 mmol / L solution to prepare a 0.5 mmol / L solution. Also, for itraconazole and ketoconazole, 8 μL of DMSO was added to 2 μL of the 1 mmol / L solution to prepare a 0.2 mmol / L solution.

[0878] Next, the dissolved human serum was collected, and 9 times the amount of phosphate buffered saline was added, and 1 / 1000 times the amount of a 1 mmol / L solution containing the test object (aripiprazole, deferasirox, lapatinib, ticagrelor, itraconazole, ketoconazole or RG12525) was added to the solution. In this way, a donor solution with a serum concentration of 10% (volume ratio) and a DMSO concentration of 0.1% (volume ratio) was prepared. The dissolved human liver microsomes (20 mg / mL) were collected, and 199 times the amount of phosphate buffered saline was added, and 1 / 1000 times the amount of a 1 mmol / L solution containing the test object (aripiprazole, deferasirox, lapatinib or ticagrelor), 1 / 1000 times the amount of a 0.5 mmol / L solution containing RG12525, or 1 / 1000 times the amount of a 0.2 mmol / L solution of the test object (itraconazole or ketoconazole) was added to the solution. In this way, a donor solution having a microsome concentration of 0.1 mg / mL and a DMSO concentration of 0.1% (volume ratio) was prepared.

[0879] The samples used were the same as those in Example 1 except for nifedipine.

[0880] [Preparation of Acceptor Solution]

[0881] A receptor solution having a serum concentration of 10% (volume ratio) and a DMSO concentration of 0.1% (volume ratio) was prepared in the same manner as the donor solution except that a DMSO solution was added instead of the 1 mmol / L solution containing each measurement target substance.

[0882] [Dialysis reaction]

[0883] The same method as in Example 1 was used except that the measurement substance was changed from nifedipine to each measurement target substance of Examples 14 to 20 and the receptor solution was uniformly made of human serum. The donor solution or receptor solution dispensed into each chamber is shown in Table 46 below.

[0884] Table 46 shows a total of 8 situations in which measurements were performed at 0.5 hours, 2 hours, 4 hours, and 20 hours, respectively, for the cases where human serum was set as the donor side and human serum was set as the recipient side (tests A to H; same conditions), and the cases where human liver microsomes were set as the donor side and human serum was set as the recipient side (tests A to H: same conditions).

[0885] [Table 46]

[0886]

[0887] [Sampling after dialysis reaction]

[0888] The same method as in Example 1 was carried out except that the collected donor solution and acceptor solution were sealed without using a quenching solution and each sample was stored at -80°C until the pretreatment.

[0889] [Pretreatment of samples after sampling]

[0890] 20 μL of the sample after sampling was added with 10 μL of DMSO and 100 μL of IS solution, and the mixture was transferred to filter plates ("MultiScreen filter plate 0.45 um, low binding", manufactured by MILLIPORE) and filtered. 70 μL of water was added to the filtered sample to prepare a sample for LC-MS / MS measurement. The composition of the IS solution is shown in Table 47 below.

[0891] [Table 47]

[0892]

[0893] [Concentration measurement of target substance by LC-MS / MS]

[0894] Preparation of calibration curve samples: 1 μL of each analyte (1 mmol / L) dissolved in DMSO was added with 99 μL of DMSO to prepare a 10 μmol / L solution of the compound. The following dilution series (1) to (8) and a blank solution were prepared.

[0895] [(1) To 60 μL of a 10 μmol / L solution of each substance to be measured, 40 μL of DMSO was added to prepare a 6000 nmol / L solution of the compound.

[0896] (2) To 60 μL of a 600 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 2000 nmol / L solution of the compound.

[0897] (3) To 60 μL of a 2000 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 600 nmol / L solution of the compound.

[0898] (4) To 60 μL of a 600 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 200 nmol / L solution of the compound.

[0899] (5) To 60 μL of a 200 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 60 nmol / L solution of the compound.

