Use of proteins in predicting in vivo efficacy

Abstract

To provide a method, in order to predict in vivo disposition and / or effect of a candidate compound, for evaluating disposition and / or effect of the candidate compound in vitro cultured product and / or suspension.SOLUTION: A method consists of: a step of providing cultured product and / or suspension of a cell; a step of exposing a candidate compound to the cultured product and / or suspension; a step of exposing the cultured product and / or suspension to culture medium providing related in vivo extracellular environment which has properties that resembles physiologic concentration or physiological concentration, in which arbitrary combination of the "step of exposing" can be performed in any order or simultaneously; and a step of evaluating disposition and / or effect of the in vitro candidate compound, and predicting in vivo disposition and / or effect of the candidate compound.SELECTED DRAWING: None

Description

Cross - reference to related applications

[0001] This application claims the priority of U.S. Provisional Patent Application 62 / 060,916, filed on October 7, 2014, the entire disclosure of which is incorporated herein by reference.

Technical Field

[0002] In some embodiments, the present invention relates to a method for evaluating the disposition and / or effect of a candidate compound in an in vitro culture and / or suspension to predict the in vivo disposition and / or effect of the candidate compound. More specifically, the present invention relates to a method for evaluating the disposition and / or effect, including but not limited to, uptake clearance, paracellular clearance, lacrimal drainage clearance, intracellular concentration, bile clearance, metabolic clearance, and the combined effects of the pharmacokinetics of the candidate compound in an in vitro culture and / or suspension to predict the in vivo disposition and / or effect of the candidate compound. In some embodiments, the method includes exposing the culture and / or suspension to a medium that provides an in vivo - relevant extracellular environment. The medium is, for example, a medium containing components such as proteins at physiological concentrations or concentrations having binding characteristics similar to physiological concentrations.

Background Art

[0003] Typically, when experiments are performed in vitro using hepatocytes or related cell lines (suspended, seeded, sandwich-cultured, or other 3D models, Caco-2, MDCK, Opti-Target® (Optivia Biotechnology, Menlo Park, California, USA)), and those available under the trademark HepaRG® (Biopredic International, Saint Gregoire, France), proteins are either absent or present only at non-physiological levels during the experiment. Therefore, only unbound drug concentrations are evaluated in all of these experiments. This is often done for experimental simplicity. To translate this to clinical or in vivo situations, separate in vitro protein binding experiments are performed to determine the fraction of drug bound to plasma proteins, and this information is used to apply the parameters obtained from the in vitro experiments to the in vivo situation. Then, the effect of the "free" compound is estimated by multiplying the results of other experiments by this percentage, in order to apply this percentage somewhat blindly to the results of other experiments. It was assumed that such corrective measures would yield reasonably accurate results related to clinical or in vivo situations. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Therefore, an approach that does not rely on such corrective measures and assumptions is needed. There is a need for a method to evaluate the disposition of candidate compounds in in vitro cultures and / or suspensions and predict the in vivo disposition of candidate compounds. [Means for solving the problem]

[0005] In some embodiments, the method of the present invention 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 a medium that provides a relevant extracellular environment in vivo (e.g., a medium containing components such as proteins at physiological concentrations or concentrations having binding properties similar to physiological concentrations); and determining the amount of the candidate compound incorporated into the culture and / or suspension, thereby evaluating the disposition and / or effect of the candidate compound and predicting the in vivo disposition and / or effect of the candidate compound.

[0006] In some embodiments, the culture and / or suspension includes an artificial membrane system adapted to mimic cells. In some embodiments, the artificial membrane system mimics supporting cells and cells in the co-culture medium, the supporting cells consisting of fibroblasts and / or Kupffer cells. In some embodiments, the cells mimicked by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells mimicked by the artificial membrane system consist of a single cell line, optionally selected from the group consisting of HepaRG® cell lines, Caco-2, and MDC. In some embodiments, the step of "determining the amount of candidate compound incorporated into the culture and / or suspension and thereby evaluating the disposition" consists of the steps of determining the intracellular concentration of the candidate compound, determining hepatic accumulation, determining bile excretion, and / or determining bile clearance.

