Utility of protein in prediction of in vivo effects

Abstract

To provide a method of evaluating disposition and / or effect of a candidate compound in an in vitro culture and / or suspension to predict in vivo disposition and / or effect of the candidate compound.SOLUTION: A method comprises the steps of: providing a cell culture and / or suspension; exposing a candidate compound to the culture and / or suspension; exposing the culture and / or suspension to media providing an in vivo relevant extracellular environment (media comprising such as a protein and / or other component, such as lipoproteins and bile acids at a physiological concentration or a concentration having characteristics similar to the physiological concentration), in which any combination of any of the exposing steps may occur in any order or simultaneously; and evaluating in vitro disposition and / or effect of the candidate compound to predict in vivo disposition and / or effect of the candidate compound.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 060,916, filed October 7, 2014. The entire disclosure of which is incorporated herein by reference. [Technical Field]

[0002] In some embodiments, the present invention provides a method for detecting the in vivo disposition of a candidate compound. and / or in vitro culture and / or suspension of this candidate compound to predict efficacy. More specifically, the present invention relates to a method for evaluating the disposition and / or effect of a compound. The invention relates to the use of uptake assays to predict the in vivo disposition and / or effect of candidate compounds. suction clearance, basolateral outflow clearance, canalicular outflow clearance, intracellular concentration, bile clearance Clearance, metabolic clearance, and candidate compound in in vitro culture and / or suspension Disposition and In some embodiments, the method comprises: exposing the sample and / or suspension to a medium that provides an in vivo relevant extracellular environment. The medium may contain, for example, a physiological concentration or a concentration that has binding properties similar to a physiological concentration. It is a medium containing components such as proteins. [Background technology]

[0003] Typically, hepatocytes or related cell lines (suspended, seeded, sandwich cultured) or other 3D models), Caco-2, MDCK, Opti-Target® (Optivia Biotechno logy, Menlo Park, California, USA), and HepaRG® (Biopredic International nal, Saint Gregoire, France) If the protein is not present or present at non-physiological levels during the experiment, Therefore, only unbound drug concentrations were evaluated in all of these experiments. This is often done for reasons of practical simplicity. To determine the extent of drug binding to plasma proteins, separate in vitro protein binding experiments were performed. The fraction of the target substance is determined and this information is used to compare the parameters obtained from the in vitro experiments with those obtained from the in vitro experiments. and to apply the ratio somewhat blindly to the results of other experiments. Then, estimate the effect of the "free" compound by multiplying the results of other experiments by this percentage. Such corrective actions will provide reasonably accurate results relevant to clinical or in vivo situations. There was a presumption that this would happen. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, there is a need for such corrective measures and approaches that do not rely on assumptions. The disposition of the candidate compounds is evaluated in vitro in culture and / or suspension to identify the candidate compounds. There is a need for methods to predict the in vivo disposition of compounds. [Means for solving the problem]

[0005] In some embodiments, the methods of the present invention provide cultures and / or suspensions of cells. exposing a candidate compound to the culture and / or suspension; The suspension is then placed in a medium (e.g., physiological concentration or biotin) that provides a relevant extracellular environment in vivo. Exposure to a medium containing components such as proteins at concentrations with binding properties similar to the physiological concentration of the target organism. determining the amount of the candidate compound taken up in the culture and / or suspension; The disposition and / or effect of the candidate compound is evaluated, and the candidate compound is The method comprises predicting the in vivo disposition and / or effect of the compound.

[0006] In some embodiments, the culture and / or suspension is prepared in a manner that mimics cells. In some embodiments, the artificial membrane system comprises: Mimicking cells in a co-culture with supporting cells, wherein the supporting cells are fibroblasts and / or Kupffer cells In some embodiments, the cell mimicked by this artificial membrane system comprises: Vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells In some embodiments, the cells are selected from the group consisting of: alveoli, adipocytes, and lung cells. The cells mimicked by the artificial membrane system consist of a cell line, and optionally, this cell line are selected from the group consisting of HepaRG® cell lines, Caco-2 and MDC. In an embodiment, the amount of the candidate compound incorporated into the culture and / or suspension is The step of "determining, thereby assessing the disposition" is carried out by determining the intracellular concentration of the candidate compound. determining hepatic accumulation, determining biliary excretion, and / or determining biliary cleavage It consists of a step to determine the clearance.

[0007] In some embodiments, a method for testing the biliary excretion sensitivity of a candidate compound is provided. In some embodiments, the method involves the use of an artificial membrane system adapted to mimic a cell. providing a culture and / or suspension comprising a stem and at least one bile canaliculus; exposing the culture and / or suspension to a co-compound; , exposing the cells to a medium that provides an in vivo relevant extracellular environment, and The amount of the candidate compound in the bile canaliculi is determined, thereby examining the susceptibility of the candidate compound to biliary excretion. In some embodiments, detecting the presence of a cytoplasmic ... The step of determining the amount of the candidate compound may involve using a labeled pre-mixture of the candidate compound and the transport protein. and a selected amount of substrate simultaneously for a sufficient time to allow for uptake by the cell culture and / or exposing the cell culture and / or suspension to a solution; washing the cell culture and / or suspension; and The amount of labeled substrate present in each bile canaliculus is detected, and the amount of labeled substrate present in each bile canaliculus is measured. assessing competition between the candidate compound and the labeled substrate for biliary excretion; the amount of the labeled substrate in the at least one bile canaliculus compared to a preselected amount of the labeled substrate. The presence of reduced amounts of the target substrate may affect the susceptibility of the candidate compound to biliary excretion by the transport protein. In some embodiments, the artificial membrane system comprises a support cell. The support cells are composed of fibroblasts and / or Kupffer cells. In some embodiments, the cells mimicked by this artificial membrane system include vesicles, Hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, lipid In some embodiments, the artificial membrane is selected from the group consisting of adipocytes and lung cells. The cells mimicked by the system consist of a cell line, and optionally, this cell line is Hepa In some embodiments, the cell line is selected from the group consisting of RG® cell line, Caco-2, and MDC. In the method, the labeled substrate may be a fluorogenic compound, a fluorescent compound, a chemiluminescent compound, a comparative a compound selected from the group consisting of a color compound, a radiolabeled compound, and combinations thereof; In some embodiments, the amount of the candidate compound in the at least one bile canaliculus is is determined by calculating the biliary clearance value of this culture and / or suspension. do.

[0008] In some embodiments, a method for testing the biliary excretion sensitivity of a candidate compound is provided. In some embodiments, the method comprises: (a) providing a first and a second cell culture and / or establishing a suspension, wherein the first and second cultures and / or suspensions are an artificial membrane system adapted to mimic a cell and at least one bile canaliculus; The first culture and / or suspension has intact bile canaliculi and the second culture and / or suspension has intact bile canaliculi. (b) a stage in which the suspension has collapsed bile canaliculi; and (c) a stage in which the candidate compound is introduced into the suspension, the stage in which the suspension has collapsed bile canaliculi; and and incubating the first culture and / or suspension and the second culture and / or suspension for a sufficient time to (c) exposing the first and second cultures and / or suspensions to in vivo relevant (d) exposing the first and second cultures and / or the cells to a medium that provides an extracellular environment associated with the cells; (e) washing and lysing the suspension; and (d) each of the cultures and / or suspensions of step (d). Determine the amount of candidate compound present in the lysate obtained from each culture and / or suspension. and evaluating the biliary excretion sensitivity of the candidate compound using the amount of the candidate compound in the sample. In some embodiments, the method comprises: (i) detecting a candidate compound in a sample containing the candidate compound; The cells are exposed to the first and second cultures and / or suspensions for a sufficient time (T) to allow for the cells to be isolated. (ii) transferring the first and second cultures and / or suspensions to the relevant cells in vivo. (iii) exposing the first and second cultures and / or suspensions to a medium that provides an external environment. (iv) washing and lysing each of the first and second fractions of step (iii); determining the amount of the candidate compound present in a lysate obtained from the culture and / or suspension; (v) The amount of bile canaliculi is compared between the first culture and / or suspension with intact bile canaliculi and the first culture and / or suspension with disrupted bile canaliculi. The second culture and / or suspension with isolated bile canaliculi contained the lysates obtained from the two cultures. (vi) calculating the amount of candidate compound present in the sample using the amount calculated in step (iv); In some embodiments, the step of evaluating the biliary excretion susceptibility of the candidate compound is This artificial membrane system mimics cells in a co-culture with feeder cells, and the feeder cells are fibroblasts. In some embodiments, the artificial membrane system includes cells and / or Kupffer cells. The more mimicked cells include vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, and cardiac muscle cells. cells, nerve cells, muscle cells, adipocytes, and lung cells. In an embodiment, the cells mimicked by this artificial membrane system comprise a single cell line. Optionally, the cell line is selected from the group consisting of HepaRG® cell line, Caco-2, and MDC. It is selected.