[0900] (6) To 60 μL of a 60 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 20 nmol / L solution of the compound.

[0901] (7) To 60 μL of a 20 nmol / L solution of each substance to be measured, 140 μL of DMSO was added to prepare a 6 nmol / L solution of the compound.

[0902] (8) To 60 μL of the 6 nmol / L solution of each substance to be measured, 120 μL of DMSO was added to prepare a 2 nmol / L solution of the compound.]

[0903] Since the dilution series of (1) to (8) were adjusted to 2 times the concentration when the actual sample was used as the reference, the concentration adjusted by the dilution series was corrected to 1 / 2 times to obtain the concentration of the calibration curve sample.

[0904] Concentrations of calibration curve samples: (8) 1nM (7) 3nM, (6) 10nM, (5) 30nM, (4) 100nM, (3) 300nM, (2) 1000nM, (1) 3000nM

[0905] For 20 μL of blank sample of human liver microsome (0.1 mg / mL) or human serum, 10 μL of calibration curve sample and 100 μL of IS solution were added, and the samples were transferred to filter plates ("Multi Screen Filter Plate 0.45 um, Low Binding", manufactured by MILLIPORE) and filtered. 70 μL of water was added to the filtered sample to prepare a measurement sample for LC-MS / MS.

[0906] [Concentration measurement of target substance by LC-MS / MS]

[0907] For each of the LC-MS / MS measurement samples, the concentration of aripiprazole, deferasirox, lapatinib, ticagrelor, ketoconazole, itraconazole or RG12525 in each chamber was measured using the following measuring instrument and measuring conditions. When the concentration of a specific target substance was measured in a plurality of wells using the same biological sample type (serum or human liver microsomes), the average value of the concentrations measured in each well of the same biological sample type group was used for the calculation of the concentration of the specific target substance. u Calculation of ratio.

[0908] (Measurement Instrument)

[0909] LC: Waters Acquity UPLC

[0910] MS: XEVO-TQS#WAA696 (Waters Corporation, Milford, Massachusetts)

[0911] Column: YMC-Triart C18 column, 30x 2.0mm ID, S-1.9um, 12nm

[0912] (Measurement conditions)

[0913] Column temperature: 50°C (set value)

[0914] Solvent: A) 0.1% formic acid aqueous solution

[0915] B) 0.1% formic acid acetonitrile solution

[0916] Gradient conditions:

[0917] [Table 48]

[0918] Time (minutes) A％ B％ 0.0 99 1 0.8 1 99 1.6 1 99 1.61 99 1

[0919] Flow rate: 0.6mL / min

[0920] Automatic sampler temperature: 10℃ (set value)

[0921] Injection Volume: 1μL

[0922] MS conditions: (ESI positive)

[0923] [Table 49]

[0924]

[0925] The same method as in Example 1 was used to obtain f except that the parameters were changed as follows. u Compare.

[0926] fu1: fu value of the substance to be measured relative to the biological sample contained in the donor solution (calculated value)

[0927] fu2: fu value (calculated value) of the substance to be measured relative to the biological sample contained in the acceptor solution. When the donor solution is human serum and the acceptor solution is human serum, fu2 is the same as fu1.

[0928] PS: Dialysis membrane clearance of the substance to be measured (fixed value, 1.5×10 -4 L / h)

[0929] Document (Model, Reaction, Rules, Definition, Estimated Parameters) and implement.

[0930] Reaction:

[0931] Eq1.Donor1<->Eq1.Acceptor1:+fu1*PS*Eq1.Donor1 / Vd1-fu1*PS*Eq1.Acceptor1 / Vd2

[0932] Eq2.Donor2<->Eq2.Acceptor2:+fu2*PS*Eq2.Donor2 / Vd1-fu1*PS*Eq2.Acceptor2 / Vd2

[0933] Eq2.Donor2->Eq2.Ad:+CL2*fu2*Eq2.Donor2 / Vd1-(Koff2*Eq2.Ad)

[0934] Rules:

[0935] Same as Example 1.