[0007] In some embodiments, a method is provided for testing the bile efflux sensitivity of a candidate compound. In some embodiments, the method comprises the steps of: providing a culture and / or suspension comprising an artificial membrane system adapted to mimic cells and at least one bile canal; exposing a candidate compound to the culture and / or suspension; exposing the culture and / or suspension to a medium providing a relevant in vivo extracellular environment; and determining the amount of the candidate compound in the at least one bile canal, thereby testing the bile efflux sensitivity of the candidate compound. In some embodiments, the step of determining the amount of the candidate compound in the at least one bile canal comprises the steps of: exposing the candidate compound and a pre-selected amount of labeled substrate for the transport protein to a cell culture and / or suspension for a sufficient time to allow simultaneous uptake; washing the cell culture and / or suspension; and detecting the amount of labeled substrate present in the at least one bile canal and evaluating the competition between the candidate compound and the labeled substrate for bile excretion by the transport protein, wherein the presence of a decrease in the amount of labeled substrate in the at least one bile canal compared to a pre-selected amount of labeled substrate indicates the sensitivity of the candidate compound to bile excretion by the transport protein. In some embodiments, the artificial membrane system mimics supporting cells and cells in a co-culture medium, the supporting cells consisting of fibroblasts and / or Kupffer cells. In some embodiments, the cells mimicked by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes and lung cells. In some embodiments, the cells mimicked by this artificial membrane system consist of a single cell line, which is optionally selected from the group consisting of HepaRG® cell lines, Caco-2, and MDC. In some embodiments, the labeled substrate includes a compound selected from the group consisting of fluorescent compounds, fluorescent compounds, chemiluminescent compounds, colorimetric compounds, radiolabeled compounds, and combinations thereof.In some embodiments, the amount of the candidate compound in at least one bile canal is determined by calculating the bile clearance value of the culture and / or suspension.

[0008] In some embodiments, a method is provided for testing the bile excretion sensitivity of a candidate compound. In some embodiments, the method is: (a) establishing first and second cell cultures and / or suspensions, each comprising an artificial membrane system adapted to mimic cells and at least one bile canal, wherein the first culture and / or suspension has intact bile canals and the second culture and / or suspension has disintegrated bile canals; (b) exposing a candidate compound to the first culture and / or suspension and the second culture and / or suspension for a sufficient time to allow uptake of the candidate compound; (c) exposing the first and second cultures and / or suspensions to a medium providing the relevant in vivo extracellular environment; (d) washing and dissolving the first and second cultures and / or suspensions; and (e) The process comprises the steps of determining the amount of candidate compound present in the lysate obtained from each culture and / or suspension in step (d), and evaluating the bile excretion sensitivity of the candidate compound using the amount of this candidate compound in each culture and / or suspension. In some embodiments, the method includes (i) exposing a candidate compound to the first and second cultures and / or suspensions for a sufficient time (T) to allow uptake of the candidate compound; (ii) exposing the first and second cultures and / or suspensions to a medium providing the relevant extracellular environment in vivo; (iii) washing and lysing the first and second fractions of the first and second cultures and / or suspensions, respectively; (iv) determining the amount of the candidate compound present in the lysates obtained from the first and second cultures and / or suspensions in step (iii); (v) calculating the amount in the bile canaliculi as the difference in the amount of the candidate compound present in two lysates obtained from a first culture and / or suspension having intact bile canaliculi and a second culture and / or suspension having disintegrated bile canaliculi; and (vi) evaluating the bile efflux sensitivity of the candidate compound using the amount calculated in step (iv). In some embodiments, the artificial membrane system mimics supporting cells and cells in a co-culture medium, wherein the supporting cells include fibroblasts and / or Kupffer cells.In some embodiments, the cells mimicked by this artificial membrane system are selected from the group consisting of vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiomyocytes, nerve cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells mimicked by this artificial membrane system consist of a single cell line, which is optionally selected from the group consisting of HepaRG® cell lines, Caco-2, and MDC.

[0009] In some embodiments, a method is provided for evaluating the effect of a candidate compound in an in vitro culture and / or suspension to predict the in vivo effect of the candidate compound. In some embodiments, the method comprises the steps of: providing a cell culture and / or suspension; exposing the culture and / or suspension to at least one candidate compound at least once; exposing the culture and / or suspension to a medium that provides a relevant extracellular environment in vivo; and evaluating the effect of exposing the culture and / or suspension to at least one candidate compound to predict the in vivo effect of the candidate compound. In some embodiments, the method includes the step of providing a culture and / or suspension comprising an artificial membrane system adapted to mimic cells and at least one bile canal. In some embodiments, the artificial membrane system mimics supporting cells and cells in a co-culture medium, the supporting cells comprising fibroblasts and / or Kupffer cells. In some embodiments, the cells mimicked by the artificial membrane system are selected from the group comprising vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells mimicked by this artificial membrane system consist of a single cell line, optionally selected from the group consisting of HepaRG® cell lines, Caco-2, and MDC. In some embodiments, the effect is selected from the group consisting of transmission and other types of studies (metabolism, induction, and toxicity); metabolic studies including metabolite identification and metabolic stability (parental lifespan); gene regulation (induction / repression); P450 and transporter drug interactions; intracellular accumulation and release or all (bound + free) intracellular concentrations (e.g., nucleus, mitochondria); and toxicological effects. In some embodiments, the culture and / or suspension is exposed to multiple candidate compounds. In some embodiments, the culture and / or suspension is repeatedly exposed to one or more candidate compounds.