[0009] In some embodiments, in vivo assays are used to predict the efficacy of candidate compounds. Methods for assessing the effect of candidate compounds in cultures and / or suspensions of tuna are provided. In some embodiments, the method includes providing a culture and / or suspension of cells; exposing the culture and / or suspension at least once to at least one candidate compound; The cultures and / or suspensions are then exposed to a medium that provides an in vivo relevant extracellular environment. and exposing the culture and / or suspension to at least one candidate compound. The step of evaluating the efficacy of candidate compounds to predict their in vivo efficacy. In an embodiment, the method comprises using an artificial membrane system adapted to mimic a cell and at least providing a culture and / or suspension containing at least one bile canaliculus. In embodiments, the artificial membrane system mimics cells in co-culture with feeder cells, and In some embodiments, the sustainer cells comprise fibroblasts and / or Kupffer cells. The cells mimicked by the artificial membrane system include vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, and cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and lung cells. In some embodiments, the cells mimicked by this artificial membrane system are and two cell lines, optionally the cell lines being HepaRG® cell line, Caco-2 and In some embodiments, the effect is selected from the group consisting of: MDCs. studies of the type of metabolite (metabolism, induction and toxicity); metabolite identification and metabolic stability (parent life span) Gene regulation (induction / repression); P450 and transporter-drug interactions; Intracellular storage bound and free or total (bound + free) intracellular concentrations (e.g., nuclear, mitochondrial); and In some embodiments, the culture and The suspension and / or the suspension are exposed to a plurality of candidate compounds. The culture and / or suspension is repeatedly exposed to one or more candidate compounds.

[0010] In some embodiments of the invention, the cells are derived from a mouse, rat, rabbit, human, Monkeys, apes, cats, dogs, piglets, hogs, cows, bulls, sheep, horses, and sheep It is isolated from a source selected from the group consisting of butterflies, chickens, fish, ducks and geese. In some embodiments of the present invention, the culture and / or suspension may be further cultured for a long period of time. In some embodiments of the present invention, the culture and / or suspension Alternatively, the suspension may comprise a bile canaliculus network. The culture and / or suspension may comprise clusters, aggregates, at least one layer of cells, and The present invention is characterized by having a configuration selected from the group consisting of these combinations. In some embodiments, the cells are embedded in a matrix. In some embodiments, the culture and / or suspension may further comprise a sandwich culture or and / or a suspension of cells, wherein the sandwich culture and / or suspension comprises at least one and optionally at least one bile canaliculus within at least one layer of cells. In some embodiments of the present invention, the sandwich culture and / or suspension Further embodiments of the present invention include long-term sandwich cultures and / or suspensions. In morphology, at least one layer of cells is sandwiched between two layers of matrix. In some embodiments of the present invention, the matrix is ​​a biological matrix. from ox medium, synthetic matrix medium, co-culture medium with feeder cell types, and combinations thereof In some embodiments of the present invention, the biological matrix is ​​selected from the group consisting of The medium contains collagen, laminin, complexes derived from basement membranes, their derivatives, and and combinations thereof.

[0011] In some embodiments of the present invention, a culture medium that provides an in vivo relevant extracellular environment consists of a medium containing components at physiological concentrations or concentrations with properties similar to physiological concentrations. In some embodiments of the invention, the component is selected from the group consisting of albumin, β-lipoprotein, α-1-acid glycoprotein; mouse, rat, rabbit, human, monkey, ape, cat, dog, Piglets, pigs, cows, bulls, sheep, horses, turkeys, chickens, fish, ducks, Plasma or serum derived from geese; bile acids or mixtures of bile acids; bilirubin; and The compound is selected from the group consisting of: In some embodiments of the invention, the method comprises: In some embodiments of the present invention, the method further comprises: Additionally, it includes screening multiple candidate compounds simultaneously. In an embodiment, a medium containing a physiological concentration of protein may modulate the properties of a candidate compound, as well as In some embodiments of the present invention, these multiple exposing steps include Any combination may be performed simultaneously or in any order.

[0012] The object of the present invention is to predict the in vivo disposition and / or effect of a candidate compound. To achieve this, the candidate compound is dispositionally assayed in an in vitro culture and / or suspension. and / or to provide a method for evaluating the effectiveness. The objectives of the subject matter disclosed herein are achieved in whole or in part by the subject matter disclosed herein. The invention has been made in accordance with the principles of the present invention and is best described below in connection with the accompanying examples. As the project progresses, other objectives will become apparent. BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The present invention will now be more fully described, where some of the inventions thereof are: However, not all embodiments are described. Indeed, the invention may be embodied in many different forms. and the present invention should not be construed as limited to the embodiments set forth herein. Rather, these embodiments should not be construed as limiting the scope of the present disclosure to any applicable legal requirements. It is offered to satisfy.

[0014] Generally, according to some embodiments of the present invention, plasma proteins and / or other components are , involved in the binding of drugs, chemicals, and endogenous compounds, to approximate the in vivo environment In particular, physiological or other relevant levels of protein (often called albumin) In particular, methods for adding extracellular components such as ATP (which may be used in vivo) are provided. The extracellular environment in which the compound is present determines the intracellular concentration and kinetics (time course) of the compound more closely related to in vivo. Both their intracellular concentrations and kinetics allow us to assess changes in cellular processes and interpret cellular behavior. These processes are the factors that guide the test. The efflux of this compound (in the case of the liver, the basolateral and canalicular), the intracellular concentration of this compound, The results obtained from this approach include the metabolism of the compound, the induction potential of this compound, and the toxicity of this compound. The experimental results are surprising and cannot be predicted using conventional methods in the art. This resulted in...

[0015] Typically, hepatocytes or their related cell lines (in suspension, plated, sandwich culture or other 3D cultures) model), Caco-2, MDCK, Opti-Target® (Optivia Biotechnology, Menlo Park, k, California, USA), Hurellux® (Hurel Corporation, North Brunswick, New Jersey, USA), and HepaRG® (Biopredic International, Saint Gregoire, CA, USA). e, France) in vitro experiments using cell lines available under the trademark During this time, components such as proteins are absent or present at non-physiological levels. Therefore, only unbound drug concentrations were assessed in all of these experiments. To apply this to clinical or in vivo situations, other studies such as in vitro protein binding experiments may be performed. Experiments can be performed to determine, for example, the fraction of drug bound to plasma proteins, and this information can be used to Used to extrapolate parameters obtained from in vitro experiments to the in vivo situation. As a further example, protein binding experiments, such as those using equilibrium dialysis, can yield "fraction free" This provides information on the proportion of unbound compounds (Fu) and can be used to compare this proportion with the results of other experiments. Applying this somewhat blindly, the results of other experiments are multiplied by this ratio to determine the "free" compound. Estimate the impact of

[0016] Typically, experiments to assess hepatic uptake and biliary excretion of test compounds are performed using extracellular This is often done for experimental simplicity and representativeness. For this reason, the separately determined free fractions are applied to these results.

[0017] According to the present invention, measurements of hepatocytes (uptake, biliary excretion, hepatobiliary clearance, or cell Blind application of a free fraction correction factor to a given concentration (e.g., intravenous concentration) can result in physiological data being misinterpreted. Indeed, in certain instances, the addition of protein may alters (often dramatically) the pharmacokinetics of compound uptake into cells, and It is observed that the interaction modifies the dynamics of proteins and changes the thermodynamic binding parameters. When determining the separation ratio separately, these parameters are not taken into account at all or are not taken into account at all. Ultimately, there is an assumption that the amount of uptake is proportional to the unbound drug concentration. However, this can often be incorrect. In fact, protein addition is not always predictable. that the observed amount of compound uptake and the specificity of the compound may not necessarily have the desired effect. Here we demonstrate that intravesicular concentrations do not match the uptake predicted in the presence of protein. This observation is unexpected.

[0018] Indeed, as demonstrated and discussed in the Examples herein, experimental data surprisingly For some compounds, the in vivo environment (such as in the presence of proteins) is approximated. In vivo biliary clearance and intrahepatic concentration under these conditions were determined in separate studies (two-stage method). By adjusting the data using the fraction free values ​​obtained from Rather, the evaluation is performed in the presence of physiological concentrations of protein (but By performing the experiments under conditions similar to the in vivo environment (including but not limited to), it is possible to Therefore, as shown herein, In contrast to the estimated or calculated biliary clearance and intrahepatic concentration, may be over- or under-predicted in surprisingly dramatic ways.

[0019] Thus, in some embodiments, the present invention provides an in vivo relevant extracellular environment (e.g., protein and / or other components) to determine hepatocyte disposition and / or toxicity Using an integrated system combined with intracellular disposition and / or efficacy evaluation such as An approach is provided.

[0020] In some embodiments, the present invention provides a method for adding a candidate compound to the culture and / or suspension. and exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment. This extracellular environment may include, for example, exposing the cells to a medium that provides a component (e.g., a protein). and / or other components) at physiological or near-physiological concentrations, Some embodiments of the present invention provide a medium containing a soluble ... Indeed, the present invention encompasses any combination of multiple exposure steps. In some embodiments, the medium comprises a combination of The candidate compound may contain a protein and may contain other components that can modulate the properties of the candidate compound. In some embodiments, the culture and / or suspension is in the presence of a medium containing the protein. The solution can be exposed to a putative inducer or inhibitor, and then the protein containing the candidate compound can be analyzed. Furthermore, in some embodiments, the evaluation can be performed in a medium that does not contain Proteins are composed of several proteins (albumin, alpha-1-acid glycoprotein in physiological concentrations, or It has been determined to have binding properties similar to those observed at physiological protein concentrations. In some embodiments, the mixture is obtained directly from the species of interest. Proteins should be at physiological concentrations or produce results similar to those observed at physiological protein concentrations. Proteins (albumin, alpha-1-acid glycoprotein) at concentrations determined to have binding properties It may also include serum or plasma obtained directly from the species of interest. Included.