[0936] Definition:

[0937] When the first biological sample is human serum and the second biological sample is human liver microsome, the following four data are used as one set for analysis.

[0938] Data 1 (Eq1.Cd1): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains human serum

[0939] Data 2 (Eq1.Ca1): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains human serum and the receptor solution contains human serum

[0940] Data 3 (Eq2.Cd2): Concentration of the substance to be measured in the donor solution in an equilibrium dialysis system in which the donor solution contains human liver microsomes and the receptor solution contains human serum

[0941] Data 4 (Eq2.Ca2): Concentration of the substance to be measured in the receptor solution in an equilibrium dialysis system in which the donor solution contains human liver microsomes and the receptor solution contains human serum

[0942] Estimated Parameters:

[0943] exp(fu1).InitialValue=0.01; Bounds=[1e-05 1];

[0944] exp(fu2).InitialValue=0.01; Bounds=[1e-05 1];

[0945] exp(Koff2).InitialValue=1; Bounds=[1e-05 10000];

[0946] exp(CL2).InitialValue=0.001; Bounds=[1e-07 100];

[0947] exp(Dose1).InitialValue=0.2115; Bounds=[0.01 0.5];

[0948] exp(Dose2).InitialValue=0.2114; Bounds=[0.01 0.5];

[0949] The f calculated by the above dynamic analysis method u (Human, serum concentration 1 / 10) The following dilution formula was used to correct the serum concentration to 1 / 1, and the f of each analyte was calculated. u Ratio (serum / microsomes).

[0950] Dilution

[0951] In the formula, D represents the dilution ratio. This time, 10-fold diluted serum was used, so D=10.

[0952] The results are shown below.

[0953] [Table 50]

[0954] <![CDATA[f u Ratio (serum / microsomes)]]> Aripiprazole 0.0076 Deferasirox 0.0030 Lapatinib 0.0060 Ticagrelor 0.0103 Ketoconazole 0.0116 Itraconazole 0.0074 RG12525 0.0002

[0955] [Comparative Examples 14 to 20]

[0956] Relative analysis of different biological samples (human serum and microsomes) of the same (human) species was obtained by static analysis. f u Ratio (Substance to be measured: the same as in Examples 14 to 20)

[0957] In the same manner as in Examples 4 to 13, f u In the case of the ratio, as a dialysis reaction, it is necessary to prepare a donor solution with a microsome concentration of 0.1 mg / mL and a DMSO concentration of 0.1% (volume ratio) and a receptor solution with a human serum concentration of 100% and a DMSO concentration of 0.1% (volume ratio), but in Examples 14 to 20, the compounds are f uFor highly protein-bound compounds with values ​​likely to be less than 0.01, it is considered difficult to obtain an equilibrium state when using 100% human serum concentration (i.e., when the serum is not diluted with a buffer). Therefore, these compounds were not subjected to the same method as in Comparative Examples 4 to 13. u Than calculation.

[0958] [Reference Examples 14 to 20]

[0959] Different biological samples (human serum and microparticles) of the same (human) species were obtained by static analysis (vs. buffer) f u Value (Measured substance: Same as Examples 14 to 20) )

[0960] Collect dissolved human serum, add 9 times the amount of phosphate buffered saline, and add 1 / 1000 times the amount of 1 mmol / L solution of the compound to the solution. In this way, a donor solution with a serum concentration of 10% (volume ratio) and a DMSO concentration of 0.1% (volume ratio) is prepared. Collect dissolved human liver microsomes (20 mg / mL), add 199 times the amount of phosphate buffered saline, and add 1 / 1000 times the amount of 1 mmol / L solution of the compound to the solution. In this way, a donor solution with a microsome concentration of 0.1 mg / mL and a DMSO concentration of 0.1% (volume ratio) is prepared.

