Genetically modified non-human animal

Abstract

The present invention provides a genetically modified non-human animal or the like that functionally lacks an endogenous ABCB1 gene and that functionally expresses the human ABCB1 gene, the genetically modified non-human animal or the like retaining a DNA construct for functionally expressing the human ABCB1 gene, wherein the DNA construct includes a coding sequence for the human ABCB1 gene, a CAR / PXR response element present in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and a sequence of the enhancer region present at the 3' end side of the human ABCB1 gene, and the DNA construct does not include at least a portion of the coding sequence for the human ABCB4 gene or includes only a portion of the human RUNDC3B gene.

Description

Genetically modified non-human animals

[0001] The present invention relates to a genetically modified non-human animal in which the endogenous ABCB1 gene is functionally deleted on the genome and the human ABCB1 gene is functionally expressed; a DNA construct for functionally expressing the human ABCB1 gene in a non-human animal; a method for creating a genetically modified non-human animal that functionally expresses the human ABCB1 gene; a method for measuring the gastrointestinal absorption of a test substance in a genetically modified non-human animal; a method for predicting the gastrointestinal absorption of a test substance in humans; and a method for screening a test substance.

[0002] ABCB1 (ATP binding cassette subfamily B member 1; also known as MDR1 or P-GP) is a member of the ATP-binding cassette (ABC) transporter superfamily. ABCB1 consists of two transmembrane domains and two cytoplasmic nucleotide-binding domains, and in the tissues where it is expressed (small intestine, blood-brain barrier, liver, kidney, reproductive organs, etc.), it actively transports substrates extracellularly using energy from ATP hydrolysis (Non-patent documents 1 and 2).

[0003] In normal tissues, ABCB1 plays a role in drug central nervous system transport, inhibition of intestinal absorption, and promotion of biliary and urinary excretion, thereby influencing drug blood concentration and tissue distribution. Because ABCB1 recognizes a wide variety of drugs as substrates, it affects the pharmacokinetics of many drugs.

[0004] The use of disease models and animal models is essential for investigating the efficacy and safety of drugs, and the development of more advanced models is desirable for accurately predicting the oral bioavailability of drugs. However, when using animal models, species differences between humans and animals pose a significant problem. For example, species differences have been shown in the drug transport activity of P-glycoprotein (Non-Patent Literature 3). Problems sometimes only surface during human clinical trials, which drives up development costs.

[0005] It is known that there are differences in expression and activity between human ABCB1 and rodent ABCB1 (Non-Patent Literature 4). In most mammals, including humans, a single gene encodes the ABCB1 protein, but in rodents, including mice, two different genes, Abbcb1a and Abbcb1b, encode the ABCB1 protein (Non-Patent Literature 5). In humans, the ABCB1 gene is expressed in the brain, liver, lungs, kidneys, intestinal epithelium, and adrenal glands, while the mouse Abbcb1a gene is expressed in the brain capillary endothelium, liver, lungs, kidneys, and intestinal epithelium, and the mouse Abbcb1b gene is expressed in the brain parenchyma, adrenal glands, and liver. Furthermore, there are differences between human ABCB1 and rodent ABCB1 in their recognition of various substrates (Non-Patent Literature 6 and 7).

[0006] Curr Med Chem. 2010;17(8):786-800.J Clin Invest. 2013 Oct;123(10):4131-3.J Pharm Sci. 2006 Dec;95(12):2673-83.Arch Toxicol. 2021 Sep;95(9):3015-3029.PLoS One. 2014 Oct 29;9(10):e111135.Neuropharmacology. 2007 Feb;52(2):333-46.J Pharmacol Exp Ther. 2001 Mar;296(3):723-35.PLoS One. 2015 May 1;10(5):e0118638.Eur J Microbiol Immunol (Bp). 2018 Jul 6;8(3):78-86.Drug Metab Dispos. 2018 Nov;46(11):1756-1766.Br J Pharmacol. 2021 Nov;178(21):4335-4351.

[0007] Given the species differences in ABCB1 expression and activity described above, it is difficult to predict the pharmacokinetics and drug interactions of ABCB1 substrates in humans using pharmacokinetic studies conducted in experimental animals that lack human ABCB1.

[0008] Therefore, attempts have been made to construct humanized model mice by replacing the ABCB1 gene in experimental mice with human ABCB1 through gene transfer. As one example, knock-in type transgenic mice in which human ABCB1 cDNA is replaced with the mouse Abbcb1 gene have been reported (Non-Patent Documents 8 and 9). However, in transgenic mice produced in this way, the expression of human ABCB1 protein in the intestines was remarkably low, and the pharmacokinetics were more similar to Abbcb1a / b knockout mice than to wild-type animals. Therefore, these mice cannot be used to predict the in vivo pharmacokinetics of drugs that are substrates of ABCB1 in humans.

[0009] Furthermore, humanized mice (hMDR1-MAC mice) have been reported in which a portion of the chromosome containing human ABCB1 has been introduced using a mouse artificial chromosome vector through chromosome engineering technology (Non-Patent Documents 10 and 11). For the plasma concentrations of several ABCB1 substrates, the ratio of the AUC when an ABCB1 inhibitor is administered to the AUC when an ABCB1 inhibitor is not administered in humans (AUCR) human ) and the ratio of the AUC of ABCB1 knockout mice to the AUC of hMDR1-MAC mice in mice (AUCR hMDR1-MAC A good correlation was observed between (Non-Patent Literature 11). This indicates that hMDR1-MAC mice are a useful method for predicting the in vivo pharmacokinetics of drugs that are substrates of ABCB1 in humans. However, the expression level of ABCB1 protein in the intestines of hMDR1-MAC mice is significantly lower than that of wild-type animals, and no difference was observed between hMDR1-MAC mice and Abbcb1a / 1b knockout mice in the area under the concentration-time curve (AUC) after oral administration of paclitaxel, a model compound that is a substrate of ABCB1 (Non-Patent Literature 10). Therefore, further improvement of the model animal is necessary to predict the in vivo pharmacokinetics or drug interactions of drugs that are substrates of ABCB1 with higher accuracy.

[0010] Therefore, the present invention aims to provide genetically modified non-human animals that functionally express the human ABCB1 gene, for example, genetically modified non-human animals that express human ABCB1 with normal function in tissues such as intestinal epithelium and liver, a method for producing such genetically modified non-human animals, and a method for screening therapeutic agents for various diseases using such genetically modified animals.

[0011] The inventors hypothesized that the reason for the significantly low expression levels of human ABCB1 in major drug absorption and excretion organs such as the intestines and liver in conventional human ABCB1-expressing transgenic mice is that the structure or sequence of the introduced gene affected the expression level and tissues of human ABCB1. Therefore, in order to solve the above problem, we attempted to create mice that functionally express human ABCB1 by deleting endogenous Abbcb1a and Abbcb1b. For the introduction of the human ABCB1 gene, we used the human ABCB1 genome sequence with the exon-intron structure preserved, which is incorporated into an Escherichia coli artificial chromosome (BAC). Regions other than those assumed to be promoters and enhancers that regulate human ABCB1 expression were removed by homologous recombination in Escherichia coli. Using a BAC containing such a human ABCB1 gene region, we created ABCB1 humanized mice in which endogenous Abbcb1a and Abbcb1b were deleted and the human ABCB1 gene was introduced. In these ABCB1 humanized mice, we confirmed that human ABCB1 protein is expressed in each organ, and that its expression pattern and function are similar to those of humans. Furthermore, when we calculated the AUCR (Autumnal Organ Correlation) for plasma substrate concentrations after oral administration of several known ABCB1 substrates to these mice, we found that the AUCR of these mice showed a high correlation with the AUCR of humans, and that the dynamic range of correlation was wider than that of hMDR1-MAC mice. In addition, the dynamic range of AUCR correlation was wider in human ABCB1 homozygous individuals than in human ABCB1 heterozygous individuals, indicating that a higher expression level of human ABCB1 leads to a wider dynamic range of AUCR correlation.

[0012] The present invention is completed based on these findings and, in specific embodiments, provides, for example, the following inventions: [1] A genetically modified non-human animal that functionally lacks the endogenous ABCB1 gene and functionally expresses the human ABCB1 gene, wherein it holds a DNA construct for functionally expressing the human ABCB1 gene, the DNA construct comprising the coding sequence of the human ABCB1 gene, a CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of the enhancer region located at the 3' end of the human ABCB1 gene, and the DNA construct either does not contain at least a portion of the coding sequence of the human ABCB4 gene or contains only a portion of the human RUNDC3B gene. [2] The genetically modified non-human animal according to [1], which functionally expresses the human ABCB1 gene in at least one organ selected from the small intestine and the liver. [3] The genetically modified non-human animal according to [2], which functionally expresses the human ABCB1 gene in the small intestine. [4] A genetically modified non-human animal according to any one of [1] to [3], wherein the DNA construct is not present on a mammalian artificial chromosome vector. [5] A genetically modified non-human animal according to any one of [1] to [3], wherein the DNA construct is not present on a mouse artificial chromosome vector. [6] A genetically modified non-human animal according to any one of [1] to [5], wherein the DNA construct does not include at least a portion of the coding sequence of the human ABCB4 gene and includes only a portion of the human RUNDC3B gene. [7] A genetically modified non-human animal according to any one of [1] to [6], wherein the DNA construct includes all the introns of the human ABCB1 gene. [8] A genetically modified non-human animal according to any one of [1] to [7], wherein the non-human animal is a non-human mammal. [9] A genetically modified non-human animal according to [8], wherein the non-human mammal is a mouse.

[10] A genetically modified non-human animal according to any one of [1] to [9] for measuring the pharmacokinetics of a test substance.

[11] A genetically modified non-human animal according to any one of [1] to [9] for predicting the bioavailability of a test substance mediated by human ABCB1 when administered orally to a human.

[12] A genetically modified non-human animal according to any one of [1] to

[11] , wherein the human ABCB1 gene is derived from human male fibroblasts or human male hematopoietic cells.

[13] A genetically modified non-human animal according to any one of [1] to

[12] , wherein the human ABCB1 gene is derived from the BAC / PAC human genome library.

[14] A genetically modified non-human animal according to

[13] , wherein the nucleotide sequence derived from the BAC / PAC human genome library is included in its genomic sequence.

[15] A DNA construct for functionally expressing the human ABCB1 gene in a non-human animal, comprising the coding sequence of the human ABCB1 gene, the CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of the enhancer region located at the 3' end of the human ABCB1 gene, and not comprising at least a portion of the coding sequence of the human ABCB4 gene, or comprising only a portion of the human RUNDC3B gene.

[16] A BAC vector comprising the DNA construct described in

[15] .

[17] A method for producing a genetically modified non-human animal that functionally expresses the human ABCB1 gene, comprising the step of introducing a DNA construct for functionally expressing the human ABCB1 gene into the cells of the non-human animal, wherein the DNA construct comprises the coding sequence of the human ABCB1 gene, a CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of an enhancer region located at the 3' end of the human ABCB1 gene, and the DNA construct either does not include at least a portion of the coding sequence of the human ABCB4 gene or includes only a portion of the human RUNDC3B gene.

[18] The method according to

[17] , further comprising the step of crossing a non-human animal into which the DNA construct has been introduced into its cells with a non-human animal of the same species that is functionally lacking the endogenous ABCB1 gene, to select a non-human animal that retains the DNA construct and is functionally lacking the endogenous ABCB1 gene.

[19] The method according to

[17] or

[18] , further comprising the step of selecting a non-human animal from which the DNA construct has been introduced into its cells that functionally expresses human ABCB1 in at least one organ selected from the intestine and the liver.

[20] A method for measuring the pharmacokinetics of a test substance in a genetically modified non-human animal, comprising the steps of: (1) administering the test substance to a genetically modified non-human animal according to any one of [1] to

[14] ; and (2) taking a biological sample from the genetically modified non-human animal after administration of the test substance in step (1) and measuring the content of the test substance in the biological sample.

[21] The method according to

[20] , wherein the biological sample is at least one selected from the group consisting of plasma, bile, feces, small intestinal contents, and large intestinal contents.