[0021] In some embodiments of the present invention, hepatocyte cultures, such as sandwich-cultured hepatocytes, are used. can be used to assess hepatic uptake and biliary excretion of compounds of interest, such as drug compounds. No. 6,780,580, which is incorporated herein by reference in its entirety. As such, screening for compounds of interest (e.g., therapeutic compositions) can be performed in this manner. These compounds are taken up and excreted extensively through the biliary excretion process. Therefore, early evaluation procedures are desirable because they have the least chance of affecting the therapeutic effect of the subject. Compounds with undesirably high susceptibility in the process may be further evaluated as therapeutic agents. The susceptibility of the compound to hepatocyte uptake and biliary excretion is assessed to facilitate its elimination from the liver. It is desirable to establish an in vivo test method for hepatocyte cultures. To maintain the desired functional properties that reflect the natural hepatocytes, the sensitivity to bile excretion was investigated. The following U.S. Patents also provide models for screening compounds of interest: No. 7,601,494; U.S. Pat. ... 682,781; U.S. Patent No. 7,604,934; U.S. Patent No. 8,367,630; and U.S. Patent Application No. US-20 Publication No. 10-0035293-A1.

[0022] As will be appreciated by those skilled in the art, in vivo biological processes can be accurately monitored at a desired level. To perform the derivation, cells (e.g., but not limited to, hepatocytes) are cultured in vitro. The cultures and / or suspensions may be in vivo cells (e.g., but not limited to, hepatocytes). ) should be structurally and functionally similar to the In the cultures and / or suspensions of the present invention, the structural and functional characteristics displayed in vivo are For example, the present invention provides a method for the treatment of the sinusoidal or bile canalicular transport system, or both the sinusoidal and bile canalicular transport systems are established. In particular, in accordance with the present invention, cultures of cells (e.g., but not limited to, hepatocytes) and / or establishing at least one bile canaliculus in the suspension. The culture and / or suspension may comprise a plurality of bile canaliculi. At least one bile duct or bile canaliculus network may be included. The establishment of the cytoplasm is achieved by culturing cells (e.g., but not limited to, hepatocytes) to form gallbladder cells in vivo. Similar to bile excretion, bile and bile components are excreted into at least one bile canaliculus. do.

[0023] In addition to the bile canalicular transport system, specific This liver-associated medium contains vesicles-like transporters. cells, or transgenic or knocked out for human-specific proteins (e.g., transporters, P450), HepaRG® cell line, Hurellux® cell line , to mimic cell lines such as Opti-Target® cell lines, Caco-2, and MDCK Exemplary transporters include Ntcp, cMoat, O These include, but are not limited to, atp1, Oatp2, Mrp2, Mrp3, Pgp, Bsep, and Mdr2. The expression and function of these hepatic transporters are substantially similar to that seen in hepatocytes in vivo. It can be similar to.

[0024] In cultures and / or suspensions of cells (e.g., but not limited to, hepatocytes) Establishment of normal metabolic capacity, including expression and activity of metabolic enzymes, is also provided in accordance with the present invention. Thus, the cultures are a substitute for in vivo cells (such as, but not limited to, hepatocytes). For example, various P450 isozymes can be expressed in a variety of ways, each of which can have a metabolic capacity that substantially reflects the metabolic capacity of the individual. Phase I metabolic enzymes such as ATP, phase II metabolic enzymes such as UDP-glucuronosyltransferase (UGT), and and in vitro cell (e.g., but not limited to, hepatocyte) cultures and / or suspensions. The normal expression of other enzymes involved in the conjugation of primary bile acids with taurine and glycine in suspension The present invention provides for the functions and activities of the present invention.

[0025] Such methods, in some embodiments, provide cultures and / or suspensions of cells. exposing the culture and / or suspension to a candidate compound; and / or exposing the suspension to a medium that provides an in vivo relevant extracellular environment; The amount of the candidate compound taken up into the culture and / or suspension of the compound is determined, thereby determining whether the candidate compound is The disposition of the complementary compound is evaluated to determine the in vivo disposition of the candidate compound. The extracellular environment can include, for example, predicting the presence of components (e.g., proteins). Proteins and / or other components) at physiological or near-physiological concentrations (e.g., The medium contains the compound at a concentration that has binding properties. The step of determining the amount of the complement compound and thereby assessing the disposition may further comprise: determining the intracellular concentration of the compound; determining liver accumulation; determining biliary excretion; and / or determining biliary clearance.

[0026] In some embodiments, a method for testing the biliary excretion sensitivity of a candidate compound is provided. do. A method for testing the susceptibility of such candidate compounds to biliary excretion is to use cell cultures and / or suspensions. fluid (e.g., an artificial membrane system adapted to mimic a cell and at least one bile canaliculus) providing a culture and / or suspension of cells containing the tube; injecting a candidate compound into the cell culture; exposing the cell culture and / or suspension to an in vivo exposing the at least one bile canaliculus to a medium that provides a relevant extracellular environment; determining the amount of the candidate compound in the bile, thereby testing the susceptibility of the candidate compound to biliary excretion. This extracellular environment can include, for example, components (e.g., proteins and / or or other components) at physiological concentrations or near physiological concentrations, with properties (e.g., binding properties) that The medium contains the ATP at a concentration of 1000 kJ / kg.

[0027] In some embodiments, the amount of the candidate compound in the at least one bile canaliculus is determined. The determining step involves combining the candidate compound and a labeled preselected amount of substrate for the transport protein. and simultaneously exposed to the cell culture and / or suspension for a sufficient time to allow uptake. washing the cell culture and / or suspension, and removing the at least one bile canaliculus. The amount of labeled substrate present in the bile is detected, and the amount of labeled substrate present in the bile is measured. and assessing competition between the candidate compound and the labeled substrate. In some embodiments, the at least one nucleotide sequence is selected from the group consisting of nucleotides, nucleotides, and nucleotides, as compared to a preselected amount of the labeled substrate. If the amount of the labeled substrate in the other bile canaliculus is reduced, it is transferred to the transport protein. The susceptibility of the candidate compound to biliary excretion by IL-1 is demonstrated. The amount of the candidate compound in the at least one bile canaliculus is determined by measuring the amount of the candidate compound in the culture and / or suspension. The biliary clearance value is determined by calculating the biliary clearance value.

[0028] Furthermore, in some embodiments, the cell culture and / or suspension in the above method comprises: Cells alone or in co-culture with support cells such as fibroblasts and / or Kupffer cells The artificial membrane system may include an artificial membrane system adapted to mimic the The cells may be, for example, vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac The cells may be cells, nerve cells, muscle cells, adipocytes and / or lung cells. In some embodiments, the cells mimicked by the artificial membrane system may consist of a single cell line. In some embodiments, the cell line is a HepaRG® cell line, Hurellux (registered trademark) cell line, Opti-Target (registered trademark) cell line, Caco-2 and / or MDC is selected from.

[0029] When a labeled substrate is used in any of the methods described herein, such label The substrates used include fluorogenic compounds, fluorescent compounds, chemiluminescent compounds, colorimetric compounds, radioactive compounds, and the like. The compound may include a compound selected from the group consisting of: ...

[0030] In some embodiments, a method for testing the biliary excretion sensitivity of a candidate compound is provided. Such methods include providing first and second cell cultures and / or suspensions and at least one establishing one bile canaliculus, wherein the first culture and / or suspension comprises an intact bile canaliculus; and the second culture and / or suspension has collapsed bile canaliculi. Such a method can further include transferring the candidate compound to a substrate that allows for uptake of the candidate compound. the first culture and / or suspension and the second culture and / or suspension for a sufficient time to exposing the first and second cultures and / or suspensions to the relevant cells in vivo. exposing the first and second cultures and / or suspensions to a medium that provides an extracellular environment; The steps of purification and lysis, and the presence of the lysates obtained from each culture and / or suspension The amount of candidate compound present in each culture and / or suspension is determined and used to The method may include a step of evaluating the biliary excretion sensitivity of the candidate compound. The boundary may be, for example, a physiological concentration or concentration of a component (e.g., a protein and / or other component). It is a medium containing a concentration that has properties (eg, binding properties) that approximate physiological concentrations.

[0031] In some embodiments, each of the first and second cultures and / or suspensions comprises a culture of cells. Further, in some embodiments, the artificial membrane system may include an artificial membrane system adapted to: The cultures and / or suspensions of the above methods may be cells alone or fibroblasts and / or Kupffer cells. It includes an artificial membrane system adapted to mimic cells in co-culture with support cells such as cells. The cells mimicked by the artificial membrane system may be, for example, vesicles, hepatocytes, liver-derived cells, etc. cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes and / or lung cells Furthermore, in some embodiments, the cells may be mimicked by an artificial membrane system. The cells may consist of a cell line. In some embodiments, the cell line is HepaRG® cell line, Caco-2 and / or MDC.