[0010] In some embodiments of the present invention, the cells are isolated from a source selected from the group consisting of mice, rats, rabbits, humans, monkeys, apes, cats, dogs, piglets, oyster pigs, cattle, bulls, sheep, horses, turkeys, chickens, fish, ducks, and geese. In some embodiments of the present invention, the culture and / or suspension further comprises a longer-term culture and / or suspension. In some embodiments of the present invention, the culture and / or suspension comprises a bile canal network. In some embodiments of the present invention, the culture and / or suspension is characterized by having a configuration selected from the group consisting of clusters, aggregates, at least one layer of cells, and combinations thereof. In some embodiments of the present invention, the cells are embedded in a matrix. In some embodiments of the present invention, the culture and / or suspension further comprises a sandwich culture and / or suspension, the sandwich culture and / or suspension comprising at least one layer of cells and optionally comprising at least one type of bile canal within at least one layer of cells. In some embodiments of the present invention, the sandwich culture and / or suspension further comprises a longer-term sandwich culture and / or suspension. In some embodiments of the present invention, at least one layer of the cells is sandwiched between two layers of the matrix. In some embodiments of the present invention, the matrix is ​​selected from the group consisting of biological matrix media, synthetic matrix media, co-culture media of supporting cell types, and combinations thereof. In some embodiments of the present invention, the biological matrix medium is selected from the group consisting of collagen, laminin, basement membrane-derived complexes, derivatives thereof, and combinations thereof.

[0011] In some embodiments of the present invention, the culture medium providing the relevant extracellular environment in vivo comprises a medium containing a component at a physiological concentration or a concentration having properties similar to a physiological concentration. In some embodiments of the present invention, this component is selected from the group consisting of albumin; β-lipoprotein; α-1-acid glycoprotein; plasma or serum derived from mouse, rat, rabbit, human, monkey, ape, cattle, dog, piglet, oyster, cattle, bull, sheep, horse, turkey, chicken, fish, duck or goose; bile acids or mixtures of bile acids; bilirubin; and combinations thereof. In some embodiments of the present invention, the method is carried out in at least one well in a multiwell plate. In some embodiments of the present invention, the method further includes a step of simultaneously screening multiple candidate compounds. In some embodiments of the present invention, the culture medium containing physiological concentrations of protein contains other compounds that modulate the properties of the candidate compounds. In some embodiments of the present invention, any combination of these multiple exposure steps may be carried out simultaneously or in any order.

[0012] The object of the present invention is to provide a method for evaluating the disposition and / or effect of a candidate compound in an in vitro culture and / or suspension in order to predict the in vivo disposition and / or effect of the candidate compound. While the objectives of the subject matter disclosed herein are achieved, either whole or in part, other objectives will become apparent as the description progresses in relation to the appended examples best described herein. [Best Mode for Carrying Out the Invention]

[0013] The present invention will be described more fully below, where some, but not all, embodiments of the invention are described. In fact, the invention can be carried out in many different forms, and the invention should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided to satisfy the legal requirements to which this disclosure applies.

[0014] According to some embodiments of the present invention, since plasma proteins and / or other components are generally involved in the binding of drugs, chemicals, and endogenous compounds, methods are provided for adding extracellular components, such as proteins (often referred to as albumin), at physiological or other relevant levels, to approximate the in vivo environment. In particular, a more in vivo-related extracellular environment better defines the in vivo-related intracellular concentrations and compound dynamics (changes over time). Both these intracellular concentrations and dynamics are factors that guide them when evaluating changes in cellular processes and testing cellular behavior. These processes include the uptake of a compound, its efflux (in the case of the liver, in the basolateral and tubules), its intracellular concentration, its metabolism, its induced potential, and its toxicity. Experimental results obtained with this approach have yielded surprising results that cannot be predicted using conventional methods in the art.