[0961] [Table 51]

[0962]

[0963] The donor solution and the acceptor solution were prepared in the same manner as in Examples 14 to 20, the dialysis reaction was started, and the concentration of the substance to be measured in the solution added to each chamber at the time when 24 hours had passed since the start of the dialysis reaction was measured. The obtained data were applied to Formula 5 of Reference Example 1 to obtain f u In addition, using the dilution formula, the diluted f u The value (human, serum concentration 1 / 10) is corrected to serum concentration 1 / 1, and f is obtained. u Value (human, serum concentration 1 / 1).

[0964] [Table 52]

[0965]

[0966] According to the obtained f u value, and find the f of each substance to be measured u Ratio (serum / microsomes).

[0967] [Table 53]

[0968] <![CDATA[f u Ratio (serum / microsomes)]]> Aripiprazole 0.0033 Deferasirox 0.0046 Lapatinib 0.0087 Ticagrelor 0.0065 Ketoconazole 0.0073 Itraconazole 0.0041 RG12525 0.0005

[0969] [Reference Examples 21 to 27]

[0970] Based on the results of Reference Examples 14 to 20 and FDA guidance u Calculation of ratio (substance to be measured: with Example 14 to 20 same)

[0971] The fu values ​​(microsomes) obtained in Reference Examples 14 to 20 and the fu values ​​(serum) of each measurement target substance based on FDA guidance = 0.01 were used as follows (serum / microsome).

[0972] [Table 54]

[0973] fu ratio (serum / microsomes) Aripiprazole 0.0160 Deferasirox 0.0099 Lapatinib 0.1972 Ticagrelor 0.0207 Ketoconazole 0.0091 Itraconazole 0.0129 RG12525 0.0129

[0974] [Evaluation Example 2]

[0975] In order to predict the risk of DDI, the inhibition constant Ki value of each compound in human liver microsomes was determined using the CYP3A4 substrate midazolam as a probe compound.

[0976] To 24 μL of dissolved commercially available human liver microsomes (20 mg / mL, manufactured by Xenotech), add 3976 μL of 0.1 mol / L phosphate buffer to prepare a total of 4000 μL of human liver microsome solution (concentration 0.12 mg / mL). Or to 37 μL of commercially available human liver microsomes (20 mg / mL, manufactured by Xenotech), add 5963 μL of 0.1 mol / L phosphate buffer to prepare a total of 6000 μL of human liver microsome solution (concentration 0.12 mg / mL). Prepare 3 μL of a DMSO solution of the test compound before dilution at a concentration of 1000 times the final addition concentration, add 87 μL of a 60% acetonitrile solution to prepare a total of 90 μL of the test compound solution. To 5 μL of a solution containing 50 mmol / L midazolam, 70 μL of a 50% acetonitrile solution was added to prepare a total of 75 μL of a solution. To 12 μL of this solution, 63 μL of an acetonitrile solution and 5925 μL of water were added to prepare a total of 6000 μL of a midazolam solution (concentration 6.6 μmol / L). 3000 μL of a 0.1 mol / L phosphate buffer was added to 25 mg of commercially available β-NADPH (Wako Pure Chemical Industries, Ltd.) to prepare an NADPH solution (10 mmol / L). The adjusted solutions were mixed in the following order to prepare a solution for inhibiting the reaction (a total of 100 μL). The reaction was allowed to react at 37°C for 5 minutes while stirring, with the reaction starting at 0 minutes immediately after the addition of the NADPH solution.