[0032] In some embodiments, first and second cell cultures and / or suspensions are used. The method further comprises exposing the candidate compound to the analyte for a sufficient time (T) to allow uptake of the candidate compound. exposing the first and second cultures and / or suspensions to the first and second cultures and / or suspensions; and / or exposing the suspension to a medium that provides an in vivo relevant extracellular environment, washing and lysing the first and second fractions of the first and second cultures and / or suspensions, respectively; the candidate compounds present in the lysates obtained from each of the first and second cultures and / or suspensions. determining the amount of bile canaliculi in the first culture and / or the second culture having intact bile canaliculi; The two samples were obtained from the suspension and the second culture and / or suspension with collapsed bile canaliculi. calculating the difference between the amounts of the candidate compound present in the two lysates, and The step of evaluating the biliary excretion sensitivity of the candidate compound may include using the extracellular environment For example, components (e.g., proteins and / or other components) may be obtained at physiological or biological concentrations. A medium containing a compound at a concentration that has properties (e.g., binding characteristics) similar to the physiological concentration of the compound.

[0033] In some embodiments, in vivo assays are used to predict the efficacy of candidate compounds. Methods are provided for assessing the effects of candidate compounds in cultures and / or suspensions of trophic ... The method of et al. comprises the steps of providing a culture and / or suspension of cells, and / or exposing the suspension to at least one candidate compound; exposing the suspension to a medium that provides an in vivo relevant extracellular environment, and The effect of exposing the sample and / or suspension to at least one candidate compound is evaluated to determine the effect of the candidate compound. This extracellular environment may include, for example, predicting the in vivo effect of the compound. components (e.g., proteins and / or other components) at or near physiological concentrations The medium is a medium containing the compound at a concentration that has the properties (e.g., binding properties) of the compound evaluated in the above method. These effects include transduction and other types of studies (metabolism, induction, and toxicity); metabolite identification and metabolic stability studies; Metabolic studies including qualitative (parental lifespan); gene regulation (induction / repression); P450 and transporters Drug interactions; intracellular accumulation and free or total (bound + free) intracellular concentrations (e.g., nuclear, mitochondrial); and / or toxicological effects.

[0034] Moreover, in some embodiments, such methods are further adapted to mimic cells. A culture and / or suspension of cells comprising an artificial membrane system and at least one bile canaliculus. In some embodiments, the culture of cells of the above method can include providing and / or the suspension is co-cultured with support cells, such as fibroblasts and / or Kupffer cells. The invention can include an artificial membrane system adapted to mimic cells in a nutrient solution or a single cell. The cells mimicked by the artificial membrane system are, for example, Examples of cells that can be used include vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, and pancreatic cells. , cardiac cells, neuronal cells, muscle cells, adipocytes, and / or lung cells. In some embodiments, the cells mimicked by the artificial membrane system are HepaRG® cells. cell lines, Hurellux® cell lines, Opti-Target® cell lines, Caco-2, and / or may consist of a single cell line, such as a cell line selected from the group consisting of MDC, In some embodiments, such cells are derived from mouse, lambda, or ovine serum albumin (OSA). rats, rabbits, humans, monkeys, apes, cats, dogs, piglets, piglets, cows, bulls, oxen A livestock selected from the group consisting of horses, turkeys, chickens, fish, ducks and geese. It may be isolated from a source.

[0035] In some embodiments, the cultures and / or suspensions of this method may be further cultured for a long period of time. The culture and / or suspension may include a bile canalicular network. The culture and / or suspension may include clusters, aggregates, cells, or the like. and combinations thereof. In some embodiments, the cells are characterized in that they are The material may be embedded in a glass tube.

[0036] Additionally, in some embodiments, the culture and / or suspension further comprises a sandwich The sandwich culture and / or suspension may comprise , comprising at least one layer of cells, and optionally at least one layer of cells Both contain one type of bile canaliculus. The sandwich culture and / or suspension may be used for further long-term storage. The cell suspension may comprise a sandwich culture and / or a suspension. The layer may be sandwiched between two layers of a matrix, the matrix being a biological Typical matrix media, synthetic matrix media, co-culture media with support cell types, and combinations thereof The biological matrix medium may be selected from the group consisting of collagen. , laminin, basement membrane-derived complexes, their derivatives, and combinations thereof. The compound may be selected from the group consisting of:

[0037] In any of the methods disclosed herein, the culture and / or suspension may be cultured in a medium containing a plurality of candidates. In some embodiments of the presently disclosed methods, the target protein can be exposed to a co-compound. In the method, the culture and / or suspension is repeatedly exposed to one or more candidate compounds. can be exposed.

[0038] In the methods disclosed herein, physiological concentrations or properties near physiological concentrations (e.g., Examples of ingredients (such as proteins) that can be added at concentrations that have the desired properties include: , albumin; α-1-acid glycoprotein; β-lipoprotein; bilirubin; bile acids or bile and / or mice, rats, rabbits, humans, monkeys, apes, cats, dogs, Piglets, pigs, cows, bulls, sheep, horses, turkeys, chickens, fish, ducks, Examples of suitable antibodies include plasma or serum derived from a representative or desired subject, such as a goose, Furthermore, in some embodiments, for example, ingredients (e.g., tan Proteins and / or other components) at physiological or near-physiological concentrations (e.g., The medium of the relevant extracellular environment in vivo, such as the medium containing the antibody at a concentration having the binding properties Other compounds that modulate the properties of the candidate compound may be included. bilirubin; β-lipoprotein; and a protein or protein mixture. The medium containing the candidate compound may contain other compounds that modulate the properties of the candidate compound. In addition to the target concentration, the concentration of components (e.g., proteins and / or other components) may also be It may be higher or lower, but at concentrations that have the same effect. In embodiments, non-physiological concentrations that have a similar effect, such as a similar binding effect, are also provided. It is served.

[0039] In the methods disclosed herein, such methods include: In this way, multiple candidates can be detected simultaneously in one well. Furthermore, in the methods disclosed herein, these compounds may be screened. Any combination of the multiple exposure steps may be performed simultaneously or in any order. Good too.

[0040] Representative calculations that can be used in the present invention include the Bile Efflux Index (BEI) and Bile Clearance. In some embodiments, the biliary excretion index is calculated based on the amount of bile taken up by hepatocytes. The percentage of compounds excreted in the bile out of compounds taken up is shown. In this study, the biliary clearance value indicates the possibility that a compound is excreted in the bile. It is the best predictor of biliary clearance of a compound in the body. In some embodiments, the biliary excretion index (BEI) is determined by measuring the uptake of a candidate compound as follows: Calculated from intake and exhaust: Bile excretion index (BEI) = 100% × ((uptake into intact bile canaliculi in culture)) - (calcium Uptake into hepatocytes in Ca2+-free medium only) / (intact hepatocytes in culture) uptake into bile ductules) In some embodiments, biliary clearance is calculated as follows: Biliary clearance = ((uptake into intact bile canaliculi in culture)) - (calcium (Ca ) Uptake into hepatocytes in free medium only)) / (candidate compound in buffer solution during incubation) multiplied by the concentration of the substance)

[0041] In some embodiments, the bile clearance value is measured in a curve corresponding to the amount of bile canaliculi in the culture medium. The area under the curve (AUC) can be calculated as the ratio of the area under the curve (AUC) from time 0 to Time T (time can be measured in any unit, but is usually measured in minutes) or The values ​​represent the integral of the amount of the candidate compound in the medium at 1000 μg / mL. In practice, this area under the curve (AUC) can be expressed as:

number

[0042] The following terms are believed to be well understood by those of ordinary skill in the art, but are used to facilitate the description of the present invention. For this purpose, the following definitions are provided:

[0043] "In vivo relevant extracellular environment" refers to the in vitro environment of a candidate compound according to the present invention. Environments that mimic or approximate in vivo conditions relevant to assessing disposition and / or efficacy For example, the relevant extracellular environment in vivo is composed of components (e.g., proteins and / or or other components) at physiological concentrations or near physiological concentrations, This binding property can be measured by the "tightness" (Ka( The term "binding constant" includes the degree of binding as well as the degree of binding (as indicated by the binding constant) or Kd (dissociation constant).

[0044] The term "disposition and / or effect" includes, but is not limited to: These include but are not limited to: uptake clearance; basolateral outflow clearance; canalicular outflow clearance Metabolic clearance; Intracellular concentration; Compound kinetics; Toxicological effects; Metabolite ID and metabolism Stability (parental lifespan); Gene regulation (induction / repression); P450 and transporter-drug interactions Intracellular accumulation and free or total (bound + free) intracellular concentration (e.g., nuclear, mitochondrial) and overall biliary clearance. This term "disposition and / or effect" Combinations also include pharmacokinetics (PK), which is the ability of an organism or system to interact with a compound of interest. and (2) the quality of all additives in culture and / or suspension systems. It can be broadly defined as the clearance (uptake, efflux, metabolism) of The combination of the terms "position and / or effect" is understood by those of skill in the art reviewing this disclosure. The evaluation may include any desired evaluation as would be apparent to a person skilled in the art.

[0045] The term "calcium-free buffer" refers to any buffer that is substantially free of calcium. A non-limiting example of a calcium-free buffer is calcium-free Hanks' equilibrium buffer. The salt solution is calcium-free Hank's balanced salt solution (HBSS). As can be appreciated, any suitable buffer that is substantially calcium-free can be used in the preparation of the present invention. The use of calcium-free buffers allows for the preparation of some embodiments of the present invention. According to the morphology, collapsed bile canaliculi are provided.

[0046] The terms "normal metabolic function," "normal metabolic activity," and "desirable metabolic characteristics" are used herein. are used interchangeably herein and are intended to mean cells (such as hepatocytes) that grow under normal basal conditions in vivo. The activity of enzymes involved in metabolic pathways and metabolic reactions in , function and / or expression.