[22] A method for predicting the human ABCB1-mediated pharmacokinetics of a test substance in humans, comprising the steps of: (1) measuring the pharmacokinetics of a test substance in a genetically modified non-human animal by the method according to

[20] or

[21] ; and (2) predicting the human ABCB1-mediated pharmacokinetics of the test substance when administered to humans, based on the pharmacokinetics of the test substance in the genetically modified non-human animal measured in (1).

[23] A method for screening a test substance, comprising the steps of: (1) measuring the pharmacokinetics of a test substance in a genetically modified non-human animal by the method described in

[20] or

[21] ; (2) predicting the human ABCB1-mediated pharmacokinetics of the test substance when administered to a human, based on the pharmacokinetics of the test substance in the genetically modified non-human animal measured in (1); and (3) selecting a test substance that is predicted in (2) to exhibit a desired pharmacokinetic in humans.

[0013] According to the present invention, by providing genetically modified non-human animals in which the endogenous ABCB1 gene (Abcb1a and Abbcb1b genes in mice) is deleted and the human ABCB1 gene is functionally expressed, it becomes possible to predict the in vivo pharmacokinetics or drug interactions of drugs that are substrates of ABCB1 in humans with higher accuracy than before.

[0014] A schematic diagram of an expression vector containing the human ABCB1 gene region with an exon-intron structure is shown. A schematic diagram of an expression vector containing human ABCB1 cDNA downstream of the human ABCB1 gene regulatory region is shown. Results of confirming the presence of the human ABCB1 gene by PCR in founder mice into which the human ABCB1 gene has been introduced are shown. Results of confirming the copy number of the human ABCB1 gene by qPCR in founder mice into which the human ABCB1 gene has been introduced are shown. Results of confirming the transfer of the human ABCB1 gene from founder mice into which the human ABCB1 gene has been introduced to the next generation of mice by qPCR are shown. Results of confirming the expression of the human ABCB1 gene in the next generation of mice obtained by crossing founder mice into which the human ABCB1 gene has been introduced with C57BL / 6J mice are shown by RT-PCR. RT: Reverse transcription present; non RT: Reverse transcription absent. The following results show the quantification of human ABCB1 gene expression in the small intestine of next-generation mice obtained by crossing founder mice into which the human ABCB1 gene has been introduced with C57BL / 6J mice, using RT-qPCR. The following results show the quantification of human ABCB1 gene expression in the brain of human ABCB1-expressing mice from the RP11-31F line, using RT-qPCR. The following results show the quantification of human ABCB1 gene expression in the heart of human ABCB1-expressing mice from the RP11-31F line, using RT-qPCR. The following results show the quantification of human ABCB1 gene expression in the liver of human ABCB1-expressing mice from the RP11-31F line, using RT-qPCR. The following results show the quantification of human ABCB1 gene expression in the kidney of human ABCB1-expressing mice from the RP11-31F line, using RT-qPCR. This document shows the results of quantifying the expression level of the human ABCB1 gene in the small intestine (ileum) of human ABCB1-expressing mice from the RP11-31F line using RT-qPCR. A schematic diagram of the method for creating double knockout mice lacking both endogenous Abcb1a and Abcb1 genes is shown. The results of analyzing the tissue distribution of human ABCB1 in ABCB1 humanized mice and double knockout mice lacking both endogenous Abcb1a and Abcb1 genes using immunohistochemical staining are also shown. IHC: Immunohistochemistry. HE: Hematoxylin / eosin.Regarding four types of ABCB1 substrate drugs, AUCR (AUCR) was conducted on ABCB1 humanized mice. humanized ) and AUCR in humans (AUCR human This shows a correlation with ABCB1. It also shows the clearance of ABCB1 substrate drugs in ABCB1 humanized mice and endogenous ABCB1 knockout mice.

[0015] 1. definition

[0016] In this specification, "gene region" means a specific region on the genome that contains a particular gene. As a non-limiting example, the human ABCB1 gene region includes at least the base sequence that codes for the human ABCB1 protein (coding region or coding region), and may also include surrounding sequences involved in the regulation of human ABCB1 gene expression. The coding region (also called the coding sequence) refers to a continuous region from the start codon to the stop codon. On the eukaryotic genome, the coding region is usually divided by intervening sequences called introns. In eukaryotic genes, the coding region and uncoding region (UTR) are divided into small fragments (exons), with intervening sequences (introns) between them. Introns are transcribed into RNA along with exons and removed by RNA splicing (Alberts et al., Molecular Biology of the Cell (6th edition), p. 316). Regions involved in the regulation of gene expression are often located upstream (5' side) of the base sequence that codes for the protein, but can also be located downstream (3' side) or in intron regions. For example, in the promoter region of the human ABCB1 gene, a CAR / PXR response element exists as a regulatory sequence approximately 8 kilobases (kb) upstream of the start codon. This sequence has the function of inducing the expression of the human ABCB1 gene by the activating molecules PXR and CAR (Drug Metab Dispos. 2015;43(11):1646-54.).

[0017] In this specification, "substrate drugs of ABCB1" refers to any compound that can be excreted extracellularly by ABCB1 (e.g., human ABCB1), including, but not limited to, Aliskiren, Betrixaban, Celiprolol, Talinolol, digoxin, everolimus, and imatinib. Peptide compounds may also be included in "substrate drugs of ABCB1".

[0018] In this specification, "peptide compound" means a compound in which two or more amino acids are linked by an amide bond. Peptides having an ester bond in part of the main chain, such as depsipeptides, are also included in the definition of "peptide compound" in this specification. The number of amino acid residues contained in the peptides in this disclosure is not particularly limited, but is preferably 5 to 30 residues, more preferably 8 to 15 residues, and even more preferably 9 to 13 residues. The peptide compounds in this disclosure preferably contain at least three N-substituted amino acids, more preferably at least five, and even more preferably at least six. These N-substituted amino acids may be present continuously or discontinuously in the peptide compound. The peptide compounds in this disclosure may be linear or cyclic, with cyclic peptide compounds being preferred.

[0019] In this specification, "cyclic peptide compound" means a peptide compound having a cyclic structure composed of four or more amino acid residues. The cyclic structure of a cyclic peptide compound may include bonds other than amide bonds, for example, bonds selected from the group consisting of COC bonds, C(O)-O bonds, C(S)-O bonds utilizing oxygen atoms, C(O)-S bonds, C(S)-S bonds, CSSC bonds, CSC bonds, CS(O)-C bonds, CS(O2)-C bonds utilizing sulfur atoms, and CNC bonds, C=NC bonds, NC(O)-N bonds, NC(S)N bonds, C(S)-N bonds, and CC bonds utilizing nitrogen atoms. In addition to the cyclic structure, a cyclic peptide compound may have amino acids or chain-like peptide structures that are not included in the cyclic structure. Furthermore, it may have structures other than amino acids or chain-like peptide structures.

[0020] "Cyclization" of a peptide compound means forming a cyclic structure containing four or more amino acid residues. The number of amino acids in the cyclic structure of a cyclic peptide compound as used herein is not particularly limited, but examples include 4-20 residues, 5-15 residues, and 6-13 residues. Methods for converting linear peptide compounds into cyclic peptide compounds can be carried out by performing an intramolecular bond formation reaction, as described in Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (by RC Larock) or March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Edition (by MB Smith, J. March). Further functional group transformation reactions can also be performed after the bond formation reaction. Examples of bond-forming reactions include those that form C(O)-N bonds from carboxylic acids and amines, COC bonds, C(O)-O bonds, and C(S)-O bonds using oxygen atoms, C(O)-S bonds, C(S)-S bonds, CSSC bonds, CSC bonds, CS(O)-C bonds, and CS(O2)-C bonds using sulfur atoms, and CNC bonds, C=NC bonds, NC(O)-N bonds, NC(S)N bonds, and C(S)-N bonds using nitrogen atoms. Furthermore, transition metal-catalyzed CC bond formation reactions such as the Suzuki reaction, Heck reaction, and Sonogashira reaction are also mentioned. Examples of functional group transformation reactions that follow bond-forming reactions include oxidation or reduction reactions. Specifically, examples include reactions that oxidize sulfur atoms to convert them into sulfoxide or sulfone groups. In addition, examples include reduction reactions that reduce triple or double carbon-carbon bonds to convert them into double or single bonds. When two amino acids are joined in the main chain of an amino acid, a ring-closed structure is formed by a peptide bond. However, a covalent bond may also be formed between the two amino acids through bonding of their side chains, or between the side chains and the main chain.

[0021] "Amino acid" as used herein includes natural amino acids and non-natural amino acids. Also, in this specification, "amino acid" may mean an amino acid residue. "Natural amino acid" as used herein refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, Pro. The non-natural amino acids are not particularly limited, and examples include β-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids with side chains different from natural amino acids, hydroxycarboxylic acids, etc. Any configuration is acceptable for the amino acids as used herein. The selection of the side chain of the amino acid is not particularly restricted, and in addition to a hydrogen atom, for example, it can be freely selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a heteroarylalkyl group, a cycloalkyl group, a spiro-bonded cycloalkyl group. Each may be provided with a substituent, and these substituents are also not restricted, and for example, one or more can be freely selected independently from any substituents containing a halogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a boron atom, a silicon atom, or a phosphorus atom. That is, examples include an optionally substituted alkyl group, an alkoxy group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, etc., or oxo, aminocarbonyl, a halogen atom, etc. In one non-limiting aspect, the amino acid as used herein may be a compound having a carboxyl group and an amino group within the same molecule (even in this case, proline, hydroxyproline, azetidine-2-carboxylic acid, etc., in which the nitrogen atom of the amino group and any atom of the side chain combine to form a ring, are also included in the amino acids).

[0022] In this specification, the "amino acid residue" constituting the peptide compound may sometimes be simply referred to as "amino acid".

[0023] In this specification, any configuration of amino acids is acceptable. There is no particular limitation on the selection of the side chains of amino acids. In addition to a hydrogen atom, for example, they can be freely selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group, a cycloalkyl group, and a spiro-bonded cycloalkyl group. Each of them may be provided with a substituent, and these substituents are also not limited. For example, they may be independently selected freely from one or more of any substituents containing a halogen atom, an O atom, an S atom, an N atom, a B atom, a Si atom, or a P atom. That is, examples include an optionally substituted alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, etc., or oxo, aminocarbonyl, a halogen atom, etc. The amino acid according to one embodiment may be a compound having a carboxy group and an amino group within the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in amino acids).

[0024] In this specification, the "ABCB1 inhibitor" refers to a compound that inhibits the function of human ABCB1 as an efflux transporter, and examples include, but are not limited to, Zosuquidar, cyclosporine, itraconazole, ritonavir, etc.

[0025] In this specification, the "absorption" of a drug refers to the process by which a drug administered to an animal migrates into the systemic circulation blood. In the case of oral administration, the transport of the drug passing through the membrane of epithelial cells in the digestive tract is sometimes referred to as gastrointestinal absorption, or simply absorption. An orally administered drug enters the systemic circulation blood from the digestive tract via the portal vein blood and the liver and is transported to the target site of action. The ratio incorporated into the systemic circulation blood is called bioavailability, which is a quantitative index of drug absorbability. The absorption of a drug can be confirmed by measuring the concentration of the drug taken up in the blood after a certain period of time has elapsed after the drug is administered.

[0026] In this specification, "Pharmacokinetics" (also referred to as "PK") refers to the change in the concentration (amount) of a drug in the living body after a series of processes of absorption, distribution, metabolism, and excretion in the living body after the drug is administered. After the drug is administered, in the living body, the processes of absorption, distribution, metabolism, and excretion proceed in parallel. As basic PK parameters for decomposing and describing these processes, (1) bioavailability (F), (2) volume of distribution (Vd or V), (3) fraction unbound in blood (fuB), (4) clearance (CL), and (5) cumulative amount of drug excreted in urine (Ae) have been established (Journal of Pharmacometrics Vol. 36, Special Issue, S 3-S 18 (2015)). As indicators of bioavailability, area under the blood concentration-time curve (AUC), maximum blood concentration (Cmax), time to reach maximum blood concentration (Tmax), etc. are known. Also, the bioavailability at oral administration can be represented by the product of the gastrointestinal absorption rate (F a F g ) and the extraction rate of the drug in the liver (F h ). F a represents the absorption rate, F g represents the rate of avoiding metabolism in the gastrointestinal tract, and F h represents the rate of avoiding metabolism in the liver. As an indicator of the volume of distribution, the volume of distribution at steady state (Vss), etc. are known. As other PK parameters, blood concentration half-life (t1 / 2), mean residence time (MRT), etc. are known. In this specification, with respect to pharmacokinetics (for example, bioavailability, clearance, etc.), "mediated by human ABCB1" means that the function of human ABCB1 is involved.