[0977] [Table 55]

[0978]

[0979] [Table 56]

[0980] Inhibition solution, concentration of compound added (μmol / L) Aripiprazole 0(control group), 0.94, 1.88, 3.75, 7.5, 15, 30, 60 Deferasirox 0 (control group), 1.56, 3.13, 6.25, 12.5, 25, 50, 100 Lapatinib 0 (control group), 1.25, 2.5, 5, 10, 20, 40, 80 Ticagrelor 0 (control group), 1.56, 3.13, 6.25, 12.5, 25, 50, 100 Ketoconazole 0(control group), 0.0012, 0.004, 0.011, 0.03, 0.1, 0.3, 0.9 Itraconazole 0(control group), 0.0014, 0.004, 0.012, 0.04, 0.11, 0.33, 1 RG12525 0(control group), 0.0823, 0.247, 0.741, 2.22, 6.67, 20, 60

[0981] After the reaction in the metabolic reaction solution, 35 μL was recovered and mixed with 140 μL of acetonitrile / isopropanol = 1 / 1 solution (containing 2.5 nmol / L 1-hydroxymidazolam [13C3] and 10 nM warfarin as IS), and centrifuged using a slow protein treatment filter to prepare the sample as an LCMS measurement solution. Two sets of LCMS measurement solutions were prepared, one for quantification of 1-hydroxymidazolam generated by the metabolic reaction and one for measuring the actual concentration of the measurement target compound (inhibitor), and each was used for LCMS quantitative analysis.

[0982] For each test compound (inhibitor), the 50% inhibitory concentration (IC 50 ).

[0983] IC 50 =10 (Log(b / a)×(50-A) / (B-A)+Log(a))

[0984] A: The nearest % greater than 50% of the control (low concentration side)

[0985] B: The nearest % less than 50% of the control (high concentration side)

[0986] a: Compound concentration at A (low concentration)

[0987] b: Compound concentration at B (high concentration)

[0988] The concentration of midazolam used as a CYP3A4 substrate was 0.33 μmol / L, which is low enough compared to the Km value of 2.06 μmol / L suggested in the literature. Therefore, the IC 50 It was used directly as the Ki value. (Non-patent document 14: Takano, J., et al. (2016). "The Prediction of the Relative Importance of CYP3A / P-glycoprotein to the Nonlinear Intestinal Absorption of Drugs by Advanced Compartmental Absorption and Transit Model." Drug Metab Dispos 44(11): 1808-1818.) The Ki values ​​of each compound are shown below.

[0989] [Table 57]

[0990] Ki value μmol / L Aripiprazole 17.8 Deferasirox 49 (maximum added concentration) or above Lapatinib 8.94 Ticagrelor 87 (maximum added concentration) or above Ketoconazole 0.034 Itraconazole 0.023 RG12525 1.49

[0991] Deferasirox and ticagrelor did not reach the concentration that showed 50% inhibition, so the concentration above the maximum added concentration was considered as the Ki value. For convenience, the maximum added concentration was used as the Ki value used in the prediction.

[0992] The f obtained in Examples 14 to 20, Reference Examples 14 to 20, and Reference Examples 21 to 27 was used. u Ratio and f u The predicted value of the area under the blood concentration-time curve (AUC) increase ratio (AUCR) of the CYP3A4 substrate midazolam was calculated by using the Ki value used in the inhibitor experiment and comparing it with the observed AUCR to predict the DDI risk.

[0993] The AUCR (observed value) of midazolam when the inhibitor is used in combination is cited in non-patent literature 10-13, as shown in the following table. In addition, when there are multiple AUCR records at the same dose (Dose), the geometric mean of the AUCR is calculated as the observed value. Since there is no numerical description of the AUCR of aripiprazole and ticagrelor, or there is a description that there is no effect on oral administration of midazolam, the AUCR (observed value) is set to 1.

[0994] [Table 58]

[0995] Dosage (mg) Cmax ng / ml AUCR(observed value) Aripiprazole 15 242 1.00 Deferasirox 210 40327 0.90 Lapatinib 1500 2470 1.45 Ticagrelor 90 403 1.00 Ketoconazole 1 200 4681.36 7.13 Ketoconazole 2 400 5877.28 10.96 Itraconazole 1 50 14.11 1.91 Itraconazole 2 200 163.93 5.05 Itraconazole 3 400 317.52 5.07 RG12525-1 100 1482 1.09 RG12525_2 600 8186 1.01

[0996] The following net effect model is used for DDI prediction.