[0047] The term "functional property" refers to a property that confers a specific function involved in the biology of an organism, cell, or biochemical reaction. According to the present invention, this functional property includes any biological property that contributes to the production of a substance, such as an enzymatic activity, Enzyme function, enzyme expression, transporter expression and transporter function, and enzyme and It may also include the regulatory pathway responsible for the expression of the transporter.

[0048] The terms "compound," "candidate compound," "target compound," or "drug compound" are used herein to refer to are used interchangeably in the context of the compound (exogenously administered or endogenously produced) Any compound for which sensitivity to metabolism, toxicity, hepatic uptake, or biliary excretion is desirable. Exemplary compounds, target compounds, or drug compounds include drugs and other therapeutic agents. , exogenous biosubstances such as carcinogens and environmental pollutants, as well as steroids, bile acids, fatty acids, and endogenous biosubstances such as prostaglandins.

[0049] The subject compounds that are therapeutic agents can be useful in the treatment of warm-blooded vertebrates. The present invention relates to mammals and birds. Here, we will look at humans, endangered and therefore important mammals (such as the Siberian tiger), Economically important animals (animals raised for human consumption) and / or socially important Animals of human importance (animals kept in zoos or as pets), e.g., humans Carnivores other than cats and dogs, pigs (pigs, piglets, wild boars), ruminants ( cattle, bulls, sheep, giraffes, deer, goats, bison, and camels), and horses Treatments for all mammals are provided. Endangered birds and animals are also treated here. Birds and fowl kept in aquatic environments, more particularly domesticated birds, i.e. poultry (e.g. Treatments for birds, including turkeys, chickens, ducks, geese, guinea pigs, etc., are provided. These are also important economically for humans. Includes, but is not limited to, domesticated pigs (pigs and domestic pigs), ruminants, horses, and poultry. Non-limiting methods of treating livestock are provided.

[0050] The phrase "assess toxicological effects" refers to cells such as, but not limited to, hepatocytes. (not applicable to the toxicological effects of a compound) This refers to any appropriate method for measuring

[0051] The term "biliary excretion" refers to the process by which substances are taken up by hepatocytes and transported via the bile canaliculi. and excretion into the bile (i.e., uptake and efflux) from the subject's circulatory system. For example, uptake into hepatocytes is achieved by Ntcp, Oatp transport systems endogenous to hepatocytes, such as, but not limited to, Oatp1 and Oatp2. Excretion into the bile canaliculi is mediated by Mrp2, Mdr3, Pgp, Bsep, etc. (but not limited to these) The bile canaliculi are the transporters of efflux from hepatocytes. bile ducts are structures within the liver tissue that receive the bile-injected components and transport them to the bile duct for removal from the subject. do.

[0052] The method of the present invention involves forming at least one hepatocyte layer between two matrix layers. The method may include establishing a sandwich culture of hepatocytes. Such a configuration is a preferred configuration for culture, but as will be apparent to those skilled in the art, Any such suitable configuration is within the scope of the present invention. For example, at least one bile canaliculus are formed, and the functional properties of cells (e.g., but not limited to, hepatocytes) are Cells (e.g., hepatocytes, but not limited to) in culture and / or suspension, as established Clusters, aggregates or other associations or clusters of (but are not limited to) are within the scope of the present invention. Other cell types, such as Kupffer cells and fibroblasts or other cell types derived from primitive mesenchyme, Cells in co-culture with other cell types (e.g., but not limited to, hepatocytes) may also be used. Optionally, the culture and / or suspension may be prepared by in vivo hepatocyte culture. This facilitates the formation of multiple bile canaliculi that reflect the cells. This culture composition facilitates the formation of bile canaliculi networks. , or a desired cell type substantially similar to an in vivo cell (e.g., but not limited to, a hepatocyte). Establishment of a culture of cells (e.g., but not limited to, hepatocytes) having the metabolic characteristics of Similarly, in vivo cells (e.g., but not limited to, hepatocytes) can be easily Desired transporter expression and function, substantially similar to that described above, is optionally established.

[0053] Furthermore, the sandwich structure can contain cells (e.g., but not limited to, hepatocytes). They can be cultured in a monolayer or scaffold between two matrix layers. Cells (e.g., but not limited to, hepatocytes) can be embedded in the matrix. Or you can arrange the matrix vertically, horizontally, diagonally, or any of these. Combinations can also be stretched heterogeneously to form one- and three-dimensional aggregates. Additionally, the culture and / or suspension may be cultured in a variety of systems, including but not limited to, a three-dimensional flow-through system. It can be established in a bioreactor system, microenvironment, or three-dimensional scaffold (not specified). See, e.g., Griffith and Naughton, (2002) Science 295:1009-1014. Cultures and / or suspensions of cells (e.g., but not limited to, hepatocytes) may be prepared by (e.g., but not limited to, hepatocytes) are mixed with a suitable matrix, and the mixture is Inserting the mixture into a suitable culture vessel such as a multi-well plate or culture chamber. It can be formed by:

[0054] Collagen can be used in cultures of cells (e.g., but not limited to, hepatocytes) and / or are exemplary substrates or scaffolds for suspensions, but other substrates, both natural and synthetic, may be used as would be apparent to one skilled in the art. Any suitable substrate or scaffold, or combination thereof, is within the scope of the present invention. Other companies include Collaborative Biomedical Products, Inc. (Bedford, Massachusetts, USA) Derived from laminin and biological cell culture substrates sold under the registered trademark MATRIGEL by Pharma, Inc. (Pty. Ltd.) Biological matrices, including basement membranes, are suitable matrix or scaffold materials. Synthetic matrix materials, substrate materials or scaffolding materials, which are typically made from various materials, are also included in the present invention. Used in culturing cells (e.g., but not limited to, hepatocytes) Various component materials having specific matrices for use in the preparation of the medicament are also provided in accordance with the method of the present invention. will be done.

[0055] Any suitable cell source (e.g., , hepatocytes, etc. are also within the scope of the present invention. Exemplary sources include: These include the warm-blooded vertebrates listed above. In particular, exemplary sources include humans, rats, mice, and Squirrels, monkeys, apes, cats, dogs, piglets, hogs, cows, bulls, sheep, horses, and cattle. Including, but not limited to, turkeys, chickens, ducks and geese.

[0056] Cultured cells (e.g., but not limited to, hepatocytes) are referred to as "long-term cultures and "Long-term culture and / or suspension" means at least Also refers to cells (e.g., but not limited to, hepatocytes) cultured for about 12 hours. Optionally, "long-term culture and / or suspension" refers to a period of at least about 24 hours, at least about 4 hours, Cells (e.g., but not limited to, hepatocytes) cultured for at least about 8 hours or for at least about 72 hours Optionally, "long-term culture and / or suspension" refers to at least about 96 hours. Cells (e.g., hepatocytes, (However, this is not limited to this.) Long-term culture refers to the formation of bile canaliculi and the growth of cultures and / or facilitates the establishment of functional properties such as metabolic pathways in suspension.

[0057] Liver cell cultures and / or suspensions are described herein as representative cultures. However, the present invention also relates to any cell or tissue of interest that is capable of modulating the function of the disclosed cell types. are provided for culturing or using cell types alone or any combination of cell types and support cells. Thus, with reference to the present disclosure, one skilled in the art can easily implement any desired cell culture and / or suspension. The above descriptions and approaches can be adapted for use in the preparation of representative cells. The cultures and / or suspensions may include hepatocytes, kidney cells, gastrointestinal cells, pancreatic cells, muscle cells, cardiac cells, a culture and / or suspension of cells comprising cells selected from the group consisting of: fibroblasts, neural cells, and lung cells; According to some embodiments of the present invention, the support The cells may be matrix (e.g., but not limited to, fibroblasts) or functional (e.g., Single or multiple cells with other cells that provide the desired effect, including, but not limited to, Kupffer cells. Co-cultures with the mold are provided.

[0058] In accordance with long-standing patent law conventions, the terms "a" and "an" are used in conjunction with the present invention, including the claims. When used in an application, it means "one or more." Unless otherwise indicated, the amounts of ingredients, reaction conditions, etc. used in the specification and claims are All numbers expressing quantities, etc. are in all instances modified by the term "about." Accordingly, unless otherwise indicated, the present specification and patent application The numerical parameters set forth in the claims are sought to be obtained by the present invention. These are approximations that may vary depending on the desired properties. As used herein, refers to a value, or mass, weight, time, volume, concentration, or percentage The term "about" when used interchangeably with "about" should be understood to encompass any variation that may be applicable to the practice of the disclosed method. In some embodiments, ±20%, and in some embodiments, ±10% of the amount specified; In some embodiments, ±5%, in some embodiments, ±1%, in some embodiments, In some embodiments, the range is ±0.5%, and in some embodiments, ±0.1%.

[0059] As used herein, "and / or" when used in the context of listing entities Thus, for example, "A, B, C, and / or "or D" includes A, B, C, and D individually, but also any combination of A, B, C, and D. includes all combinations and subcombinations. "Comprising" can be used in conjunction with "including," "containing," or Synonymous with "characterized by," but inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. "Comprising" means that the named elements and / or steps are present. Although the term "component" refers to a claim, other elements and / or steps may be added and still fall within the scope of the claim. Enter. As used herein, the term "consisting of" means specifically This "exclusion" refers to the exclusion of any element, step, or ingredient not specifically listed. The term "consisting of" does not follow the preamble and is included in the body of the claim. When used in a claim, it is limited to only the elements recited therein and does not include other elements from the entire claim. are excluded. "Consisting essentially of" extends the claim to the specified material. or steps that substantially affect the basic and novel characteristics of the claimed subject matter. Limit the materials or steps that are not used. "comprising," "consisting of," and "consisting With respect to "essentially of," one of these three terms is used herein. When used herein, the invention can include the use of either of the other two terms.