[0027] 2. Genetically Modified Non-Human Animals In one embodiment, the present invention relates to a genetically modified non-human animal (hereinafter also referred to as the genetically modified non-human animal of the present invention) that functionally lacks the endogenous ABCB1 gene and functionally expresses the human ABCB1 gene.

[0028] ABCB1 is one of the ABC transporters, also known as MDR1 or P-GP. In this specification, unless otherwise specified, ABCB1 is used as the gene name or protein name. In most mammals, including humans, the ABCB1 protein is encoded by a single ABCB1 gene. The human ABCB1 gene is assigned Gene ID 5243 (updated on 5-Mar-2024) in the NCBI database, for example, and corresponds to the sequence 87,503,017–87,713,295 (complementary strand) in the human genome assembly GRCh38 / hg38. The polynucleotide sequence of human ABCB1 is registered in the database as, for example, NM_001348946.2 (RefSeq registry number), and the polypeptide sequence as NP_001335875.1 (RefSeq registry number). In this specification, "human ABCB1 gene" means a human gene expressing a functional ABCB1 protein, unless otherwise specified. In rodents, including mice, it is known that two different genes, Abbcb1a and Abbcb1b, encode the ABCB1 protein.

[0029] In this specification, "endogenous ABCB1 gene" refers to the ABCB1 gene that encodes a functional ABC transporter, which is inherently present in the non-human animal species to which the genetically modified non-human animal of the present invention belongs. Normally, the "endogenous ABCB1 gene" is located on a chromosome (or genome), but in this invention, it is not a matter of whether it is located on a chromosome, outside of a chromosome, or introduced into a cell from an external source.

[0030] In this specification, "functionally deficient in the endogenous ABCB1 gene" means a state in which the endogenous ABCB1 gene does not express its original function in a non-human animal. Such a state is not particularly limited as long as it does not interfere with the use of the genetically modified non-human animals of the present invention in studies of the pharmacokinetics or drug interactions of drugs that are substrates of human ABCB1, and includes, for example, a state in which the endogenous ABCB1 gene on the genome of a non-human animal is manipulated so that it is not normally expressed. For example, the endogenous ABCB1 gene may be deleted (knocked out) on the genome of a non-human animal using genetic engineering techniques such as homologous recombination technology, CRISPR / Cas9, zinc finger nucleases, or genome editing technologies such as TALEN, which are widely known in the art, or the functional expression of the endogenous ABCB1 gene may be suppressed (knocked down) using methods that suppress gene expression using siRNA or the like. For example, to delete the endogenous ABCB1 gene in the genome of a non-human animal, the genomic region containing the endogenous ABCB1 gene can be deleted by inserting the substrate sequence of a sequence-specific recombinant enzyme into the 5' and 3' ends of the genomic region encoding the endogenous ABCB1 gene, and then applying the recombinant enzyme. Examples of sequence-specific recombinant enzymes that can be used include Cre, Dre, and Flp. A specific recombinant enzyme can be used depending on the substrate sequence to be inserted into the genomic region. For example, the loxP sequence is used for Cre, the Rox sequence for Dre, and the Frt sequence for Flp. Alternatively, to prevent the endogenous ABCB1 gene from being functionally expressed, a foreign gene may be inserted at the location of the endogenous ABCB1 gene in the non-human animal genome, for example, using knock-in technology. The method for producing the genetically modified non-human animal of the present invention is not particularly limited. For example, in a non-human animal that functionally expresses the human ABCB1 gene, a procedure may be performed to functionally delete the endogenous ABCB1 gene, or in a non-human animal that functionally lacks the endogenous ABCB1 gene, a procedure may be performed to functionally express the human ABCB1 gene.Furthermore, by crossbreeding a non-human animal that functionally expresses the human ABCB1 gene with a non-human animal that functionally lacks the endogenous ABCB1 gene, it is possible to create a genetically modified non-human animal that functionally lacks the endogenous ABCB1 gene and functionally expresses the human ABCB1 gene.

[0031] In animals with a functional deficiency of the endogenous ABCB1 gene, if exogenous ABCB1, such as the human ABCB1 gene, is not expressed, the amount of drug efflux by transporters expressed on the luminal side of the cell membrane of gastrointestinal epithelial cells decreases, which is thought to increase the amount of drug absorbed from the intestines. In addition, in normal animals, multiple efflux transporters such as ABCB1 and BCRP are expressed on the blood side of the blood-brain barrier, limiting the cross-section of drugs into the brain. Therefore, in animals with a functional deficiency of the endogenous ABCB1 gene, the cross-section of drugs into the brain may increase. If exogenous ABCB1, such as the human ABCB1 gene, is not expressed, the amount of drug absorbed from the intestines into the bloodstream via enterocytes in animals with a functional deficiency of the endogenous ABCB1 gene may increase compared to levels in wild-type animals or levels considered normal.

[0032] If the non-human animal from which the genetically modified non-human animal of the present invention is derived has multiple endogenous ABCB1 genes, the genetically modified non-human animal of the present invention may have at least one endogenous ABCB1 gene functionally deficient, or may have all endogenous ABCB1 genes functionally deficient, as long as this does not prevent the use of the genetically modified non-human animal of the present invention in pharmacokinetic or drug interaction studies of drugs that are substrates of human ABCB1. For example, if the study involves a specific organ (small intestine, liver, kidney, brain, etc.), it is sufficient for the endogenous ABCB1 gene to be functionally deficient in that organ. For example, if the non-human animal in the present invention is a mouse, the genetically modified non-human animal of the present invention may have one or both of the Abbcb1a and Abbcb1b genes functionally deficient. In a preferred embodiment, the genetically modified non-human animal of the present invention may have all endogenous ABCB1 genes functionally deficient.

[0033] If a non-human animal functionally expresses an ABCB1 gene derived from a non-human species, this can interfere with the pharmacokinetics or drug-drug interaction studies of drugs that are substrates for human ABCB1. Therefore, in a preferred embodiment, the genetically modified non-human animal of the present invention does not functionally express an ABCB1 gene derived from a non-human species. In this case, "not functionally expressing an ABCB1 gene derived from a non-human species" means that in the genetically modified non-human animal of the present invention, the ABCB1 gene derived from a non-human species does not express its original function in that species, or is absent. Such a state is not particularly limited as long as it does not interfere with the use of the genetically modified non-human animal of the present invention in studies of the pharmacokinetics or drug-drug interaction studies of drugs that are substrates for human ABCB1, and includes, for example, a state in which the endogenous ABCB1 gene on the non-human animal genome is manipulated so that it is not normally expressed, as well as a state in which an exogenous ABCB1 gene derived from a non-human species has not been introduced into the genetically modified non-human animal of the present invention. In a preferred embodiment, the genetically modified non-human animal of the present invention does not have an exogenous ABCB1 gene derived from a non-human species introduced into it.

[0034] In this specification, “functionally expressing the human ABCB1 gene” means that the human ABCB1 gene is expressed in at least one organ of a non-human animal, and that the human ABCB1 protein functions as a drug efflux transporter in that organ. This organ is one in which the human ABCB1 gene is expressed in humans, and may be, for example, the small intestine (duodenum, jejunum, and ileum), liver, kidney, and brain. In a preferred embodiment, the genetically modified non-human animal of the present invention functionally expresses the human ABCB1 gene in at least one organ selected from the small intestine and liver; more preferably, functionally expresses the human ABCB1 gene in the small intestine; and even more preferably, functionally expresses the human ABCB1 gene in both the small intestine and the liver. The expression of the human ABCB1 gene can be confirmed by detecting or quantifying mRNA or protein expression using techniques known in the art, such as reverse transcription PCR (RT-PCR), quantitative reverse transcription PCR (RT-qPCR), protein mass spectrometry, and immunohistochemical staining. The function of human ABCB1 as a drug efflux transporter can be confirmed by pharmacokinetic studies (see, for example, Non-Patent Document 11). For example, pharmacokinetics can be evaluated by measuring the absorption of a drug that is a substrate of ABCB1 (e.g., digoxin) from the duodenum (see, for example, Pharm Res. 2003 Apr;20(4):552-6).

[0035] The genetically modified non-human animal of the present invention possesses a DNA construct for functionally expressing the human ABCB1 gene, the DNA construct comprising the coding sequence of the human ABCB1 gene, the CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of the enhancer region located at the 3' end of the human ABCB1 gene, but not including at least a portion of the coding sequence of the human ABCB4 gene, or including only a portion of the human RUNDC3B gene.

[0036] In this invention, a DNA construct means a DNA segment that does not naturally exist in non-human animals.

[0037] The "coding sequence of the human ABCB1 gene" refers to the sequence of the coding region, which is the region of the human ABCB1 gene excluding the untranslated region, where all exons of the human ABCB1 gene are linked together. In a preferred embodiment, the DNA construct of the present invention may include the untranslated region in addition to the "coding sequence of the human ABCB1 gene." In this case, the DNA construct of the present invention may include all exons of the human ABCB1 gene so that the human ABCB1 protein is produced in vivo. In the DNA construct of the present invention, the "coding sequence of the human ABCB1 gene" is interrupted by "at least one intron of the human ABCB1 gene." When the human ABCB1 gene is expressed, the intron is removed by splicing, so that a normal human ABCB1 protein is expressed. In a preferred embodiment, the DNA construct of the present invention may include all exons and introns of the human ABCB1 gene so that the human ABCB1 protein is produced in vivo.

[0038] The "CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene" refers to the CAR / PXR response element (Drug Metab Dispos. 2015;43(11):1646-54.) located approximately 8kb upstream of the start codon of the wild-type human ABCB1 gene and known as a regulatory sequence. In the chromosome 7 sequence GRCh38 / hg38, the CAR / PXR response element is contained in the sequence 87600185 to 87608764. In one embodiment, the DNA construct of the present invention may include the region from the start codon of the human ABCB1 gene to approximately 8kb upstream thereof as the region containing the "CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene," and preferably may include the sequence 87600185 to 87608764 of GRCh38 / hg38.

[0039] Two regions of GRCh38 / hg38 are known as "sequences of enhancer regions located at the 3' end of the wild-type human ABCB1 gene": 87497491-87498800 and 87499001-87500200. In one embodiment, the DNA construct of the present invention may include at least one or both of 87497491-87498800 and 87499001-87500200 of GRCh38 / hg38 as "sequences of enhancer regions located at the 3' end of the wild-type human ABCB1 gene," preferably both.

[0040] The "at least one intron of the human ABCB1 gene" included in the DNA construct of the present invention is located at a position that is naturally present in the human genome, between exons in the coding sequence of the human ABCB1 gene. The "at least one intron of the human ABCB1 gene" is removed by splicing when the human ABCB1 gene is expressed. In a preferred embodiment, the DNA construct of the present invention may include all introns of the human ABCB1 gene.

[0041] In one embodiment, the genetically modified non-human animal of the present invention may include the full-length nucleotide sequence of the human ABCB1 gene. The "full-length nucleotide sequence" of the human ABCB1 gene may include the coding sequence, untranslated regions, introns, and expression regulatory regions such as promoters and enhancers, and may include, for example, the sequence of GRCh38 / hg38 from 87,493,091 to 87,631,371 (the human ABCB1 gene is encoded in the complementary strand).

[0042] In one embodiment, the human ABCB1 gene in the genetically modified non-human animal of the present invention may be derived from the BAC / PAC human genome library. That is, the sequence of the human ABCB1 gene in the DNA construct of the present invention may be derived from the BAC / PAC human genome library. Examples of BAC / PAC human genome libraries include, but are not limited to, the RPCI-11 library, the CalTech D (CTD) library, the CalTech C (CTC) library, and the CalTech B (CTB) library. Furthermore, these BAC / PAC human genome libraries may include, but are not limited to, sequences such as replication origins (ori), drug resistance genes such as chloramphenicol resistance gene (cmR) and kanamycin resistance gene (kanR), and restriction enzyme sites for inserting target DNA fragments (Proc Natl Acad Sci US A. 89(18): 8794-8797; Genomics. 1999 Jun 15; 58(3): 250-253; Proc Natl Acad Sci US A. 1992 Mar 15; 89(6): 2056-2060., etc.). In a preferred embodiment, the genome sequence of the genetically modified non-human animal of the present invention may include nucleotide sequences derived from the BAC / PAC human genome library.