[0997]

[0998] (Non-patent literature 15: FDA DDI guidance (2020))

[0999] The symbols in the above formula have the following meanings.

[1000]

[1001]

[1002] K i，u [μmol / L] = Ki value × fu, mic

[1003] k a 6[h -1 ], f a Use 1.

[1004] Dose is the dosage of each inhibitor administered [μmol].

[1005] Cmax, total [μmol / L]

[1006] Q ent 18 [L / h / 70kg] was used (Non-patent document 16: Yang, Jamei et al., 2007).

[1007] Regarding Fg, in order to verify the prediction accuracy of the fu ratio obtained by dynamic analysis, 1 is used as the input value this time.

[1008] (Non-patent document 16: Yang, J., et al. (2007). "Prediction of intestinal first-pass drug metabolism." Curr Dmg Metab 8(7): 676-684.)

[1009]

[1010] [I]h[μmol / L] (inhibitor concentration in hepatocytes)

[1011]

[1012] K i，u [μmol / L] = Ki value × fu, mic

[1013] K a [h-1] is 6, f a , R b Use 1.

[1014] Dose is the dosage of each inhibitor administered [μmol].

[1015] Cmax, total [μmol / L]

[1016] The Fm used is 0.93.

[1017] Q h 97 [L / h / 70 kg] was used (Non-patent document 17: Yang, Jamei et al., 2007).

[1018] (Non-patent document 17: Yang, J., et al. (2007). "Misuse of the well-stirred model of hepatic drug clearance." Drug Metab Dispos 35(3): 501-502.)

[1019] (Non-patent document 18: Obach, RS, et al. (2006). "The utility of in vitro cytochrome P450 inhibition data in the prediction of drug-drug interactions." J Pharmacol Exp Ther 316 (1): 336-348.)

[1020] also,

[1021] [I]h / Ki,u

[1022] It can be expressed as {f u血清 / (f umic ×K i )}×{Cmax,total+(K a ×f a ×Dose / Q h )÷R b}, for f u血清 / f umic For the fractions, the fu ratio (serum / microsomes) was used.

[1023] Bg·Bh, which showed irreversible inhibition in the intestine and liver, and Cg·Ch, which showed induction, were considered as inhibitors with no effect at this time, and 1 was used as the input value for each.

[1024] The predicted results are as follows Figure 14-1 to Figure 14-3 And shown in the following table.

[1025] [Table 59]

[1026] AUCR Prediction Embodiments 14 to 20 Reference Examples 14 to 20 Reference Examples 21 to 27 AAFE 1.12 1.17 1.37

[1027] Using the relative f obtained by the method of the present invention (dynamic analysis) u The accuracy of DDI prediction of the ratio is superior to that of prediction shown by the conventional method. That is, by using the method of the present invention, it is known that the relative f can be obtained with good accuracy even for the measurement object substance that is difficult to reach the equilibrium state. u Ratio, can be used for DDI prediction.

[1028] Industrial Applicability

[1029] For predictions of human pharmacokinetics based on nonclinical studies (assays using mice, rats, dogs, monkeys, etc.), the fraction unbound in plasma of each animal species (fraction unbound, f u ) is considered to be an important parameter.

[1030] The present invention is a method for directly detecting the relative protein unbound fraction ratio (f) between heterogeneous biological samples of drugs. u By combining rapid equilibrium dialysis and dynamic analysis, a highly accurate f u By using the pharmacokinetic parameters of various animal species based on the ratio of free drug concentrations in plasma, etc., it is expected that the accuracy of human pharmacokinetics estimation will be improved. In addition, it is believed that this technology can be widely used not only for compounds with high protein binding rates but also for compounds with medium to very low protein binding rates, and it is expected to be an indispensable technology in the overall research of low and medium molecule drugs in the future.