[0060] As used herein, "significant" or "significant" refers to two or more entities. It is a statistical analysis of the probability that a non-random association exists between two or more variables. To determine whether a certain condition exists, statistical manipulation of the data is performed to calculate the probability and obtain a "p-value." Those p-values ​​below the user-defined cutoff point are called " In some embodiments, a value of 0.05 or less is considered "significant / prominent." In some embodiments, less than 0.01, in some embodiments, less than 0.005, In this case, a p-value of less than 0.001 is considered significant. Therefore, a p-value of 0.05 or greater is considered significant. The values ​​are not considered "significant / remarkable." [Example]

[0061] Hepatobiliary dissociation of multiple compounds in the absence and presence of different types of proteins The results of some exemplary experiments comparing positions are described below. It is provided to illustrate exemplary embodiments of the present invention. Those skilled in the art will recognize that the following examples are merely representative and that no deviations from the spirit and scope of the present invention may occur. It will be understood that many variations, modifications and variations may be employed without departing from the spirit and scope of the present invention.

[0062] Example 1 Evaluation of free fraction values ​​for selected compounds First, equilibrium dialysis is used to identify a range of free (also called unbound) fractions. Several compounds were identified. The free fraction was determined to be thermodynamically bound to the albumin protein. The amount of compound that is thermodynamically free in solution compared to the amount that is likely to combine For example, a free fraction of 0.428 means that approximately 43% of the compound is unbound, and under these conditions These values ​​are shown in Table 1.

[0063] [Table 1]

[0064] Example 2 Evaluation of measurements of endogenous bile clearance in rat hepatocytes. Physiologically concentrated protein (4% bovine serum albumin, BSA) and obtained from Wistar rats The liver uptake, efflux, and cellular activity of the nine compounds listed in Table 1 were measured in the presence and absence of serum. The intravenous concentration and biliary clearance were evaluated. Each compound was tested in rat serum at a concentration of 1 μM and for 10 minutes. These data were measured in rat hepatocytes cultured at different temperatures. reflects uptake under conditions; cellular accumulation (intracellular concentration under -Ca (calcium-free) conditions) Bile excretion index (BEI): the ratio of compounds taken up by hepatocytes to those excreted in the bile and intrinsic biliary clearance (the percentage of the compound excreted in the bile). It indicates the likelihood of being cleaved into the bile and is the best predictor of biliary clearance of a compound in vivo. The parameters of endogenous biliary clearance, which considers both the uptake and efflux of compounds, were determined. The results of the comparison are shown in Table 2. Taken together, these data suggest that the addition of extracellular proteins It has been shown that this can produce very different results compared to traditional results measured in buffer. It certainly shows.

[0065] [Table 2]

[0066] Example 3 Comparison of measured (observed) and predicted (calculated) bile clearance in rat hepatocytes. Next, we investigated the biliary clearance of these compounds in rat hepatocytes in the presence of extracellular proteins ( The values ​​(measured) were measured in the absence of protein and adjusted using the appropriate percent free values ​​shown in Table 1. and compared with predicted endogenous biliary clearance, the latter of which is the current industry standard. Table 3 summarizes these results and compares them with a two-step process based on separate experiments. We also demonstrate the effectiveness of using a one-stage integrated system to measure biliary clearance. If the multi-stage process is equivalent to this one-stage integrated system, these values ​​should match. However, this was not the case with pitavastatin and rosuvastatin, which increased biliary clearance. The predicted values ​​of the serotonin levels were underestimated compared to the measured values, which may be due to the unexpected effect of albumin in the presence of serotonin. The predicted biliary clearance of DPDPE (1 μM) was higher than the measured value. Please note that the clearance is overestimated in this case. or essentially these values ​​are not equivalent, the interaction is not as expected. and demonstrates that it is a non-invasive material that can be integrated when evaluating cell disposition. This indicates that it is desirable to use the method described above.

[0067] [Table 3]

[0068] Normally, no difference is expected between these values ​​for biliary clearance (this value (This is because the degree of protein binding is taken into account.) As can be seen from Table 2, this is Trexate, valsartan, DPDPE, pravastatin, digoxin and taurocholic acid This is true for many of the compounds evaluated, including salts. However, for the two compounds, pitavastatin and rosuvastatin, The predicted intrinsic biliary clearance values ​​in the presence of the protein are significantly lower than those observed in the absence of the protein. The effects observed in serum were similar, though not identical. This suggests that protein composition also influences compound clearance prediction. do.

[0069] Example 4 Comparison of measured (observed) and predicted (calculated) intracellular concentrations in rat hepatocytes The intrahepatic concentration (ICC) of a compound, which reflects the balance between cellular uptake, metabolism, and efflux, was also measured. It can also be determined in the absence and presence of protein. In a similar manner, a one-step method using proteins in the system was performed to measure intrahepatic concentration (ICC). The free fraction values ​​in Table 1 can be used to determine the free fraction and compared to the adjusted two-stage data. Table 4 shows the intracellular concentrations observed using the one-stage integrated system for various compounds. First, the experiment is performed in the absence of protein, and then the protein binding data from another experiment is used to determine the binding. If the methods were comparable, these values ​​would be strictly It should match.

[0070] [Table 4]

[0071] For some compounds, intracellular concentrations from separate experiments obtained in the absence of protein The protein binding information of the concentration data (ICC prediction) is calculated based on the observed intracellular concentration. However, four compounds (valsartan, pravastatin, and pitavastatin) were For statins (vastatin and rosuvastatin), the observed intracellular concentrations differed from the predicted values, and Measurements were greater than 50% overestimated (valsartan) or underestimated (pravastatin, pitavastatin) Without the integrated system, how much It is impossible to predict whether a compound is taken up by cells and the resulting intracellular concentration. It would be possible.

[0072] For some compounds, these data, i.e., in vivo biliary clearance and The intracellular concentration of riboflavin in the presence of protein (physiological conditions) was calculated based on the free fraction obtained from a separate study. It cannot be predicted by adjusting the data using the sum (two-step method). The in vivo relevant values ​​of these parameters are obtained in the presence of physiological concentrations of the protein. This effect can only be determined by conducting experiments in the presence of valsartan. , pitavastatin, and rosuvastatin all bind to the same extent as BSA, The biliary clearance of valsartan cannot be predicted based on protein binding. The effect of protein addition on the ATP-dependent ATP binding was easily determined from protein binding data obtained in a separate study. However, the biliary clearance of pitavastatin and rosuvastatin was The protein effect on The biliary effects of pitavastatin and rosuvastatin were not predicted by the Accurate prediction of serum clearance parameters requires experiments in the presence of physiological concentrations of protein. It was only obtained if you went

[0073] Accurate estimation of biliary clearance is required for in vivo clearance in human studies. This is useful when trying to predict true in vivo clearance. Prediction leads to better clinical study design, reduced need for human experiments, and clinical This can result in a reduction in development time.

[0074] The intrahepatic concentration is the driving force for any process occurring within the hepatocyte. Changes in concentration can affect all types of interactions that occur inside the liver cells. This includes transporter-based drug interactions; the extent of metabolism of the compound; interactions; the compound's induced potential (metabolism or transport); and the effects of the compound or its metabolites. These may include, but are not limited to, toxicity produced by

[0075] The observed effect of protein on intracellular concentration may result in over- or underprediction of the compound's effect. This could lead to new predictions and dramatically change expected clinical outcomes. This was observed in the case of telmisartan. Although it was thought to have potential for human cholestasis due to its role in albumin transporters, data suggest that it may be associated with a potential role in human cholestasis. Intracellular concentrations in human hepatocytes in the presence of protein were Using data predicted from studies conducted in the absence of ATP, a 100-fold increase in the free fraction was used. However, the intracellular concentration was higher than the predicted value adjusted for hepatotoxicity. The in vivo data never achieved levels high enough to produce a significant effect. These conclusions are based on the fact that telmisartan has no known toxicities associated with its use. The effects of proteins are unexpected and unpredictable, so cells may be affected during the experiment. It is desirable to expose the cells to the protein.

[0076] Further examples include the use of physiological concentrations of two compounds to understand the differences in in vivo toxicity. There is a liver accumulation experiment in the presence and absence of BSA. In vitro toxicity tests (performed in the absence of substances) and other pharmacological tests are two closely related It has been shown that compounds related to I have the same potential for in vivo toxicity. In vivo studies have shown that both compounds have significant rodent toxicity (liver) profiles. The researchers could not explain the differences based on systemic (blood) exposure. The non-toxic compound (AMG-A) had a Cmax concentration six times higher than that of the toxic compound (AMG-B). and area under the curve (AUC) values. Therefore, the researchers were able to estimate the intracellular liver concentrations. The toxic compound (AMG-B) was found to accumulate more in the liver and to have a cell proliferation rate approximately 15 times higher than that of AMG-A. This difference in intracellular concentration may explain the difference in toxicity. (Hamadeh et. al Chem. Res. Toxicol., 2010, 23 (6), pp 1025-1033).