[0015] Typically, when performing in vitro experiments using hepatocytes or related cell lines (suspends, plated, sandwich cultures, or other 3D models, Caco-2, MDCK, Opti-Target® (Optivia Biotechnology, Menlo Park, California, USA)), Hureflux® (Hurel Corporation, North Brunswick, New Jersey, USA), and HepaRG® (Biopredic International, Saint Gregoire, France), components such as proteins are either absent or present at non-physiological levels during the experiment. Therefore, only unbound drug concentrations are evaluated in all of these experiments. To apply this to clinical or in vivo situations, separate experiments, such as in vitro protein binding experiments, are performed to determine, for example, the fraction of drugs bound to plasma proteins, and this information is used to estimate parameters obtained from in vitro experiments and apply them to in vivo situations. As a further example, protein binding experiments, such as those using equilibrium dialysis, provide information on the "free" percentage or the percentage of unbound compounds (Fu). This percentage is then applied somewhat blindly to the results of other experiments, and the influence of the "free" compounds is estimated by multiplying these results by this percentage.

[0016] Typically, experiments to evaluate the hepatic uptake and bile excretion of test compounds are performed under conditions where the protein is not present extracellularly. This is often done for experimental simplicity and typicality, and separately determined free percentages are applied to these results.

[0017] According to the present invention, blindly applying a free ratio correction factor to hepatocyte measurements (such as uptake, bile excretion, hepatic bile clearance, or intracellular concentration) is deemed not to provide physiological data to that extent. In fact, in certain cases, the addition of protein is observed to alter (often dramatically) the pharmacokinetics of the uptake of the compound into cells, change the dynamics of the uptake protein, and alter the thermodynamic binding parameters. When the free ratio is determined separately, these parameters are not considered at all, or cannot be considered. Ultimately, there is an assumption that the amount of uptake is proportional to the unbound drug concentration, which can often be incorrect. In fact, it is demonstrated here that the addition of protein does not always have a predictable effect; that is, the observed amount of compound uptake and the intracellular concentration of the compound do not coincide with the amount of uptake predicted in the presence of protein. This observation is unexpected.

[0018] Indeed, as demonstrated and discussed in the examples herein, experimental data surprisingly show that for some compounds, in vivo bile clearance and intrahepatic concentrations under conditions approximating the in vivo environment (such as in the presence of protein) cannot be predicted by adjusting the data using free percentage values ​​obtained from separate studies (two-step methods). Rather, the in vivo-related values ​​of these parameters are determined by performing the evaluation under conditions close to the in vivo environment, such as (but not limited to) in the presence of physiological concentrations of protein. Consequently, estimated or calculated bile clearance and intrahepatic concentrations (as opposed to observed) may, in some cases, be surprisingly and dramatically overestimated or underestimated.

[0019] Accordingly, in some embodiments, an approach is provided using an integrated system that combines the in vivo-associated extracellular environment (e.g., protein and / or other component binding effects) with intracellular disposition and / or effect assessment, such as hepatocyte disposition and / or toxicity.

[0020] In some embodiments, the present invention includes the steps of exposing a candidate compound to the culture and / or suspension, and exposing the culture and / or suspension to a medium that provides a relevant extracellular environment in vivo. This extracellular environment is, for example, a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations having properties (e.g., binding properties) similar to physiological concentrations. Some embodiments of the present invention include simultaneous exposure of the candidate compound and this component in an uptake medium. In fact, the present invention includes any combination of the multiple exposure steps, in any order or simultaneously. In some embodiments, this medium contains a protein and another component that can modulate the properties of the candidate compound. Thus, in some embodiments, the culture and / or suspension can be exposed to a putative inducer or inhibitor in the presence of a medium containing a protein, and then evaluation can be performed in a medium containing the candidate compound but without protein. Furthermore, in some embodiments, this protein is obtained directly from a species of interest consisting of multiple proteins (albumin, α-1-glycoprotein at physiological concentrations, or a mixture at concentrations determined to have binding properties similar to those observed at physiological protein concentrations). Furthermore, in some embodiments, the protein may be a mixture of proteins (albumin, α-1-glycoprotein) at concentrations determined to have binding properties similar to those observed at physiological protein concentrations, or at concentrations similar to those observed at physiological protein concentrations. This may also include serum or plasma obtained directly from the species of interest.