[1031] For example, the FCIM method (Non-Patent Document 6), which is currently the most accurate method for predicting human clearance, uses the clearance values ​​of various animal species and f u However, for compounds with high protein binding rate, it is difficult to obtain f u value, so its verification is insufficient. By u Compared with the application of FCIM method, it can be expected that the accuracy of human clearance prediction can be further improved.

[1032] In addition, the present invention can be widely used not only for comparing heterogeneous sera, but also for comparing heterogeneous biological samples. u For example, the protein-unbound fraction of liver microsomes vs. the protein-unbound fraction of serum (f u mic vs f u Thus, it is believed that the refinement of clinical drug-drug interaction (DDI) prediction can be achieved.

[1033] The U.S. FDA's guidance on drug interactions requires that the maximum unbound drug concentration in plasma (Imax, u) and the unbound inhibition constant (Ki, u) be used to predict the extent of drug interactions using the following formula for drugs that have inhibitory effects on drug-metabolizing enzymes.

[1034] R1=1+(Imax,u / Ki,u)

[1035] This formula can be used to calculate the maximum total plasma concentration (I max ), the total concentration-based inhibition constant (K i ), unbound fraction in plasma (f u ), the fraction of unbound microsomes (f u，mic ) is transformed as follows.

[1036] R1=1+(Imax / Ki)*(fu / fumic)

[1037] In addition, the guidance takes into account the inaccuracy of protein binding determination and requires that u If it is less than 0.01, it is calculated as f u =0.01, which overestimates the extent of the interaction and leads to the risk of having to conduct unnecessary clinical drug interaction trials.

[1038] If f is directly and precisely obtained by using the method of the present invention u / f u,mic If the clinical DDI prediction can be refined, unnecessary clinical DDI trials can be skipped, and necessary drugs can be provided to patients more quickly, which is also highly useful in reducing clinical development costs.

[1039] Explanation of symbols

[1040] I: Dialysis Room System I

[1041] II: Dialysis Room System II

[1042] M: Semipermeable membrane

[1043] A: First biological sample (A)

[1044] B: Second biological sample (B)

[1045] 1: Donor side chamber

[1046] 2: Receptor side chamber

[1047] 3: Target substance

[1048] 4: Non-specific proteins from the first biological sample (A)

[1049] 5: Non-specific proteins from the second biological sample (B)

[1050] 10: Donor Side Chamber

[1051] 20: Receptor side chamber

Claims

1. A method for determining the relative unbound fraction ratio of a substance to be measured between different biological samples, comprising the following steps: Step 1: a step of preparing a chamber system I in which adjacent chambers are separated from each other by a semipermeable membrane through which a substance to be measured can pass; Step 2: In the chamber system I, a step of adding a donor solution containing the biological sample A and the substance to be measured to the donor side chamber; Step 3: a step of adding an acceptor solution containing the biological sample B to an acceptor side chamber different from the donor side chamber in the chamber system I; Step 4: a step of measuring the concentration of the substance to be measured in the donor solution of step 2 and the receptor solution of step 3 over time; and Step 5: a step of calculating the relative unbound fraction ratio of the substance to be measured between the different biological samples using the time-dependent data on the concentration of the substance to be measured measured in Step 4, in, The process 5 is a dynamic analysis. The dynamic analysis refers to an analysis method of measuring the concentration of the analyte in the donor solution and the receptor solution over time, performing convergence calculation on the measured values, and thereby calculating the relative unbound fraction ratio of the analyte between the different biological samples.