[0077] Example 5 The effects of a non-toxic compound (AMG-A) and a toxic compound ( Evaluation of hepatic uptake and intracellular concentration of AMG-B Experiments to determine the hepatic uptake and intracellular concentrations of AMG-A and AMG-B were performed in accordance with the present invention. The cells were sandwich-cultured in the absence and presence of physiological concentrations of protein (4% BSA). The results were obtained in rat hepatocytes cultured in the absence and presence of physiological concentrations of protein. Significant differences were observed in the hepatic accumulation and intracellular concentrations of the two compounds. In experiments performed in the absence of a non-toxic compound (AMG-A), the intracellular concentrations of 3 and 10 μM doses were , which was higher than the intracellular concentration of the more toxic compound (AMG-B) (Table 5). Only when experiments were performed in the presence of 4% BSA, was the intracellular concentration of The intracellular concentration of the more toxic compound (AMG-B) was higher than that of the non-toxic compound (AMG-A). These results were not an accurate predictor of in vivo efficacy. This indicates that physiological concentrations of protein are required for the two chemicals to function. The binding parameters of the two compounds in the presence / absence of the protein were the same. The differences in liver accumulation and intracellular concentrations of the two compounds could not be predicted from protein binding data. It was.

[0078] [Table 5]

[0079] [Table 6]

[0080] The present invention provides a method for predicting in vivo relevant biliary clearance and intracellular concentrations for the hepatic system. Proteins are used to assess hepatic biliary disposition and / or the effects of compounds to Furthermore, the present invention provides a method for the detection of HIV-1-associated ... The present invention provides for the use of proteins in cell proliferation and other types of research (metabolism, induction and toxicity). Thus, in some embodiments, metabolite identification and metabolic stability (parent lifespan) (Kilford et al. t al., Drug Metab Dispos, 36(7): 1 194-1 197, July 2008); Gene regulation (induction / repression ) (Jackson et al. Chemico-Biological Interactions, 179, 263-272, 2009); P450 and Transporter-drug interactions (including herb-drug interactions); intracellular accumulation (Pfeifer e t al. Drug Metab Dispos 41 :1949-1956, November 2013) and free or total (bound + free) Metabolic studies including intracellular concentrations (e.g., nuclear, mitochondrial) are provided. As will be apparent to those skilled in the art, these are regulators of metabolism, inhibition, induction, regulation and toxicity. All of the above can be used to achieve in vivo relevant intracellular concentrations.

[0081] Non-hepatic (e.g., cell lines such as Caco-2 and MDCK, as well as kidney, gastrointestinal, pancreatic, cardiac, and neuronal) For example, the present invention provides a method for mimicking the physiological conditions of lung organ-specific cell lines and administering compounds To derive a protected methodology for measuring intracellular concentrations of Knowledge of transporters and the ability to measure intracellular volume are integrated By using this method, the intracellular concentration and disposition and / or effect of the compound can be determined. (e.g., exposure, spillage, etc.)

[0082] Example 6 IC of P450 drug-metabolizing enzymes 50 Assessment of the effect of protein addition on the determination of Freshly isolated or cryopreserved hepatocytes were used to prepare sandwich cultured hepatocytes (SCs). Freshly isolated hepatocytes were placed on a 24-well cell culture plate and incubated with Qualyst T Transporter Solutions (QTS, Durham, North Carolina, USA) species-specific appropriate culture medium (QualGro™) and cultured. The cells were then stored appropriately until consumed in the study. The cells were maintained in a species-specific medium.

[0083] Cryopreserved hepatocytes were thawed according to the manufacturer's instructions. The hepatocytes were cultured in QTS hepatocyte medium (QualGro™ culture medium) on 24-well cell culture plates. After plating, cells were suspended at a density of 0.7-0.8 million viable cells / mL. After plating, cells were incubated for 2-4 hours. After 18-24 hours, the cells were incubated in warmed (37°C) seeding medium. ECM, supplemented with Matrigel® (0.25 mg / mL), appropriate species-specific QT The cells were fed and covered with QualGro™ medium (S). The cells were then stored in QualGro™ (commercially available) until consumed in the study. The cells were maintained in target hepatocyte culture medium.

[0084] The cells were cultured as described above until day 6. On day 7, the spent medium was aspirated and the cells were resuspended in 4 ml of PBS. The medium was replaced with HBSS containing or not containing 100% bovine serum albumin (BSA). Substrate and either fluconazole or ketoconazole The culture medium containing the α-amyloid β ... In situ incubation was performed in a cell culture incubator (37°C; 5% CO; humidity The incubation was carried out in 100% CO₂ at 120 rpm for 20-30 minutes. The cells were collected and stored at -80°C until use in bioanalysis.

[0085] In situ incubation: midazolam to hydroxymidazolam of ibuprofen and detection of P450-mediated metabolite formation from ibuprofen to hydroxyibuprofen Briefly, the internal standard solution (25 nM triazolam and d3-ibuprofen) was Add 300 μl of HBSS or HBSS (+ 4% BSA) to a 96-deep well plate. Protein precipitation plates (Millipore MDRPNP4; EDM Millipore, Bille) stacked on a rocker The plate was shaken for 1-2 minutes before centrifugation. The sample filtrate was evaporated to dryness, and the sample was diluted. Reconstitute in 200 µL of dilution solution (40 / 60 methanol / 10 mM ammonium acetate) and shake on a plate shaker. The reconstituted sample was filtered through a Millipore 0.45 μm filter. The mixture was transferred to a plate (Millipore MSHVN45) and centrifuged to a Costar 39 Filter into 57 plates and seal with silicone cap mats.

[0086] Enzyme activity of CYP2C9 (3-hydroxybutyrophene) and CYP3A4 (hydroxymidazolam) Fluconazole (CYP2C9) and ketoconazole (CYP3A4), inhibitors of SCHH, The direct inhibitory effect of fluconazole (CYP2C9) was evaluated in the presence and absence of 4% BSA. Ketoconazole (CYP3A4) increased the enzyme activity of CYP2C9 and CYP3A4 by 24.4% compared with the control, respectively. Positive control inhibitors of CYP2C9 and CYP3A4 reduced the expected Fluconazole (CYP2C9) reduced the enzyme activity in a dose-dependent manner. The difference in the effect of adding protein (4% BSA) to the sample was the estimated IC value of fluconazole (CYP2C9). 50 but The CYP3A4 activity decreased from 56.2 μM to 27.1 μM (Tables 7 and 8). Estimated IC of nazole (CYP3A4) 50 increased from 0.0455 μM to 0.117 μM (Tables 9 and 10). This means that the protein reacts with various compounds (in this case, probe inhibitors or probe substrates). Specificity of hepatic uptake and intracellular concentration of either midazolam or ibuprofen This strongly indicates that the

[0087] [Table 7]

[0088] [Table 8]

[0089] [Table 9]

[0090] [Table 10]

[0091] References References cited herein are intended to be illustrative of the methods, techniques and / or methods employed herein. are cited to supplement, explain, provide background to, or teach the composition. To the extent possible, all such disclosures are incorporated herein by reference.

[0092] Griffith and Naughton, (2002) Science, 295:1009-1014 Hamadeh et al., (2010) Chem. Res. Toxicol., 23(6):1025-1033 Jackson et al., (2009) Chemico-Biological Interactions, 179:263-272 Kilford et al., (2008) Drug Metab. Dispos., 36(7):1194-1197 Pfeifer et al., (2013) Drug Metab. Dispos., 41:1949-1956 US Patent No. 6,780,580 US Patent No. 7,601,494 US Patent No. 7,604,934 US Patent No. 7,682,781 US Patent No. 8,367,630 US Patent Application Publication No. US-2010-0035293-A1

[0093] Various details of the invention disclosed herein may be changed without departing from the scope of the invention. It will be understood that the foregoing description is by way of example only. and is not intended to be limiting.

Claims

1. In vitro cultures and / or Or a method for evaluating the disposition of this candidate compound in a suspension, comprising: (a) providing a culture and / or suspension of cells; (b) exposing a candidate compound to the culture and / or suspension; (c) exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment; the exposure stage, and (d) determining the amount of candidate compound taken up in the culture and / or suspension, thereby The disposition of the candidate compound is evaluated to determine the in vivo disposition of the candidate compound. Predicting the situation A method consisting of:

2. The culture and / or suspension comprises an artificial membrane system adapted to mimic cells. The method according to claim 1.

3. The artificial membrane system mimics cells in a co-culture with feeder cells, and the feeder cells are fibroblasts. and / or Kupffer cells.

4. The cells mimicked by the artificial membrane system include vesicles, hepatocytes, liver-derived cells, kidney cells, stomach cells, and the like. Selected from the group consisting of intestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes and lung cells.

3. The method of claim 2, wherein

5. The cells mimicked by the artificial membrane system comprise a cell line, and optionally, the cells 3. The method of claim 2, wherein the cell line is selected from the group consisting of HepaRG® cell line, Caco-2 and MDC. The method described below.