[0021] In some embodiments of the present invention, hepatocyte cultures, such as sandwich cultured hepatocytes, can be used to evaluate the hepatic uptake and bile excretion of compounds of interest, such as drug compounds. Screening of compounds of interest (e.g., therapeutic compositions) is desirable because such compounds are taken up and extensively excreted through the bile excretion process, minimizing their potential to provide therapeutic effects to subjects, as disclosed in U.S. Patent No. 6,780,580, which is incorporated herein by reference in its entirety. Therefore, it is desirable to establish in vivo testing methods for the sensitivity of compounds to hepatocyte uptake and bile excretion so that compounds with undesirable high sensitivity can be easily excluded from further evaluation as therapeutic agents in the initial evaluation process. Accordingly, hepatocyte cultures provide a model for screening compounds of interest for their sensitivity to bile excretion in order to maintain desired functional properties that reflect in vivo hepatocytes. The following U.S. patent publications are also incorporated herein by reference in their entirety: U.S. Patent No. 7,601,494; U.S. Patent No. 7,682,781; U.S. Patent No. 7,604,934; U.S. Patent No. 8,367,630; and the published publication of U.S. Patent Application No. US-2010-0035293-A1.

[0022] As is understood by those skilled in the art, in order to accurately model in vivo biological processes at desired levels, in vitro cultures and / or suspensions of cells (e.g., hepatocytes, but not limited thereto) should be structurally and functionally similar to in vivo cells (e.g., hepatocytes, but not limited thereto). Thus, in some embodiments, in the cultures and / or suspensions of the present invention, structural and functional characteristics exhibited in vivo are established. For example, according to the present invention, transport systems such as sinusoidal capillary or bile ductule transport systems, or both sinusoidal capillary and bile ductule transport systems, are established. In particular, according to the present invention, it is provided to establish at least one bile ductule in a culture and / or suspension of cells (e.g., hepatocytes, but not limited thereto). One culture and / or suspension can include a plurality of bile ductules. The plurality of bile ductules can include a bile ductule network. Establishment of at least one bile duct or bile ductule network enables cultured cells (e.g., hepatocytes, but not limited thereto) to excrete bile and bile components into at least one bile ductule, similar to bile excretion in vivo.

[0023] In addition to the bile ductule transport system, establishment of specific transporters in in vitro liver or liver-related culture fluids is provided. This liver-related culture fluid can include artificial membranes complete for mimicking cells such as vesicles, or cell lines such as HepaRG® cell line, Hurelflux® cell line, Opti-Target® cell line, Caco-2, and MDCK, which are genetically engineered or knocked out (e.g., transporters, P450) for human-specific proteins. system Typical transporters include, but are not limited to, Ntcp, cMoat, Oatp1, Oatp2, Mrp2, Mrp3, Pgp, Bsep, and Mdr2. The expression and function of these liver transporters can be substantially similar to those found in in vivo hepatocytes.

[0024] The establishment of normal metabolic capacity, including the expression and activity of metabolic enzymes in a culture and / or suspension of cells (such as, but not limited to, hepatocytes), is also provided according to the present invention. Thus, this culture can have a metabolic capacity that substantially reflects the metabolism of in vivo cells (such as, but not limited to, hepatocytes). For example, phase I metabolic enzymes such as various P450 isozymes, phase II metabolic enzymes such as UDP-glucuronosyltransferase (UGT), and the normal expression, function, and activity of other enzymes involved in the conjugation of primary bile acids with taurine and glycine in a culture and / or suspension of in vitro cells (such as, but not limited to, hepatocytes) are provided in the present invention.

[0025] Such a method can, in some embodiments, include providing a culture and / or suspension of cells, exposing a candidate compound to this culture and / or suspension, exposing this culture and / or suspension to a medium that provides an in vivo relevant extracellular environment, and determining the amount of the candidate compound incorporated into this culture and / or suspension, thereby evaluating the disposition of this candidate compound to predict the in vivo disposition of this candidate compound. This extracellular environment is, for example, a medium containing components (such as proteins and / or other components, etc.) at physiological concentrations or concentrations having properties (such as binding properties) approximating physiological concentrations. The step of determining the amount of the candidate compound incorporated into the culture and / or suspension and thereby evaluating the disposition can further include determining the intracellular concentration of the candidate compound, determining liver accumulation, determining bile excretion, and / or determining bile clearance.

[0026] In some embodiments, a method for testing the bile excretion sensitivity of a candidate compound is provided. A method for testing the bile efflux sensitivity of such candidate compounds may include the steps of: providing a cell culture and / or suspension (e.g., a cell culture and / or suspension comprising an artificial membrane system adapted to mimic cells and at least one bile canal); exposing a candidate compound to this cell culture and / or suspension; exposing this cell culture and / or suspension to a medium providing a relevant in vivo extracellular environment; and determining the amount of the candidate compound in this at least one bile canal, thereby testing the bile efflux sensitivity of the candidate compound. This extracellular environment may be, for example, a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations having properties (e.g., binding properties) that approximate physiological concentrations.