[0043] On the human genome, the human RUNDC3B gene is located upstream of the start codon of the human ABCB1 gene, and the human ABCB4 gene is located downstream of the stop codon. In GRCh38 / hg38, the human RUNDC3B gene has the sequence 87,628,398–87,832,296. In GRCh38 / hg38, the human ABCB4 gene has the sequence 87,365,896–87,476,027 (complementary strand). The polynucleotide sequence of human RUNDC3B is registered in the database, for example, as NM_001134405.2 (RefSeq registry number), and the polypeptide sequence as NP_001127877.1 (RefSeq registry number). The polynucleotide sequence of human ABCB4 is registered in the database as, for example, NM_000443.4 (RefSeq registry number), and the polypeptide sequence as NP_000434.1 (RefSeq registry number). The coding sequence of the human ABCB4 gene is registered in the CCDS database as CCDS5605.1.

[0044] The DNA construct in this invention either does not contain at least a portion of the coding sequence of the human ABCB4 gene, or contains only a portion of the human RUNDC3B gene. If the DNA construct in this invention does not contain at least a portion of the coding sequence of the human ABCB4 gene, it does not contain the full length of the human ABCB4 gene having an exon-intron structure, and therefore it is considered that the genetically modified non-human animal of this invention will not functionally express the human ABCB4 gene. If the DNA construct in this invention contains only a portion of the human RUNDC3B gene, it does not contain the full length of the human RUNDC3B gene having an exon-intron structure, and therefore it is considered that the genetically modified non-human animal of this invention will not functionally express the human RUNDC3B gene. In one embodiment, the genetically modified non-human animal of this invention does not functionally express the human ABCB4 gene, or does not functionally express the human RUNDC3B gene. In a preferred embodiment, the genetically modified non-human animal of this invention does not functionally express the human ABCB4 gene and the human RUNDC3B gene.

[0045] In a preferred embodiment, the DNA construct of the present invention does not contain at least a portion of the coding sequence of the human ABCB4 gene, but contains only a portion of the human RUNDC3B gene. In this case, neither the human ABCB4 gene nor the human RUNDC3B gene is expressed from the DNA construct of the present invention, and only the human ABCB1 gene is functionally expressed.

[0046] In a preferred embodiment, the DNA construct of the present invention does not include at least a portion of the coding sequences located within GRCh38 / hg38 87,402,096 to 87,475,465, or does not include at least a portion of the sequences located within GRCh38 / hg38 87,631372 to 87,832,296. In a more preferred embodiment, the DNA construct of the present invention does not include at least a portion of the coding sequences located within GRCh38 / hg38 87,402,096 to 87,475,465, and does not include at least a portion of the sequences located within GRCh38 / hg38 87,631372 to 87,832,296. In a more preferred embodiment, the DNA construct of the present invention does not include all of the coding sequences present in GRCh38 / hg38 between 87,402,096 and 87,475,465, or does not include the sequences 87,631372 to 87,832,296 of GRCh38 / hg38. In an even more preferred embodiment, the DNA construct of the present invention does not include all of the coding sequences present in GRCh38 / hg38 between 87,402,096 and 87,475,465, and does not include the sequences 87,631372 to 87,832,296 of GRCh38 / hg38.

[0047] In a preferred embodiment, the genetically modified non-human animal of the present invention does not contain the full length of the human RUNDC3B gene, which has all exon-intron structures, upstream of the start codon of the human ABCB1 gene, on its intracellular DNA. In a preferred embodiment, the genetically modified non-human animal of the present invention does not contain a portion of the human ABCB4 gene in the region up to 10kb, 20kb, 30kb, 40kb, 50kb, or 100kbp downstream of the stop codon of the human ABCB1 gene on its intracellular DNA, and more preferably, the genetically modified non-human animal of the present invention does not contain a portion of the human ABCB4 gene on its intracellular DNA.

[0048] The DNA constructs in the present invention may be incorporated into chromosomal DNA or into extrachromosomal DNA (e.g., artificial chromosome vectors), as long as they are maintained within the cells of a non-human animal so as to functionally express the human ABCB1 gene. In preferred embodiments, the DNA constructs in the present invention are not present on artificial chromosome vectors (e.g., mammalian artificial chromosome vectors such as mouse artificial chromosome vectors), and more preferably, are incorporated into chromosomal DNA.

[0049] In this invention, "non-human animals" are not intended to be particularly limited, but are preferably non-human mammals. Rodents such as mice, rats, and hamsters; non-human primates such as monkeys and chimpanzees; other mammals such as rabbits, sheep, cows, and pigs; as well as birds, amphibians, reptiles, and fish can also be used as non-human animals in this invention. In this invention, non-human animals are particularly preferably rodents, and most preferably mice.

[0050] In one embodiment, the genetically modified non-human animal of the present invention can be used to measure the pharmacokinetics of a test substance, such as gastrointestinal absorption or bioavailability. For example, the gastrointestinal absorption of a test substance is measured by administering the test substance to the genetically modified non-human animal of the present invention and then measuring the concentration of the test substance taken up into the blood after a certain period of time. In a preferred embodiment, the test substance may be a drug that is a substrate of ABCB1, but it is not necessary to know that it is a substrate of ABCB1 at the time of pharmacokinetic measurement. The route of administration can be appropriately selected depending on the type of test substance, and examples include oral administration (po), intravenous administration (iv), subcutaneous administration (sc), intraperitoneal administration (ip), intramuscular administration (im), tumor administration (it), transpulmonary administration, transnasal administration, etc. In a preferred embodiment, the pharmacokinetics of the test substance may be bioavailability, and more preferably, bioavailability after oral administration.

[0051] As shown in the examples below, the ratio of the AUC when the ABCB1 inhibitor is administered to the AUC when the ABCB1 inhibitor is not administered (AUCR) is obtained using the genetically modified non-human animals of the present invention. hABCB1 ) can be calculated. AUCR hABCB1 This may also be the ratio of the AUC in the genetically modified non-human animal of the present invention to the AUC in the control (a similar non-human animal control that does not express the human ABCB1 gene and has a functional deficiency of the endogenous ABCB1 gene). hABCB1 AUCR in humans (AUCR human It shows a high correlation with ABCB1, and the dynamic range of the correlation is wider than that of conventional ABCB1 humanized mice. Therefore, in one embodiment, the genetically modified non-human animal of the present invention can be used to predict the bioavailability of human ABCB1-mediated bioavailability when a test substance is orally administered to humans. In a preferred embodiment, the test substance may be a drug that is a substrate of ABCB1, but it does not have to be known to be a substrate of ABCB1 at the time of bioavailability measurement.

[0052] Furthermore, the genetically modified non-human animals of the present invention exhibit higher clearance (e.g., bile excretion clearance and gastrointestinal secretion clearance) compared to controls (non-human animal controls of the same species that do not express the human ABCB1 gene and functionally lack the endogenous ABCB1 gene). Therefore, in one embodiment, the genetically modified non-human animals of the present invention can be used to predict the human ABCB1-mediated clearance of a test substance in humans.

[0053] The genetically modified non-human animals of the present invention may possess the human ABCB1 gene in a heterozygous or homozygous state. As shown in the examples below, the AUCR of animals possessing the human ABCB1 gene in a homozygous state. hABCB1 AUCR in animals that carry the gene heterozygously hABCB1 The dynamic range of correlation with AUCR in humans is wider than that. Therefore, in a preferred embodiment, the genetically modified non-human animal of the present invention may possess the human ABCB1 gene in a homozygous state.

[0054] 3. DNA constructs and vectors - In one embodiment, the present invention relates to a DNA construct for functionally expressing the human ABCB1 gene in a non-human animal, comprising the coding sequence of the human ABCB1 gene, the CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of the enhancer region located at the 3' end of the human ABCB1 gene, and not comprising at least a portion of the coding sequence of the human ABCB4 gene, or only a portion of the human RUNDC3B gene (hereinafter also referred to as the DNA construct of the present invention).

[0055] The DNA constructs of the present invention do not include those integrated into the human genome, but may be integrated into the genome of a non-human animal or isolated.

[0056] The DNA construct of the present invention can be used to create genetically modified non-human animals of the present invention. The composition of the DNA construct of the present invention is as described above in "2. Genetically Modified Non-Human Animals". The sequences included in and not included in the DNA construct of the present invention, namely, "the coding sequence of the human ABCB1 gene", "the CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene", "at least one intron of the human ABCB1 gene", and "the sequence of the enhancer region located at the 3' end of the human ABCB1 gene", as well as "the human ABCB4 gene" and "the human RUNDC3B gene", are also as described above in "2. Genetically Modified Non-Human Animals".

[0057] In one embodiment, the DNA construct of the present invention may be contained in a vector. The vector is not particularly limited as long as it is used in genetic engineering, and examples include viral vectors such as bacteriophages, adenoviruses, retroviruses, poxviruses, and herpesviruses, and vectors derived from artificial chromosomes. Examples of DNA vectors include plasmid vectors, viral vectors, cosmid vectors, mammalian artificial chromosomes such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and mouse artificial chromosomes (MACs), and other non-plasmid vectors. In a preferred embodiment, the DNA construct of the present invention may be contained in a BAC vector.

[0058] In one embodiment, the present invention relates to a vector comprising the DNA construct of the present invention. Such a vector can be constructed, for example, by inserting the DNA construct of the present invention into any vector comprising the above vector.

[0059] In one embodiment, a vector containing the DNA construct of the present invention may further contain a drug selection marker. The drug selection marker can be used, for example, when creating the DNA construct of the present invention by genetic engineering techniques using Escherichia coli, cultured cells, or the like as a host. As drug selection markers, those known in the art can be used. For example, for positive selection, the neomycin resistance gene (neo) or the hygromycin B phosphotransferase gene can be used, and for negative selection, the herpesvirus thymidine kinase gene (HSV-tk) or the diphtheria toxin A gene can be used.

[0060] In a preferred embodiment, the vector containing the DNA construct of the present invention may be a BAC vector.

[0061] 4. Method for Producing Genetically Modified Non-Human Animals In one embodiment, the present invention relates to a method for producing a genetically modified non-human animal that functionally expresses the human ABCB1 gene, comprising the step of introducing a DNA construct for functionally expressing the human ABCB1 gene into the cells of a non-human animal, wherein the DNA construct includes the coding sequence of the human ABCB1 gene, a CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of an enhancer region located at the 3' end of the human ABCB1 gene, and the DNA construct does not include at least a portion of the coding sequence of the human ABCB4 gene, or includes only a portion of the human RUNDC3B gene (hereinafter also referred to as the method for producing the present invention).

[0062] In the method for producing the present invention, the "DNA construct for functionally expressing the human ABCB1 gene" has the same configuration as the DNA construct described in "3. DNA construct and vector" above, i.e., the DNA construct of the present invention.

[0063] The "step of introducing a DNA construct for functionally expressing the human ABCB1 gene into the cells of a non-human animal" (hereinafter also referred to as step (i)) is not particularly limited in the gene transfer method used, as long as the DNA construct is maintained in the cells of the non-human animal so that the human ABCB1 gene is functionally expressed. For example, known methods such as microinjection of a vector containing the DNA construct into the pronucleus of a fertilized egg, introduction into germline stem cells such as embryonic stem cells, spermatogonial stem cells, and induced pluripotent stem cells (iPS cells) by electroporation or lipofection can be used as appropriate. The DNA construct in this invention may be incorporated into chromosomal DNA or maintained in the cell in the form of being incorporated into extrachromosomal DNA (for example, an artificial chromosome vector). The vector is not particularly limited as long as it is used in genetic engineering, and examples include viral vectors such as bacteriophages, adenoviruses, retroviruses, poxviruses, and herpesviruses, and vectors derived from artificial chromosomes. Examples of DNA vectors include plasmid vectors, viral vectors, cosmid vectors, mammalian artificial chromosomes such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and mouse artificial chromosomes (MACs), as well as other non-plasmid vectors. In a preferred embodiment, a vector containing the DNA construct of the present invention may be used in the method of production of the present invention, preferably a BAC vector containing the DNA construct of the present invention may be used.