2. The method according to claim 1, further comprising the following steps: Step 6: a step of preparing a chamber system II different from the chamber system I, in which adjacent chambers are separated from each other by a semipermeable membrane that allows the substance to be measured to permeate; Step 7: In the chamber system II, a step of adding a receptor solution containing the biological sample A to the receptor side chamber; Step 8: a step of adding a donor solution containing the biological sample B and the substance to be measured to a donor side chamber different from the acceptor side chamber in the chamber system II; Step 9: a step of measuring the concentration of the substance to be measured in the acceptor solution of step 7 and the donor solution of step 8 over time; and Step 10: a step of calculating the relative unbound fraction ratio of the substance to be measured between the different biological samples using the time-dependent data on the concentration of the substance to be measured measured in step 9, in, The process 10 is a dynamic analysis. The dynamic analysis refers to an analysis method of measuring the concentration of the analyte in the donor solution and the receptor solution over time, performing convergence calculation on the measured values, and thereby calculating the relative unbound fraction ratio of the analyte between the different biological samples.

3. The method according to claim 1 or 2, wherein: The biological sample A and / or the biological sample B is diluted with a buffer.

4. The method according to claim 1 or 2, wherein: The molecular weight cutoff of the semipermeable membrane is 50 kDa or less.

5. The method according to claim 1 or 2, wherein: The protein binding rate of the substance to be measured in the biological sample A is 95% or more.

6. The method according to claim 1 or 2, wherein: The protein binding rate of the substance to be measured in the biological sample B is 95% or more.

7. The method according to claim 1 or 2, wherein: The biological sample A is one selected from the group consisting of a microsomal fraction, blood, plasma, and serum.

8. The method according to claim 1 or 2, wherein: The biological sample B is one selected from the group consisting of a microsomal fraction, blood, plasma, and serum.

9. The method according to claim 1 or 2, wherein: The biological sample A and the biological sample B are serum.

10. The method according to claim 1 or 2, wherein: The biological sample A is from a mammal.

11. The method according to claim 1 or 2, wherein: The biological sample B is from a mammal.

12. The method according to claim 1 or 2, wherein: The CLogP of the substance to be measured is 25 or less.

13. The method according to claim 1 or 2, wherein: The molecular weight of the substance to be measured is 5000 or less.

14. The method according to claim 1 or 2, wherein: The dynamic analysis is performed using the following equations 1 to 3: Formula 1: Formula 2: Formula 3: In the formula, Formula 1 represents the change in the amount of the analyte in the donor solution per unit time, Formula 2 represents the change in the amount of the analyte in the receptor solution per unit time, Formula 3 represents the change in the amount of the measurement target substance of the adsorption fraction in the donor side chamber per unit time; V1 represents the volume of the donor solution, V2 represents the volume of the receptor solution, fu1 represents the f of the analyte relative to the biological sample contained in the donor solution. u value, fu2 represents the f of the analyte relative to the biological sample contained in the receptor solution. u value, C1 represents the total concentration of the bound and unbound analyte in the donor solution, excluding the analyte adsorbed in the equilibrium dialysis device. C2 represents the total concentration of the bound and unbound analyte in the receptor solution, PS represents the clearance rate of the unbound analyte through the dialysis membrane. CLad represents the adsorption clearance rate of the analyte in the donor side chamber, koff represents the dissociation rate constant of the analyte of the adsorption fraction in the donor side chamber, Xad represents the adsorption amount of the analyte in the donor side chamber.

15. The method according to claim 1 or 2, wherein: The measurement over time is performed during the time during which the biological sample A, the biological sample B, and / or the substance to be measured is denatured.

16. The method according to claim 1 or 2, wherein: The time measurement was performed over 24 hours.

17. The method according to claim 14, wherein: The biological sample A and the biological sample B are serum.

18. A method for predicting drug clearance rate, which uses the relative unbound fraction ratio of the measured object substance between the different biological samples obtained by the method described in any one of claims 1 to 17 and the drug clearance rate data in the species derived from either the biological sample A or the biological sample B to predict the drug clearance rate in the species derived from the other biological sample.

19. A method for predicting drug distribution volume, which uses the relative unbound fraction ratio of the test object substance between the different biological samples obtained by the method described in any one of claims 1 to 17 and the drug distribution volume data in the species derived from either the biological sample A or the biological sample B to predict the drug distribution volume in the species derived from the other biological sample.

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