[0027] In some embodiments, the step of determining the amount of the candidate compound in the at least one bile canal may include the steps of exposing the cell culture and / or suspension to the candidate compound and a pre-selected amount of labeled substrate for the transport protein for a sufficient time to allow simultaneous uptake, washing the cell culture and / or suspension, and detecting the amount of labeled substrate present in the at least one bile canal and evaluating the competition between the candidate compound and the labeled substrate for bile excretion by the transport protein. In some embodiments, a decrease in the amount of labeled substrate in the at least one bile canal compared to a pre-selected amount of the labeled substrate is considered to indicate the sensitivity of the candidate compound to bile excretion by the transport protein. In some embodiments, the amount of the candidate compound in the at least one bile canal may be determined by calculating the bile clearance value of the culture and / or suspension.

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

[0029] When a labeled substrate is used in any of the methods described herein, such labeled substrates may include compounds selected from the group consisting of fluorescence-generating compounds, fluorescent compounds, chemiluminescent compounds, colorimetric compounds, radiolabeled compounds, and combinations thereof.

[0030] In some embodiments, a method is provided for testing the bile efflux sensitivity of a candidate compound. Such a method may include the steps of establishing first and second cell cultures and / or suspensions and at least one bile canal, wherein the first culture and / or suspension has intact bile canals and the second culture and / or suspension has disintegrated bile canals. Such a method may further include the steps of exposing the candidate compound to the first culture and / or suspension and the second culture and / or suspension for a sufficient time to allow uptake of the candidate compound, exposing the first and second cultures and / or suspensions to a medium providing a relevant in vivo extracellular environment, washing and lysing the first and second cultures and / or suspensions, and determining the amount of the candidate compound present in the lysates obtained from each culture and / or suspension, and using the amount of this candidate compound in each culture and / or suspension, evaluating the bile efflux sensitivity of the candidate compound. This extracellular environment is, for example, a culture medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations having properties (e.g., binding properties) that approximate physiological concentrations.

[0031] In some embodiments, each of the first and second cultures and / or suspensions may include an artificial membrane system adapted to mimic cells. Furthermore, in some embodiments, the cultures and / or suspensions of the above method may include an artificial membrane system adapted to mimic cells alone or cells in a co-culture medium with supporting cells such as fibroblasts and / or Kupffer cells. Cells mimicked by the artificial membrane system may be, for example, vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes and / or lung cells. Furthermore, in some embodiments, the cells mimicked by the artificial membrane system may consist of a single cell line. In some embodiments, this cell line is selected from the group consisting of HepaRG® cell lines, Caco-2 and / or MDC.

[0032] In some embodiments, the method using first and second cell cultures and / or suspensions may further include the steps of: exposing the candidate compound to the first and second cultures and / or suspensions for a sufficient time (T) to allow uptake of the candidate compound; exposing the first and second cultures and / or suspensions to a medium providing the relevant in vivo extracellular environment; washing and lysing the first and second fractions of the first and second cultures and / or suspensions; determining the amount of the candidate compound present in the lysates obtained from the first and second cultures and / or suspensions; calculating the amount in the bile canaliculi as the difference in the amount of the candidate compound present in two lysates obtained from a first culture and / or suspension having intact bile canaliculi and a second culture and / or suspension having disintegrated bile canaliculi; and using this calculated amount to evaluate the bile excretion sensitivity of the candidate compound. This extracellular environment is, for example, a culture medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations having properties (e.g., binding properties) that approximate physiological concentrations.

[0033] In some embodiments, methods are provided for evaluating the effects of a candidate compound in an in vitro culture and / or suspension to predict the in vivo effect of the candidate compound. These methods may include the steps of: providing a cell culture and / or suspension; exposing the culture and / or suspension to at least one candidate compound at least once; exposing the culture and / or suspension to a medium that provides a relevant extracellular environment in vivo; and evaluating the effect of exposing the culture and / or suspension to at least one candidate compound to predict the in vivo effect of the candidate compound. This extracellular environment is, for example, a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations having properties approximating physiological concentrations (e.g., binding properties). Effects evaluated in the above methods may include: transmission and other types of studies (metabolism, induction and toxicity); metabolic studies including metabolite identification and metabolic stability (parental lifespan); gene regulation (induction / repression); P450 and transporter drug interactions; intracellular accumulation and release or all (bound + free) intracellular concentrations (e.g., nucleus, mitochondria); and / or toxicological effects.