[0064] In a preferred embodiment, the DNA construct of the present invention is not present on an artificial chromosome vector (e.g., a mammalian artificial chromosome vector such as a mouse artificial chromosome vector), but more preferably can be incorporated into chromosomal DNA. Methods for introducing a DNA construct into cells so as to be incorporated into chromosomal DNA are known in the art, and any such method may be used, but examples include microinjection of a suitable vector, such as a BAC vector containing the DNA construct, into a pronuclear stage embryo. Furthermore, in one embodiment, zinc finger nucleases (US Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, 6,479,626) and TALEN (US Patent Nos. 8,420,782 B2, US) are used. The DNA constructs of this invention can also be incorporated into predetermined sites of chromosomal DNA using the following technologies (US8697359, US8795965, US8771945, WO2013 / 142578, WO2013 / 176772, WO2014 / 065596, WO2014 / 089290, WO2014 / 093595). Note that CAS9 proteins are commercially available (e.g., products from Takara Bio, Fujifilm Wako Pure Chemical Industries, New England Biolab Japan, Thermo Fisher Scientific, etc.).

[0065] In one embodiment, the method for producing the present invention may further include a step (i) after step (i) to select a non-human animal that functionally expresses the human ABCB1 gene (hereinafter also referred to as step (ii)). When the DNA construct of the present invention is used, a non-human animal that functionally expresses the human ABCB1 gene can be obtained with a high probability. On the other hand, for example, when the DNA construct of the present invention is incorporated into chromosomal DNA, the position of integration and the copy number can vary. Step (ii) allows for the selection of an appropriate non-human animal from among such diverse candidates, depending on the subsequent application.

[0066] Functional expression of the human ABCB1 gene can be confirmed by methods known in the art, for example, by pharmacokinetic studies (see, e.g., Non-Patent Document 11, Bio Pharm Bull. 2020 Mar 1;43(3):384-392, etc.). Depending on the intended use of the non-human animals produced by the method of the present invention, the pharmacokinetics in the desired organs may be investigated. These organs are those in which the human ABCB1 gene is expressed in humans, and may be, for example, the small intestine (duodenum, jejunum, and ileum), liver, kidney, and brain. For example, absorption from the duodenum can be measured as described in Pharm Res. 2003 Apr;20(4):552-6. In a preferred embodiment, the method of the present invention may further include the step of selecting non-human animals from which the DNA construct of the present invention has been introduced intracellularly that functionally express human ABCB1 in at least one organ selected from the small intestine, liver, kidney, and brain (more preferably the small intestine and liver).

[0067] In one embodiment, the method for producing the present invention may further include a step of functionally deleting the endogenous ABCB1 gene in a non-human animal (hereinafter also referred to as step (iii)). Methods for deleting genes on the chromosomes of non-human animals are known in the art, and any of these methods may be used, for example, the zinc finger nuclease, Teylen, or CRISPR-Cas techniques described above. Step (iii) may be performed before or after step (i).

[0068] Furthermore, in the method for producing the present invention, steps (i) and (iii) are not particularly limited as long as individuals lacking the endogenous ABCB1 gene and individuals functionally expressing the human ABCB1 gene are obtained, respectively, and this includes obtaining such individuals by crossbreeding, for example. For example, individuals lacking the endogenous ABCB1 gene can be newly obtained by crossing an individual possessing the endogenous ABCB1 gene with a similar individual lacking the endogenous ABCB1 gene one or more times. Also, individuals that do not functionally express the human ABCB1 gene can be newly obtained by crossing an individual that does not functionally express the human ABCB1 gene with a similar individual (donor) that functionally expresses the human ABCB1 gene one or more times. For example, by crossing an individual functionally lacking the endogenous ABCB1 gene with an individual that functionally expresses the human ABCB1 gene, it is possible to obtain offspring that functionally lack the endogenous ABCB1 gene and functionally express the human ABCB1 gene. Thus, steps (i) and (iii) each include functionally expressing the human ABCB1 gene through crossbreeding and deleting the endogenous ABCB1 gene through crossbreeding, respectively. Furthermore, steps (i) and (iii) each include newly obtaining a non-human animal that functionally expresses the human ABCB1 gene through crossbreeding and newly obtaining a non-human animal that is deficient in the endogenous ABCB1 gene through crossbreeding, respectively. In one embodiment, the production method of the present invention may further include the step of crossing a non-human animal into which the DNA construct of the present invention has been introduced into its cells with a non-human animal of the same species that is functionally deficient in the endogenous ABCB1 gene, and selecting a non-human animal that retains the DNA construct and is functionally deficient in the endogenous ABCB1 gene.

[0069] In a preferred embodiment, the manufacturing method of the present invention may be used to produce genetically modified non-human animals of the present invention.

[0070] 5. Applications of the Genetically Modified Non-Human Animals of the Present Invention Regarding the in vivo pharmacokinetics of drugs that are substrates of ABCB1, the results obtained in the genetically modified non-human animals of the Present Invention show a high correlation with the results obtained in humans. In particular, regarding the bioavailability of human ABCB1-mediated bioavailability when drugs that are substrates of ABCB1 are administered orally, the dynamic range of correlation between the AUCR in the genetically modified non-human animals of the Present Invention and the AUCR in humans is wider than that between the AUCR in conventional ABCB1 humanized mice and the AUCR in humans. Due to these characteristics, the genetically modified non-human animals of the Present Invention can be used for various evaluations, such as the pharmacokinetics of drugs that are substrates of ABCB1. Therefore, the Present Invention also relates to applications of the genetically modified non-human animals of the Present Invention, such as methods for evaluating drugs that are substrates of ABCB1 and methods for screening drugs that are substrates of ABCB1.

[0071] 5-1. Method for Measuring Pharmacokinetics In one embodiment, the present invention relates to a method for measuring the pharmacokinetics of a test substance in a genetically modified non-human animal, comprising the following steps: (1) administering the test substance to the genetically modified non-human animal of the present invention, and (2) taking a biological sample from the genetically modified non-human animal after administration of the test substance in step (1) and measuring the content of the test substance in the biological sample, the method (hereinafter also referred to as Method I of the present invention).

[0072] The test substance in Method I of the present invention may be a compound that is a candidate for drug development. In a preferred embodiment, the test substance may be a drug that is a substrate of ABCB1, but it is not necessary for it to be known to be a substrate of ABCB1 at the time of pharmacokinetic measurement.

[0073] For administration in step (1), the test substance is dissolved in a suitable solvent. Examples of such solvents include phosphate-buffered saline, Tris-buffered saline, 20 mM His-HCl, 150 mM NaCl, pH 6.0 buffer, 20 mM histidine-aspartate buffer containing 150 mM arginine-aspartate and 0.5 mg / mL kolliphor P188, pH 6.0, Hank's equilibrium salt solution (HBSS), and 10% DMSO / 60% PEG300 in saline. When administering peptide compounds, the solvent may contain 0.05% tween 20. The solvent may also contain pharmaceutically acceptable carriers such as buffers, excipients, stabilizers, and preservatives.

[0074] The "administration" in step (1) can be appropriately selected depending on the type of test substance, the type of pharmacokinetics to be evaluated, etc. Examples include intravenous administration (IV), subcutaneous administration (SC), oral administration (PO), intraperitoneal administration (IP), intramuscular administration (IM), tumor administration (IT), pulmonary administration, and nasal administration. The administration of the test substance may be a single dose or repeated multiple times as needed. The dosage and administration interval can also be appropriately adjusted depending on the type of test substance. In a preferred embodiment, the "administration" in step (1) may be oral administration.

[0075] In step (2), a biological sample is collected from the genetically modified non-human animal of the present invention after administration of the test substance. The biological sample may include any tissue and organ, such as blood, plasma, serum, bile, urine, feces, brain, testes, ovaries, lungs, heart, stomach, ileum, large intestine, jejunum, kidney, liver, spleen, pancreas, muscle, and skin. The biological sample may be collected by a method normally used depending on its type. For example, blood may be collected from a non-human animal by venous collection. Serum may be prepared by collecting blood in a blood collection tube without an anticoagulant, agglutinating the blood clot, and removing the clot by centrifugation. Plasma may be prepared by collecting blood in a blood collection tube containing an anticoagulant such as heparin, and then removing the blood cell components by centrifugation. Solid tissues or solid organs may be collected by methods such as using a puncture needle or by surgical incision.

[0076] Biological samples are collected after a certain period of time has elapsed following the administration of the test substance. The time between administration of the test substance and sample collection can be appropriately set depending on the type of test substance and the type of pharmacokinetics to be evaluated, and can be set from a few minutes to several days, for example. For example, for low molecular weight compounds and peptide compounds, it can be set from 2 minutes to 7 days. The number of biological sample collections can be appropriately set depending on the pharmacokinetics to be evaluated, and may be a single sample or multiple samples. For example, when measuring the changes in the amount of a drug present in a specific tissue or organ (e.g., changes in blood concentration), biological samples are collected multiple times over time. Also, when measuring the distribution of a drug in a tissue or organ, a single biological sample is usually collected at a specific point in time after drug administration.

[0077] The content of a test substance in a biological sample can be measured by methods known in the art, depending on the type of test substance. Such methods include, for example, mass spectrometry (LC-MS / MS, etc.), HPLC, and radioactivity measurement (in the case of a test substance labeled with a radioactive material).

[0078] The pharmacokinetics of a test substance can be measured and evaluated from the content of the test substance in a biological sample. Method I of the present invention may further include a step of quantifying the pharmacokinetics in order to evaluate the pharmacokinetics. "Quantifying the pharmacokinetics" includes calculating any of the PK parameters described in "1. Definitions" above, and the methods for calculating these parameters are well known to those skilled in the art. Alternatively, the evaluation of pharmacokinetics can be performed based on a numerical value (e.g., a ratio such as AUC) compared with the content of the test substance in a biological sample obtained by administering an ABCB1 inhibitor before administering the test substance. Alternatively, the evaluation of pharmacokinetics can be performed based on a numerical value (e.g., a ratio such as AUC) compared with the content of the test substance in a biological sample obtained by administering the test substance in a similar manner to a control (a non-human animal control of the same species that does not express the human ABCB1 gene and has a functional deficiency of the endogenous ABCB1 gene).

[0079] The administration in step (1), the collection of biological samples in step (2), and the measurement and evaluation of pharmacokinetics can be carried out in accordance with publicly known guidelines such as the "Guidelines for Nonclinical Clinical Trial Pharmacokinetic Testing" (Pharmaceutical Affairs Bureau Notification No. 496, June 26, 1998).

[0080] In a preferred embodiment, pharmacokinetics may be bioavailability or clearance. In this case, the test substance is administered orally, and the biological sample may be at least one selected from the group consisting of blood, plasma, serum, bile, feces, small intestinal contents, and large intestinal contents.

[0081] In a more preferred embodiment, the pharmacokinetic is bioavailability, the biological sample is blood, plasma, or serum, and step (2) is a step of taking a biological sample at different time intervals at least twice from a genetically modified non-human animal after administration of the test substance in step (1) and measuring the content of the test substance in the biological sample, and (3) may further include a step of determining the blood concentration profile of the test substance based on the content of the test substance measured in step (2) (hereinafter also referred to as Method I-1 of the present invention).

[0082] In Method I-1 of the present invention, the biological sample is preferably plasma.

[0083] Furthermore, the administration of the test substance in step (1) may be limited to just one dose.

[0084] In step (2), the number of times biological samples are collected can be adjusted as appropriate depending on the type of substance being tested, for example, it may be three or more times, four or more times, five or more times, or six or more times. The interval between each collection can also be adjusted as appropriate depending on the type of substance being tested, for example, it may be set to a few minutes to several days, and does not have to be constant.

[0085] In step (3), the blood concentration profile of the test substance is determined from the amount of the test substance present. This may provide an indicator for selecting a test substance with the desired pharmacokinetics.

[0086] Method I-1 of the present invention may further include a step of quantifying the pharmacokinetics based on the blood concentration profile determined in step (3). Hereinafter, the quantification includes PK parameters based on the blood concentration profile, such as clearance (CL), volume of distribution (Vd or V), area under the blood concentration-time curve (AUC), peak blood concentration (Cmax), time to peak blood concentration (Tmax), and blood concentration half-life (t 1 / 2 This includes calculating parameters such as the following. These parameters are calculated according to methods known in the art. In a preferred embodiment, AUC may be calculated based on the blood concentration profile determined in step (3).