6. "Determining the amount of the candidate compound taken up in the culture and / or suspension, thereby The "disposition evaluation stage" (a) determining the intracellular concentration of the candidate compound; (b) determining liver accumulation; (c) determining biliary excretion; and / or (d) determining biliary clearance; The method according to any one of claims 1 to 5, comprising:

7. 1. A method for testing the biliary excretion sensitivity of a candidate compound, comprising: (a) an artificial membrane system adapted to mimic a cell and at least one bile canaliculus; providing a culture and / or suspension of cells; (b) exposing a candidate compound to the culture and / or suspension of cells; (c) culturing and / or suspending said cells in a medium that provides a relevant extracellular environment in vivo; exposure to the ground, and (d) determining the amount of the candidate compound in the at least one bile canaliculus, thereby determining the amount of the candidate compound. Testing bile excretion sensitivity of A method consisting of:

8. (d) determining the amount of the candidate compound in at least one bile canaliculus, (a) simultaneously administering a candidate compound and a preselected amount of a labeled substrate for the transport protein; exposing the cell culture and / or suspension to the compound for a sufficient time to allow uptake; (b) washing the cell culture and / or suspension; and (c) detecting the amount of labeled substrate present in the at least one bile canaliculus and determining the transport protein. assessing competition between a candidate compound and a labeled substrate for biliary excretion by a protein; a preselected amount of the labeled substrate relative to the at least one bile canaliculus; The presence of reduced amounts of the labeled substrate in the biliary tract indicates that the substrate is a candidate for biliary excretion by a transport protein. A stage indicating the sensitivity of a compound The method of claim 7, comprising:

9. The artificial membrane system mimics cells in a co-culture with feeder cells, and the feeder cells are fibroblasts. and / or Kupffer cells.

10. The cells mimicked by the artificial membrane system include vesicles, hepatocytes, liver-derived cells, kidney cells, stomach cells, and the like. Selected from the group consisting of intestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes and lung cells. The method of claim 7, wherein

11. The cells mimicked by the artificial membrane system comprise a cell line, and optionally, the cells The lines were HepaRG® cell line, Caco-2, Opti-Target® cell line, and MDC.

8. The method of claim 7, wherein the compound is selected from the group consisting of:

12. The labeled substrate may be a fluorogenic compound, a fluorescent compound, a chemiluminescent compound, a colorimetric compound, and combinations thereof. Item 7. The method according to item 7.

13. The amount of the candidate compound in the at least one bile canaliculus is determined by measuring the bile content of the culture and / or suspension.

8. The method of claim 7, wherein the juice clearance value is determined by calculating the juice clearance value.

14. 1. A method for testing the biliary excretion sensitivity of a candidate compound, comprising: (a) establishing first and second cell cultures and / or suspensions, Artificial membrane systems in which the two cultures and / or suspensions are adapted to mimic cells, respectively. and at least one bile canaliculus, wherein the first culture and / or suspension comprises an intact bile canaliculus. wherein the second culture and / or suspension has collapsed bile canaliculi; (b) incubating the first culture and the second culture for a sufficient time to allow for uptake of the candidate compound. and / or suspension and said second culture and / or suspension; (c) subjecting the first and second cultures and / or suspensions to an environment that provides a relevant extracellular environment in vivo; exposing the cells to a medium containing the cells; (d) washing and lysing the first and second cultures and / or suspensions; and (e) candidates present in the lysate obtained from each culture and / or suspension of step (d). The amount of compound is determined, and the amount of candidate compound in each culture and / or suspension is used to identify the candidate compound. assessing the biliary excretion susceptibility of the compound; A method consisting of:

15. 15. The method of claim 14, comprising: (i) injecting the candidate compound into the first and second cells for a period of time (T) sufficient to allow uptake of the candidate compound. exposing to a second culture and / or suspension; (ii) subjecting the first and second cultures and / or suspensions to a relevant extracellular environment in vivo; exposing the cells to a medium provided by (iii) washing and lysing the first and second fractions of the first and second cultures and / or suspensions, respectively; The stage of (iv) present in the lysate obtained from each of the first and second cultures and / or suspensions of step (iii). determining the amount of the candidate compound; (v) comparing the amount of bile canaliculi in a first culture and / or suspension with intact bile canaliculi and a second culture and / or suspension with disrupted bile canaliculi; The second culture and / or suspension contained bile canaliculi. calculating the difference between the amounts of the candidate compound present in the sample; and (vi) assessing the biliary excretion susceptibility of the candidate compound using the amount calculated in step (iv).

16. The artificial membrane system mimics cells in a co-culture with feeder cells, and the feeder cells are fibroblasts. and / or Kupffer cells.

17. The cells mimicked by the artificial membrane system include vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, and the like. cells, pancreatic cells, cardiac muscle cells, nerve cells, muscle cells, adipocytes, and lung cells.

15. The method of claim 14, wherein

18. The cells mimicked by the artificial membrane system comprise a cell line, and optionally, the cell line is selected from the group consisting of HepaRG® cell line, Caco-2, and MDC. The method described below.

19. in vitro cultures and / or suspensions to predict the in vivo effects of candidate compounds A method for evaluating the effect of a candidate compound of (a) providing a culture and / or suspension of cells; (b) exposing the culture and / or suspension at least once to at least one candidate compound; The stage (c) exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment; the exposure stage, and (d) assessing the effect of exposing the cultures and / or suspensions to at least one candidate compound; and predicting the in vivo effects of the candidate compound. A method consisting of:

20. Artificial membrane system adapted to mimic a cell and a culture comprising at least one bile canaliculus 20. The method of claim 19, comprising providing a substance and / or a suspension.

21. The artificial membrane system mimics cells in a co-culture with feeder cells, and the feeder cells are fibroblasts. and / or Kupffer cells.

22. The cells mimicked by the artificial membrane system include vesicles, hepatocytes, liver-derived cells, kidney cells, stomach cells, and the like. Selected from the group consisting of intestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes and lung cells.

21. The method of claim 20, wherein

23. The cells mimicked by the artificial membrane system comprise a cell line, and optionally, the cells The lines were HepaRG® cell line, Caco-2, Opti-Target® cell line, and MDC.

21. The method of claim 20, wherein the compound is selected from the group consisting of:

24. The effects are investigated through transduction and other types of studies (metabolism, induction and toxicity); metabolite identification and metabolic stability studies. Metabolic studies including qualitative (parental lifespan); gene regulation (induction / repression); P450 and transporters Drug interactions: intracellular accumulation and free or total (bound + free) intracellular concentrations (e.g., nuclear, mitochondrial); and toxicological effects. The method described.

25. 25. Any of claims 19 to 24, wherein the culture and / or suspension is exposed to a plurality of candidate compounds.

1. The method according to claim 1.

26. The culture and / or suspension is repeatedly exposed to one or more candidate compounds. Item 26. The method according to any one of Items 19 to 25.

27. The cells may be derived from a mouse, rat, rabbit, human, monkey, ape, cat, dog, piglet, or edible From pigs, cows, oxen, sheep, horses, turkeys, chickens, fish, ducks and geese 27. The method of any one of claims 2 to 26, wherein the compound is isolated from a source selected from the group consisting of: 。

28. The culture and / or suspension further comprises a long-term culture and / or suspension according to claims 2 to 5.

28. The method of any one of 27.

29. Any of claims 2 to 28, wherein the culture and / or suspension contains a bile canaliculus network.

10. The method according to claim 1.

30. The culture and / or suspension comprises clusters, aggregates, at least one layer of cells, and The present invention is characterized in that the configuration is selected from the group consisting of these combinations.

30. The method of any one of 29.

31. The method according to any one of claims 2 to 30, wherein the cells are embedded in a matrix. Law.

32. The culture and / or suspension further comprises a sandwich culture and / or suspension, The sandwich culture and / or suspension comprises at least one layer of cells, and optionally Any of claims 2 to 31, comprising at least one bile canaliculus in at least one layer of cells.

1. The method according to claim 1.

33. The sandwich culture and / or suspension may be further processed into a long-term sandwich culture and / or 33. The method of claim 32, wherein the liquid comprises a suspension.

34. 2. The method of claim 1, wherein at least one layer of cells is sandwiched between two layers of matrix.

34. The method according to any one of claims 1 to 33.

35. The matrix may be a biological matrix medium, a synthetic matrix medium, a feeder cell type 35. The method of claim 34, wherein the cell surface is selected from the group consisting of a co-culture medium, ... and combinations thereof.

36. The biological matrix medium is a complex derived from collagen, laminin, and basement membrane, 36. The method of claim 35, wherein the compound is selected from the group consisting of: Law.

37. The medium that provides the relevant extracellular environment in vivo is physiological or physiologically similar. The method according to any one of claims 1 to 36, comprising a medium containing components at specific concentrations. 。

38. The components are albumin; β-lipoprotein; α-1-acid glycoprotein; mouse, rat , rabbit, human, monkey, ape, cat, dog, piglet, hog, cow, bull, sheep plasma or serum derived from horses, turkeys, chickens, fish, ducks or geese; bile a mixture of cholic acid or bile acids; bilirubin; and combinations thereof. The method according to any one of claims 1 to 37,

39. Any of claims 1 to 38, carried out in at least one well of a multi-well plate.

1. The method according to claim 1.

40. Any of claims 1 to 39 further comprising the step of screening multiple candidate compounds simultaneously. The method according to any one of claims 1 to 5.

41. The medium containing physiological concentrations of proteins may contain other compounds that modulate the properties of the candidate compound.

41. The method of any one of claims 1 to 40.

42. Any combination of the multiple exposing steps may be performed simultaneously or in any order.

42. The method according to any one of claims 1 to 41,

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