[0034] In some embodiments, such a method may further include a step of providing a cell culture and / or suspension comprising an artificial membrane system adapted to mimic cells and at least one bile canal. In some embodiments, the cell culture and / or suspension of the above method may include an artificial membrane system adapted to mimic cells in co-culture with supporting cells such as fibroblasts and / or Kupffer cells, or cells alone. Cells mimicked by the artificial membrane system may include, for example, vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, nerve cells, muscle cells, adipocytes, and / or lung cells. In some embodiments, the cells mimicked by the artificial membrane system may consist of, but are not limited to, a single cell line, such as a cell line selected from the group consisting of HepaRG® cell line, Hureflux® cell line, Opti-Target® cell line, Caco-2, and / or MDC. In some embodiments, such cells may be isolated from a source selected from a group consisting of mice, rats, rabbits, humans, monkeys, apes, cats, dogs, piglets, slaughter pigs, cattle, bulls, sheep, horses, turkeys, chickens, fish, ducks, and geese.

[0035] In some embodiments, the culture and / or suspension of this method may include a longer-term culture and / or suspension. This culture and / or suspension may include a bile canal network. This culture and / or suspension may be characterized by having a configuration selected from the group consisting of clusters, aggregates, at least one layer of cells, and combinations thereof. In some further embodiments, these cells may be embedded in a matrix.

[0036] Furthermore, in some embodiments, the culture and / or suspension may further comprise a sandwich culture and / or suspension comprising at least one layer of cells and optionally containing at least one type of bile canaliculi within that layer of cells. The sandwich culture and / or suspension may further comprise a long-term sandwich culture and / or suspension. The at least one layer of cells may be sandwiched between two layers of a matrix, the matrix of which may be selected from the group consisting of biological matrix media, synthetic matrix media, co-culture media of supporting cell types, and combinations thereof. The biological matrix media of which may be selected from the group consisting of collagen, laminin, basement membrane-derived complexes, derivatives thereof, and combinations thereof.

[0037] In any of the methods disclosed herein, the culture and / or suspension can be exposed to multiple candidate compounds. In some embodiments of the methods disclosed herein, the culture and / or suspension can be repeatedly exposed to one or more candidate compounds.

[0038] In the methods disclosed herein, components (such as proteins) that can be added at physiological concentrations or concentrations having properties (e.g., binding properties) approximating physiological concentrations include, but are not limited to, albumin; α-1-acid glycoprotein; β-lipoprotein; bilirubin; bile acids or mixtures of bile acids; and / or plasma or serum derived from representative or desired subjects such as mice, rats, rabbits, humans, monkeys, apes, cats, dogs, piglets, slaughter pigs, cattle, bulls, sheep, horses, turkeys, chickens, fish, ducks, or geese. Furthermore, in some embodiments, a culture medium of the relevant extracellular environment in vivo, such as a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations having properties (e.g., binding properties) approximating physiological concentrations, may contain other compounds that modulate the properties of the candidate compound. Furthermore, a culture medium containing bile acids or mixtures of bile acids; bilirubin; β-lipoprotein; and proteins or protein mixtures may contain other compounds that modulate the properties of the candidate compound. Furthermore, in addition to physiological concentrations, the concentrations of components (e.g., proteins and / or other components) may also be higher or lower, but still produce similar effects. Thus, in some embodiments, non-physiological concentrations that produce similar effects, such as similar binding effects, are also provided.

[0039] In the method disclosed herein, such a method can be carried out in at least one well in a multiwell plate. In such a method, multiple candidate compounds may be screened simultaneously. Furthermore, in the method disclosed herein, any combination of these multiple exposure steps may be carried out simultaneously or in any order.

[0040] Representative calculations that can be used in this invention include the calculation of the bile efflux index (BEI) and the bile clearance value. In some embodiments, the bile efflux index represents the proportion of compounds taken up by hepatocytes that are excreted into the bile. In some embodiments, the bile clearance value indicates the likelihood that a compound will be excreted into the bile and is the factor that best predicts the removal of a compound into the bile in vivo. In some embodiments, the bile excretion index (BEI) is calculated from the uptake and excretion of candidate compounds as follows: Bile excretion index (BEI) = 100% × ((Uptake into intact bile canaliculi in culture) - (Uptake into hepatocytes in calcium (Ca2+)-free medium only)) / (Uptake into intact bile canaliculi in culture) In some embodiments, bile clearance is calculated as follows: Bile clearance = ((Uptake into intact bile canaliculi in culture) - (Uptake into hepatocytes in calcium (Ca2+)-free medium only)) / (Culture time multiplied by the concentration of the candidate compound in the buffer)

[0041] In some embodiments, the bile clearance value can be calculated as the ratio of the volume of bile canaliculi in the culture medium to the area under the curve (AUC), where the area under the curve (AUC) represents the integral of the amount of candidate compound in the culture medium from time 0 to time T (time can be measured in any unit, but is usually measured in "minutes"). In practice, the area under this curve (AUC) can be expressed by the following equation:

number

[0042] The following terms are expected to be well understood by those skilled in the art, but for the sake of facilitating the explanation of the present invention, the following definitions are provided.