[0087] 5-2. Method for Predicting the Pharmacokinetics of a Test Substance in Humans In one embodiment, the present invention relates to a method for predicting the human ABCB1-mediated pharmacokinetics of a test substance in humans, comprising the following steps: (1) measuring the pharmacokinetics of a test substance in a genetically modified non-human animal using Method I of the present invention; and (2) predicting the human ABCB1-mediated pharmacokinetics when the test substance is administered to humans, based on the pharmacokinetics of the test substance in the genetically modified non-human animal measured in (1) above (hereinafter also referred to as Method II of the present invention).

[0088] Step (1) in Method II of the present invention is carried out in accordance with the above description relating to Method I of the present invention.

[0089] In step (2), the correlation between the pharmacokinetics in the genetically modified non-human animals of the present invention and the pharmacokinetics in humans, which has been calculated in advance based on known ABCB1 substrate drugs, can be used to predict the human ABCB1-mediated pharmacokinetics of the test substance in humans from the pharmacokinetics of the test substance obtained in step (1). Such a correlation can be confirmed, for example, by calculating the ratio of the pharmacokinetic values ​​when an ABCB1 inhibitor is administered to the pharmacokinetic values ​​when an ABCB1 inhibitor is not administered, for both the genetically modified non-human animals of the present invention and humans, and comparing them. Alternatively, the correlation can be confirmed by obtaining similar data using multiple ABCB1 substrate drugs, creating a regression line, and calculating the correlation coefficient. For example, for multiple ABCB1 substrate drugs, the ratio of AUC when an ABCB1 inhibitor is administered to the pharmacokinetic values ​​when an ABCB1 inhibitor is not administered (AUCR) can be calculated for both the genetically modified non-human animals of the present invention and humans, and the correlation coefficient can be calculated by analyzing it using a linear regression method. By utilizing this correlation coefficient, the AUCR in humans can be calculated from the AUCR in genetically modified non-human animals of the present invention for the test substance, and this can be used as a predicted value for humans.

[0090] In a preferred embodiment, pharmacokinetics may be bioavailability or clearance.

[0091] According to Method II of the present invention, it is possible to predict the human ABCB1-mediated pharmacokinetics of a test substance in humans with higher accuracy in tests using non-human animals.

[0092] 5-3. Screening Method for Test Substances In one embodiment, a screening method for test substances comprises the following steps: (1) measuring the pharmacokinetics of a test substance in a genetically modified non-human animal using Method I of the present invention; (2) predicting the human ABCB1-mediated pharmacokinetics when the test substance is administered to a human, based on the pharmacokinetics of the test substance in the genetically modified non-human animal measured in (1); and (3) selecting a test substance that is predicted to exhibit desired pharmacokinetics in humans in (2), the method (hereinafter also referred to as Method III of the present invention).

[0093] Steps (1) and (2) are carried out in accordance with the above description relating to steps (1) and (2) of Method II of the present invention.

[0094] In step (3), a test substance capable of exhibiting the desired pharmacokinetics in humans is selected. The desired pharmacokinetics may vary depending on the type of test substance and its intended use. For example, a test substance with higher bioavailability or lower clearance may be selected. Each selected test substance can be used for an application (such as a pharmaceutical product) appropriate to its characteristics and may be subjected to further testing.

[0095] In one embodiment, Method III of the present invention may further include a step of preparing two or more test substances prior to step (1). In this embodiment, the two or more test substances may be compounds that are candidates for drug development. The two or more test substances are compounds of the same type, have the same target, and are different from each other. In a preferred embodiment, the test substance may be a drug that is a substrate of ABCB1, but it is not necessary to know that it is a substrate of ABCB1 at the time of pharmacokinetic measurement. Step (2) is carried out for each of the two or more test substances according to the description of Method I of the present invention. In step (3), the predicted pharmacokinetics for each of the two or more test substances can be compared with each other, and a test substance that can exhibit the desired pharmacokinetic in humans can be selected.

[0096] In a preferred embodiment, pharmacokinetics may be bioavailability or clearance.

[0097] According to Method III of the present invention, a test substance exhibiting the desired pharmacokinetics can be selected with greater precision.

[0098] In this specification, unless a term is defined with a limitation indicating a quantity, such as "one" or "more," terms used herein are not interpreted as having a specific quantity limitation, but rather as having the meaning of "one or more."

[0099] It will be understood by those skilled in the art that any combination of one or more embodiments described herein is included in the present invention, insofar as it does not contradict the common technical knowledge of those skilled in the art.

[0100] Furthermore, all prior art documents cited herein are incorporated herein by reference.

[0101] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0102] [Example 1] Creation of humanized ABCB1 mice (1) Construction of an expression vector containing the human ABCB1 gene region having an exon-intron structure (Figure 1A) An Escherichia coli artificial chromosome (BAC) clone (Thermo Fisher Scientific Inc.) in which the genomic region where the human ABCB1 gene is located was cloned was used. The BAC clone was selected from RPCI-11 and CTD clone collections of different origins and was used to contain the promoter region and the ABCB1 gene region of the human ABCB1 gene (Figure 1A shows the case using RP11-831P22). By homologous recombination using the Red / ET system (GeneBridges), a partial sequence of the ABCB4 gene contained on the BAC DNA was replaced with a Neo gene cassette. At that time, from among the E. coli clones that could be grown in kanamycin-supplemented medium, clones in which the Neo gene was inserted as expected were selected by PCR.

[0103] (2) Construction of an expression vector containing human ABCB1 cDNA downstream of the human ABCB1 gene regulatory region (Figure 1B) For comparison with an expression vector containing the human ABCB1 gene region having an exon-intron structure, a vector for expressing human ABCB1 cDNA under the human ABCB1 promoter was constructed. To obtain the human ABCB1 promoter, a BAC clone in which the genomic region where the human ABCB1 gene is located has been cloned was used (CTD-3002K8, RP11-784L5; Figure 1B shows the case using CTD-3002K8). The human ABCB1 cDNA was obtained by artificial gene synthesis of the base sequence of GenBank NM000927. hABCB1 cDNA, with an intron structure and kozak sequence added immediately before the start codon, and a Neo gene cassette flanked by a polyA sequence and loxP sequence added after the stop codon, was introduced into the start codon downstream of the human ABCB1 gene regulatory region of a BAC clone (CTD-3002K8) by homologous recombination using the Red / ET system (GeneBridges). At that time, clones in which the Neo gene was inserted as expected were selected from among the E. coli clones that could be grown in kanamycin-supplemented medium by PCR.

[0104] (3) Injection of human ABCB1 gene region vector into mouse pronuclear stage embryos The circular vectors prepared in (1) and (2) above were linearized by restriction enzyme treatment with BsiWI or NruI, then subjected to column purification (QIAGEN) and ethanol precipitation, and dissolved in 10-fold diluted TE buffer (pH 8.0). The obtained DNA solution was injected into pronuclear stage embryos derived from C57BL / 6J mice by microinjection. The injected embryos were transplanted into the fallopian tubes of ICR-type recipient females at 0.5 days of pseudopregnancy, and founder offspring were obtained 19 days later.

[0105] (4) DNA was extracted from the tails of founder offspring after selection and weaning of founder mice into which the human ABCB1 gene had been introduced, using Maxwell 16 (Promega), and the introduced human ABCB1 gene was detected by PCR. The PCR reaction mixture was prepared by mixing 1 μl of DNA sample prepared from founder offspring, 2 μl of 5× PrimeSTAR GXL Buffer, 0.8 μl of dNTPs (2.5 mM), 0.1 μl each of primers (20 μM each), 0.2 μl of PrimeSTAR GXL DNA Polymerase (TAKARA), and 5.8 μl of distilled water (total 10 μl). The PCR conditions consisted of 35 cycles of 10 seconds at 98°C, 15 seconds at 60°C, and 2 minutes at 68°C. The primers used were as follows: To confirm the presence of the human ABCB1 gene, hMDR1208F (SEQ ID NO: 1), which recognizes the sequence of exon 12 of the human ABCB1 gene, was used as a forward primer, and hMDR163338R (SEQ ID NO: 2), which recognizes the sequence of exon 14, was used as a reverse primer. In samples from founder mice into which the human ABCB1 gene region with an exon-intron structure was introduced, a 514 bp band was amplified. In samples from founder mice into which human ABCB1 cDNA was introduced, a 227 bp band was amplified. To amplify the Neo gene sequence, the forward primer neoF (SEQ ID NO: 3) and the reverse primer neoR (SEQ ID NO: 4) were used. In both samples from founder mice into which the human ABCB1 gene region with an exon-intron structure was introduced and from founder mice into which human ABCB1 cDNA was introduced, a 663 bp band was amplified. Furthermore, to confirm the presence of the human ABCB1 gene, hMDR112246F (SEQ ID NO: 5), which recognizes the sequence of exon 1 of the human ABCB1 gene, was used as a forward primer, and hMDR113181R (SEQ ID NO: 6), which recognizes the sequence of exon 2, was used as a reverse primer. In the founder mouse samples into which the human ABCB1 gene region with an exon-intron structure was introduced, a 936 bp band was amplified.In samples from founder mice into which human ABCB1 cDNA was introduced, a 1635 bp band was amplified. The results are shown in Figure 2A. In each sample, the DNA of the expected size was amplified, confirming that the founder mice possessed the human ABCB1 gene. In this specification, for mice into which a human ABCB1 gene region with an exon-intron structure was introduced, "Ex-Int_" may be added before the strain number (e.g., RP-31F), and for mice into which human ABCB1 cDNA was introduced, "cDNA_" may be added before the strain number (e.g., RP-30F).

[0106] Furthermore, using samples from founder mice that produced amplified signals with the above primer set, the copy number of the transgene in the genome was confirmed by quantitative PCR (qPCR). The following primer / probe sets that recognize exons 5, 10, 17, 24, and 29 were used. GnRHR was also used as an internal standard. Exon 5 Forward primer: hMDR-Ex5-TaqF (SEQ ID NO: 7) Reverse primer: hMDR-Ex5-TaqR (SEQ ID NO: 8) Probe: hMDR-Ex5-TaqPro (SEQ ID NO: 9) Exon 10 Forward primer: hMDR-Ex10-TaqF (SEQ ID NO: 10) Reverse primer: hMDR-Ex10-TaqR (SEQ ID NO: 11) Probe: hMDR-Ex10-TaqPro (SEQ ID NO: 12) Exon 17 Forward primer: hMDR-Ex17-TaqF (SEQ ID NO: 13) Reverse primer: hMDR-Ex17-TaqR (SEQ ID NO: 14) Probe: hMDR-Ex17-TaqPro (SEQ ID NO: 15) Exon 24 Forward primer: hMDR-Ex24-TaqF (SEQ ID NO: 16) Reverse primer: hMDR-Ex24-TaqR (SEQ ID NO: 17) Probe: hMDR-Ex24-TaqPro (SEQ ID NO: 18) Exon 29 Forward Primer: hMDR-Ex29-TaqF (SEQ ID NO: 19) Reverse Primer: hMDR-Ex29-TaqR (SEQ ID NO: 20) Probe: hMDR-Ex29-TaqPro (SEQ ID NO: 21) GnRHR Forward Primer: GnRHR-TaqF (SEQ ID NO: 33) Reverse Primer: GnRHR-TaqR (SEQ ID NO: 34) Probe: GnRHR-TaqPro (SEQ ID NO: 35)

[0107] The qPCR reaction mixture was prepared by mixing 1 μl of DNA sample prepared from the founder's offspring, 5 μl of 2×TaqMan Fast Master Mix (Thermo Fisher Scientific Inc.), 0.05 μl each of primers (20 μM each), 0.5 μl each of probes (2 μM), and 3.4 μl of distilled water (total 10 μl). The PCR conditions were 40 amplification cycles: preheating at 50°C for 2 minutes and 95°C for 20 seconds, followed by 1 second at 95°C and 68°C for 20 seconds. The 31F line of founder mice into which the human ABCB1 gene region with an exon-intron structure was introduced was used as the reference sample, and comparative quantification was performed using the ΔΔCt method with GnRHR as the internal standard. The results are shown in Figure 2B. The similar copy numbers of different exons of the introduced human ABCB1 gene in the genome suggest that the entire length of the gene was inserted into the genome without being truncated.

[0108] (5) Selected founder mice expressing the human ABCB1 gene were mated with C57BL / 6J mice after sexual maturity, and the transfer of the human ABCB1 gene to the next generation of mice was confirmed by PCR using genomic DNA extracted from the tissues of the next generation of mice as a template. In addition, the amount of transgene transferred to siblings of the next generation of mice obtained from the same founder mouse was quantified by qPCR. PCR and qPCR were performed using the same method as used for selecting the founder mice described above. As shown in Figure 3, it was observed that the copy number of the introduced human ABCB1 gene was consistent among siblings of the next generation of mice. From these results, it was confirmed that the transgene is stably transferred to the next generation.

[0109] Next, the expression of the transgene in the next generation of mice was analyzed. Brains and small intestines were collected from the next generation of mice, and RNA and proteins were prepared. For RNA, total RNA was extracted from each tissue using Isogen (Nippon Gene), purified with RNeasy (QIAGEN) after DNase treatment. Using 1 μg of total RNA as a template, cDNA was synthesized by reverse transcription using the SuperScript III First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc.) with Oligo dT(20) primers. Human ABCB1 was detected by RT-PCR and RT-qPCR using the synthesized cDNA as a template.

[0110] Detection of human ABCB1 by RT-PCR was performed using a combination of a forward primer hMDR34F (SEQ ID NO: 22) that recognizes the sequence immediately below the start codon and a reverse primer hMDR3324R (SEQ ID NO: 23) that recognizes the sequence of exon 27. In human ABCB1 mRNA expression samples, a 3291 bp band was amplified. The PCR reaction mixture was prepared by mixing 1 μl of sample, 2 μl of 5×PrimeSTAR GXL Buffer, 0.8 μl of dNTPs (2.5 mM), 0.1 μl each of primers (20 μM each), 0.2 μl of PrimeSTAR GXL DNA Polymerase (TAKARA), and 5.8 μl of distilled water (total 10 μl). Furthermore, the PCR conditions consisted of 35 cycles of 10 seconds at 98°C, 15 seconds at 60°C, and 4 minutes at 68°C. The results are shown in Figure 4A. Human ABCB1 mRNA expression of the expected size was confirmed only in mouse lines into which the human ABCB1 gene region with an exon-intron structure was introduced (strain line numbers: Ex-Int_RP-31F, Ex-Int_RP-32M).

[0111] For the detection of human ABCB1 by RT-qPCR, the primer / probe sets that recognize exon 5 and exon 29, as described above, were used. As a control for endogenous expression by qPCR, GAPDH was quantified using TaqMan Rodent GAPDH Control Reagents (Thermo Fisher Scientific Inc.) and analyzed by the ΔΔCt method. The results are shown in Figure 4B. In mouse lines into which the human ABCB1 gene region with an exon-intron structure was introduced (strain line numbers: Ex-Int_RP-31F, Ex-Int_RP-32M), high mRNA expression levels were confirmed in both exon 5 and exon 29. Furthermore, since similar expression levels were observed in exon 5 and exon 29, it was suggested that the entire length of the gene was inserted into the genome and expressed without being cleaved.

[0112] The expression level of human ABCB1 protein in the small intestine (duodenum, jejunum, and ileum) was quantified by the following method. Collected tissue samples were homogenized with hypotonic buffer. This homogenate was subjected to dithiothreitol reduction and iodoacetamidoalkylation under denaturation with guanidine hydrochloride, followed by methanol-chloroform precipitation. The extracted protein was denatured with urea, digested with lysyl endopeptidase for 3 hours, and then digested overnight with trypsin. This digest was measured by selective reaction monitoring (SRM) using a high-performance liquid chromatography-connected electrospray ionization mass spectrometer (I-class UPLC / Xevo TQ-S, Waters). Human ABCB1-specific peptide FYDPLAGK (CosmoBio; SEQ ID NO: 24) was used as a standard substance, and stable isotope-labeled FYDPL[A_ 13 C 15 A calibration curve was created using N]GK peptide (CosmoBio) as the internal standard, and the human ABCB1 concentration (fmol / μg protein) was calculated. For the SRM transition of FYDPLAGK peptide, [M+2H] was used as the precursor ion. 2+ (m / z 455.7) was used, and y5 ion (m / z 485.3) was used as the product ion. Stable isotope label FYDPL[A_13 C 15 For the SRM transition of N]GK peptide, [M+2H] is used as the precursor ion. 2+ (m / z 457.7) was used, and y5 ion (m / z 489.3) was used as the product ion. The results are shown in Table 1. In mouse lines into which the human ABCB1 gene region with an exon-intron structure was introduced (strain numbers: Ex-Int_RP-31F, Ex-Int_RP-32M), higher human ABCB1 expression was observed than in mouse lines into which human ABCB1 cDNA was introduced (strain number: RP-30F). Human ABCB1 was not detected in samples from wild-type mice; only mouse endogenous ABCB1 was detected.

[0113] (6) Confirmation of human ABCB1 gene expression level and tissue expression pattern in human ABCB1-expressing mouse Ex-Int_RP-31F line - Confirmation by RT-qRCR method using RNA from brain, heart, kidney, liver, and ileum - Brain, heart, kidney, liver, and ileum were collected from male and female human ABCB1-expressing heterozygous mice. RNA purification and reverse transcription were performed using the method described in (5). The cDNA copy numbers of human ABCB1, mouse Abcb1a, and mouse Abcb1b in the total RNA (25 μg) contained in the qPCR reaction system were measured and the absolute values ​​were compared. Plasmids containing synthetic genes formed by concatenating fragment sequences containing the probe recognition sequences of each gene were linearized using restriction enzymes and used as standard samples for comparison. The copy number of the synthetic gene was calculated from the DNA concentration measured by a spectrophotometer using the following formula: Y (molecules / μl) = (X [g / μl] / plasmid length x 660) x 6.022 x 10^23. A calibration curve was created by preparing samples of the above nucleic acid diluted 10-fold in steps from 10^8 copies to 1 copy, and plotting the results with the nucleic acid copy number on the X axis and Ct on the Y axis. qPCR was performed using the method described in (5). For the expression level of human ABCB1 in humans, data from ABCB1 ATP binding cassette subfamily B member 1 [Homo sapiens (human)] Gene ID: 5243, updated on 13-May-2024 (https: / / www.ncbi.nlm.nih.gov / gene / 5243) was referenced. For the expression levels of mouse Abbcb1a in mice, we referred to the data from Abbcb1a ATP-binding cassette, sub-family B member 1A [Mus musculus (house mouse)] Gene ID: 18671, updated on 12-May-2024 (https: / / www.ncbi.nlm.nih.gov / gene / 18671).For the expression levels of mouse Abbcb1b in mice, we referred to data from Abbcb1b ATP-binding cassette, sub-family B member 1B [Mus musculus (house mouse)] Gene ID: 18669, updated on 13-May-2024 (https: / / www.ncbi.nlm.nih.gov / gene / 18669). The results are shown in Figure 5. The expression patterns of human ABCB1 mRNA in each organ were found to be similar to those of humans, not mice. mRNA expression levels in the brain and small intestine were found to be similar to those of mouse Abbcb1a.

[0114] (7) Creation of double knockout mice lacking endogenous Abcb1a and Abcb1b genes (Figure 6) The CRISPR / Cas9 system was used (Methods. 2017 May 15;121-122:16-28.). To delete exons 8-9 of endogenous Abcb1a in mice, MDR1a-5', which recognizes intron 7, and MDR1a-3', which recognizes intron 9, were used as crRNAs, and 1683 bp between them was deleted. To delete exons 3-4 of endogenous Abcb1b in mice, MDR1b-5', which recognizes intron 2, and MDR1b-3', which recognizes intron 4, were used as crRNAs, and 1218 bp between them was deleted. MDR1a-5';5'-AGGUGCAUAGACCACCUCUCAAGG-3' (SEQ ID NO: 25) MDR1a-3';5'-GCACAGCAAUGAAGUAUCAAUGGGG-3' (SEQ ID NO: 26) MDR1b-5';5'-UAUGGAGGUCAGCCAGAAAACGUGG-3' (SEQ ID NO: 27) MDR1b-3';5'-UUAUGGGGCAAUUAGCAAUCUAGG-3' (SEQ ID NO: 28)

[0115] Commercially available Cas9 protein and crRNA + tracrRNA synthesized by a contract manufacturer were introduced into mouse pronuclear stage embryos by electroporation. The Cas9 reaction mixture was prepared by mixing 224 μl of OPTI-MEM (Thermo Fisher Scientific Inc.), 18 μl each of crRNA + tracrRNA (final concentration 6 μM), and 4 μl of Cas9 protein (final concentration 1.2 μM) (total 300 μl). PCR screening was performed to select founder offspring lacking both genes. The PCR reaction mixture was prepared by mixing 1 μl of DNA sample prepared from the founder's offspring, 2 μl of 5×PrimeSTAR GXL Buffer, 0.8 μl of dNTPs (2.5 mM), 0.1 μl each of primers (20 μM each), 0.2 μl of PrimeSTAR GXL DNA Polymerase (TAKARA), and 5.8 μl of distilled water (total 10 μl). The PCR conditions consisted of 35 cycles of 10 seconds at 98°C, 15 seconds at 60°C, and 2 minutes at 68°C. The primers used are as follows: To confirm the deletion of the mouse Abcb1a gene, mMDR1a-117512F (SEQ ID NO: 29), which recognizes the sequence of intron 7 of the mouse Abcb1a gene, was used as a forward primer, and mMDR1a-119976R (SEQ ID NO: 30), which recognizes the sequence of intron 9, was used as a reverse primer. To confirm the deletion of the mouse Abcb1b gene, mMDR1b-6227F (SEQ ID NO: 31), which recognizes the sequence of intron 2 of the mouse Abcb1b gene, was used as a forward primer, and mMDR1b-9378R (SEQ ID NO: 32), which recognizes the sequence of intron 4, was used as a reverse primer. For the mouse Abcb1a gene, a band of 2465 bp was amplified in the wild-type allele and approximately 800 bp in the knockout allele. For the mouse Abcb1b gene, a band of 3152 bp was amplified in the wild-type allele and approximately 2000 bp in the knockout allele. Founder offspring with the expected size of amplified DNA were obtained, and these were designated as double knockout mice lacking both the endogenous Abcb1a and Abcb1 genes.

[0116] (8) Production of mice lacking both endogenous Abcb1a and Abcb1b genes and into which the human ABCB1 gene has been introduced A mouse line expressing the human ABCB1 gene (line number: EX-Int_RP-31F) was crossed with double knockout mice lacking both endogenous Abcb1a and Abcb1b genes to obtain mouse individuals that are homozygous for the endogenous Abcb1a and Abcb1b gene regions and into which the human ABCB1 gene region having an exon-intron structure has been introduced, i.e., ABCB1 humanized mice. By crossing mice lacking both the endogenous Abbcb1a and Abbcb1b genes with human ABCB1 gene-transfected mice (mice heterozygous for the human ABCB1 gene; strain number: Ex-Int_RP-31F), mice lacking both the endogenous Abbcb1a and Abbcb1b genes and heterozygous for the human ABCB1 gene (sometimes referred to as human ABCB1 heterozygous Tg individuals or hABCB1 (hetero) in this specification) were obtained. Furthermore, by crossing these mice with each other, mice lacking both the endogenous Abbcb1a and Abbcb1b genes and homozygous for the human ABCB1 gene (sometimes referred to as hABCB1 (homo) in this specification) were obtained.

[0117] -Analysis by Immunohistochemical Staining- Using an anti-ABCB1 antibody (Cat No. ab170903, abcam) as the primary antibody, the tissue distribution of human ABCB1 in ABCB1 humanized mice (human ABCB1 heterozygous transgenic individuals) was investigated. Immunohistochemical staining was performed according to standard procedures, and samples for observation were prepared from the ileum, liver, kidney, and brain tissue. The results are shown in Figure 7. Human ABCB1-specific staining was observed in ABCB1 humanized mice. This indicates that the staining in ABCB1 humanized mice is due to the expression of the introduced human ABCB1 gene. Furthermore, the detection of ABCB1 in major organs was consistent with reports in humans.

[0118] [Example 2] Evaluation of human extrapolation in ABCB1 humanized mice Three types of ABCB1 substrate drugs [Betrixaban (Cayman Chemical Company), Celiprolol (Toronto Research Chemicals, Inc.), and Talinolol (Toronto Research Chemicals, Inc.)] were orally administered to ABCB1 humanized mice (hABCB1 (hetero) and hABCB1 (homo)). Similarly, three types of substrate drugs (Betrixaban, Celiprolol, and Talinolol) were orally administered to endogenous ABCB1 knockout mice. On the other hand, Aliskiren (FUJIFILM Wako Pure Chemical Corporation) was orally administered to ABCB1 humanized mice (hABCB1 (hetero) and hABCB1 (homo)). In addition, the ABCB1 inhibitor Zosuquidar (MedKoo Biosciences) was administered to ABCB1 humanized mice 60 minutes before Aliskiren administration, after which Aliskiren was orally administered. The experimental conditions are shown in the table below.

[0119] Blood samples were collected using a heparinized syringe at 0.167, 0.5, 1, 2, 4, 7, and 24 hours after substrate drug administration. The collected blood samples were centrifuged at 13000xg at 4°C for 5 minutes to prepare plasma, which was then stored frozen at -30°C until analysis.

[0120] The plasma concentrations of each ABCB1 substrate were quantified using LC-MS / MS (ACQUITY UPLC I Class System, Waters Corp) to calculate the AUC, and the AUC ratio (AUCR) during ABCB1 inhibition in ABCB1 humanized mice was calculated. humanized The value of ) was calculated using the following formula. Here, AUC hABCB1 This refers to the AUC in ABCB1 humanized mice, AUC hABCB1+ZOS This refers to the AUC in ABCB1 humanized mice when Zosuquidar was used in combination, AUC ABCB1-KOThis is the AUC in endogenous ABCB1 knockout mice. For Betrixaban, Celiprolol, and Talinolol, the AUC is... ABCB1-KO / AUC hABCB1 Using this method, for Aliskiren, AUC ABCB1+Zos / AUC hABCB1 I used it.

[0121] Clinical information related to ABCB1 inhibition (compound name, route of administration, dosage, inhibitor, and target AUC change rate) was collected using the University of Washington Database (DIDB, https: / / didb.druginteractionsolutions.org / ). The maximum AUC change rate (AUCR) was primarily used for comparison with results from ABCB1 KO mice or with combination therapy using potent inhibitors. human ) was calculated using the following formula, similar to Non-Patent Document 11. Here, AUC human This is the AUC in humans. AUCR of Aliskiren, Betrixaban, Celiprolol and Talinolol human These are 6.3, 3.1, 1.8, and 1.5, respectively.

[0122] Figure 8 shows the calculated AUCR of ABCB1 humanized mice (AUCR hABCB1 ) and AUCR human The data for hMDR-MAC mice were those described in Non-Patent Literature 11. The AUCR of the ABCB1 humanized mouse of the present invention showed a high correlation with the human AUCR, similar to the AUCR of hMDR-MAC mice. However, the dynamic range of the AUCR of the ABCB1 humanized mouse of the present invention was found to be wider than that of hMDR-MAC mice. Furthermore, mice possessing the human ABCB1 gene homozygously had a wider dynamic range than mice possessing it heterozygously.

[0123] [Example 3] Evaluation of human ABCB1-mediated bile excretion and gastrointestinal secretion function in ABCB1 humanized mice. ABCB1 substrate drugs [Digoxin (Sigma-Aldrich, Inc.) and Fexofenadine (Sigma-Aldrich, Inc.)] were administered intravenously to ABCB1 humanized mice (hABCB1 (homo)) and endogenous ABCB1 knockout mice (ABCB1 KO) that had undergone gallbladder cannulation. Gallbladder cannulation was performed according to the method described in Pharmacol Exp Ther. 2023 Nov;387(2):135-149. Specifically, the following was done: Gallbladder cannulation was performed aseptically under isoflurane anesthesia. Flunixin meglumine (2.5 mg kg) was administered for analgesia. -1 After subcutaneous injection of the drug, each mouse was dissected, the common bile duct was ligated, and the gallbladder wall was opened using microscises. A polyurethane catheter (0.2 mm inner diameter x 0.4 mm outer diameter, 60 cm length) was inserted into the incised gallbladder and fixed to it. The other end of the catheter passed through the subcutaneous tissue and exited through the incision on the mouse's back. The incisions on the back and abdomen were closed, 0.25% bupivacaine was applied directly to the incisions, and 500 mL of warm saline was subcutaneously injected to hydrate the mice. The catheter was threaded through a tether and integrated with each mouse's harness. Finally, the catheter was fixed to a bile collection tube. To avoid blood contamination of the bile due to the surgery, the mice were used at least one hour after they had woken up. Mice that secreted more than 25 microliters of bile within 30 minutes post-surgery were assigned to the study. Bile samples were collected every 30 minutes from conscious mice for up to 6 hours after administration. The experimental conditions are shown in the table below.

[0124] Blood samples were collected using a heparinized syringe at 0.05, 0.167, 0.5, 1, 2.5, 6, and 24 hours after administration of the substrate drug. The collected blood samples were centrifuged at 13000xg at 4°C for 5 minutes to prepare plasma, which was then stored frozen at -30°C until analysis. Bile samples were collected every hour until 6 hours after administration of the substrate drug, and again at 24 hours after administration. Feces were collected 24 hours after administration of the substrate drug. Small and large intestinal contents were collected 24 hours after administration of the substrate drug.

[0125] The concentrations of ABCB1 substrates in plasma, bile, feces, small intestinal contents, and large intestinal contents were quantified using LC-MS / MS (ACQUITY UPLC I Class System, Waters Corp), and the clearances for each were determined using the following formula. Here, CL total CL stands for Total Clearance. renal CL is renal clearance. bile This refers to bile excretion clearance, CL secretion This is secretory clearance, X urine X is the amount of renal excretion of the substrate drug 24 hours after administration. bile X is the amount of biliary excretion of the substrate drug 24 hours after administration. feces X is the amount of substrate drug excreted in the feces 24 hours after administration. intestine This refers to the amount of substrate drug in the small intestinal contents 24 hours after administration, X colon This refers to the amount of substrate drug in the colonic contents and AUC 24 hours after administration. 0-24h This is the AUC for up to 24 hours.

[0126] Figure 9 shows the clearance of ABCB1 substrate drugs in ABCB1 humanized mice and endogenous ABCB1 knockout mice. Comparison with endogenous ABCB1 knockout mice confirmed the presence of biliary excretion clearance and gastrointestinal secretion clearance of Digoxin (Figure 9A) and Fexofenadine (Figure 9B) by ABCB1 in ABCB1 humanized mice. Therefore, it was shown that the biliary excretion mechanism by ABCB1 in the liver and the secretion mechanism by ABCB1 in the gastrointestinal tract are functioning in the ABCB1 humanized mice of the present invention.

[0127] This invention makes it possible to predict the pharmacokinetics or drug-drug interactions of ABCB1 substrates in humans with higher accuracy than before.

Claims

1. A genetically modified non-human animal that functionally lacks the endogenous ABCB1 gene and functionally expresses the human ABCB1 gene, wherein it possesses a DNA construct for functionally expressing the human ABCB1 gene, the DNA construct comprising the coding sequence of the human ABCB1 gene, the CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of the enhancer region located at the 3' end of the human ABCB1 gene, and the DNA construct either does not contain at least a portion of the coding sequence of the human ABCB4 gene or contains only a portion of the human RUNDC3B gene.

2. A genetically modified non-human animal according to claim 1, wherein the human ABCB1 gene is functionally expressed in at least one organ selected from the small intestine and the liver.

3. A genetically modified non-human animal according to claim 2, which functionally expresses the human ABCB1 gene in the small intestine.

4. The genetically modified non-human animal according to any one of claims 1 to 3, wherein the DNA construct is not present on a mammalian artificial chromosome vector.

5. The genetically modified non-human animal according to any one of claims 1 to 3, wherein the DNA construct is not present on the mouse artificial chromosome vector.

6. The genetically modified non-human animal according to any one of claims 1 to 5, wherein the DNA construct does not contain at least a portion of the coding sequence of the human ABCB4 gene and contains only a portion of the human RUNDC3B gene.

7. The genetically modified non-human animal according to any one of claims 1 to 6, wherein the DNA construct comprises all the introns of the human ABCB1 gene.

8. The genetically modified non-human animal according to any one of claims 1 to 7, wherein the non-human animal is a non-human mammal.

9. The genetically modified non-human animal according to claim 8, wherein the non-human mammal is a mouse.

10. A genetically modified non-human animal according to any one of claims 1 to 9 for measuring the pharmacokinetics of a test substance.

11. A genetically modified non-human animal according to any one of claims 1 to 9 for predicting the human ABCB1-mediated bioavailability of a test substance upon oral administration in humans.

12. A genetically modified non-human animal according to any one of claims 1 to 11, wherein the human ABCB1 gene is derived from human male fibroblasts or human male hematopoietic cells.

13. A genetically modified non-human animal according to any one of claims 1 to 12, wherein the human ABCB1 gene is derived from the BAC / PAC human genome library.

14. The genetically modified non-human animal according to claim 13, wherein its genome sequence contains a base sequence derived from the BAC / PAC human genome library.

15. A DNA construct for functionally expressing the human ABCB1 gene in a non-human animal, comprising the coding sequence of the human ABCB1 gene, the CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of the enhancer region located at the 3' end of the human ABCB1 gene, and not comprising at least a portion of the coding sequence of the human ABCB4 gene, or comprising only a portion of the human RUNDC3B gene.

16. A BAC vector comprising the DNA construct described in claim 15.

17. A method for producing a genetically modified non-human animal that functionally expresses the human ABCB1 gene, comprising the step of introducing a DNA construct for functionally expressing the human ABCB1 gene into the cells of the non-human animal, wherein the DNA construct includes the coding sequence of the human ABCB1 gene, a CAR / PXR response element located in the 5' promoter region of the human ABCB1 gene, at least one intron of the human ABCB1 gene, and the sequence of an enhancer region located at the 3' end of the human ABCB1 gene, and the DNA construct does not include at least a portion of the coding sequence of the human ABCB4 gene, or includes only a portion of the human RUNDC3B gene.

18. The method according to claim 17, further comprising the step of crossing a non-human animal into which the DNA construct has been introduced into its cells with a non-human animal of the same species that is functionally deficient in the endogenous ABCB1 gene, and selecting a non-human animal that retains the DNA construct and is functionally deficient in the endogenous ABCB1 gene.

19. The method according to claim 17 or 18, further comprising the step of selecting a non-human animal from which the DNA construct has been introduced intracellularly that functionally expresses human ABCB1 in at least one organ selected from the intestine and the liver.

20. A method for measuring the pharmacokinetics of a test substance in a genetically modified non-human animal, comprising the following steps: (1) administering a test substance to a genetically modified non-human animal according to any one of claims 1 to 14; and (2) taking a biological sample from the genetically modified non-human animal after administration of the test substance in step (1) and measuring the content of the test substance in the biological sample.

21. The method according to claim 20, wherein the biological sample is at least one selected from the group consisting of plasma, bile, feces, small intestinal contents, and large intestinal contents.

22. A method for predicting the human ABCB1-mediated pharmacokinetics of a test substance in humans, comprising the steps of: (1) measuring the pharmacokinetics of a test substance in a genetically modified non-human animal by the method of claim 20 or 21; and (2) predicting the human ABCB1-mediated pharmacokinetics of the test substance when administered to humans, based on the pharmacokinetics of the test substance in the genetically modified non-human animal measured in (1).

23. A method for screening a test substance, comprising the following steps: (1) measuring the pharmacokinetics of a test substance in a genetically modified non-human animal by the method described in claim 20 or 21; (2) predicting the human ABCB1-mediated pharmacokinetics of the test substance when administered to a human, based on the pharmacokinetics of the test substance in the genetically modified non-human animal measured in (1); and (3) selecting a test substance that is predicted to exhibit a desired pharmacokinetic in humans in (2).

Images