[0043] "In vivo relevant extracellular environment" refers to an environment that mimics or approximates the in vivo state, relating to the evaluation of the disposition and / or effects of a candidate compound in vitro according to the present invention. For example, the in vivo relevant extracellular environment can provide components (e.g., proteins and / or other components) at physiological concentrations or concentrations having properties (e.g., binding properties) that approximate physiological concentrations. These binding properties include the degree of binding in addition to the "tension" of protein binding (indicated by Ka (binding constant) or Kd (dissociation constant)).

[0044] The combination of terms “disposition and / or effect” includes, but is not limited to,: uptake clearance; basal efflux clearance; lacrimal canaliculi efflux clearance; metabolic clearance; intracellular concentration; compound kinetics; toxicological effects; metabolite ID and metabolic stability (parental lifespan); gene regulation (induction / repression); P450 and transporter drug interactions; intracellular accumulation and release or total (bound + free) intracellular concentration (e.g., nucleus, mitochondria); and global bile clearance. This combination of terms “disposition and / or effect” also includes pharmacokinetics (PK), which can be broadly defined as (1) the way in which an organism or system acts on the compound of interest, and (2) the clearance (uptake, efflux, metabolism) of all additives in a culture and / or suspension system. In fact, this combination of terms “disposition and / or effect” may include any desired assessment as will be apparent to a person skilled in the art reviewing this disclosure.

[0045] The term "calcium-free buffer" means any buffer that is substantially free of calcium. A non-limiting example of a calcium-free buffer is calcium-free Hank's balanced salt solution (HBSS). As can be understood by those skilled in the art, any suitable buffer that is substantially free of calcium falls within the scope of the present invention. By using a calcium-free buffer, disintegrated bile canaliculi are provided according to several embodiments of the present invention.

[0046] The terms “normal metabolic function,” “normal metabolic activity,” and “desired metabolic characteristics” are used interchangeably herein and mean the activity, function, and / or expression of enzymes involved in metabolic pathways and metabolic reactions in cells (such as, but not limited to, hepatocytes) under normal, basic in vivo conditions.

[0047] The term "functional property" includes any biological property that confers a specific function to an organism, cell, or biochemical reaction that is involved in the biology of that organism. According to the present invention, this functional property may include enzyme activity, enzyme function, enzyme expression, transporter expression and transporter function, as well as regulatory pathways responsible for the expression of enzymes and transporters.

[0048] The terms “compound,” “candidate compound,” “target compound,” or “drug compound” are used interchangeably herein and refer to any compound (either administered exogenously or produced endogenously) for which sensitivity to metabolism, toxicity, hepatic uptake, or bile excretion is desirable. Typical compounds, target compounds, or drug compounds include exogenous biomolecules such as drugs and other therapeutic agents, carcinogens and environmental pollutants, as well as endogenous biomolecules such as steroids, bile acids, fatty acids, and prostaglandins.

[0049] The target compound, which is a therapeutic agent, can be useful in the treatment of warm-blooded vertebrates. Therefore, this invention relates to mammals and birds. This section provides treatments for mammals such as humans, critically endangered mammals (e.g., Siberian tigers), economically important animals (animals raised for human consumption), and / or socially important animals to humans (animals kept in zoos or as pets), such as non-human carnivores (e.g., cats and dogs), pigs (piglets, slaughter pigs, wild boars), ruminants (e.g., cattle, bulls, sheep, giraffes, deer, goats, bison, and camels), and horses. It also provides treatments for birds, including critically endangered birds, zoo birds, and avian, more specifically domesticated birds, i.e., poultry (e.g., turkeys, chickens, ducks, geese, guinea pigs), as they are economically important to humans. Therefore, this section provides treatments for livestock, including, but not limited to, domesticated pigs (piglets and farm pigs), ruminants, horses, and poultry.

[0050] The phrase "evaluate toxicological effects" refers to any appropriate method for quantitatively and / or qualitatively measuring the toxic effects of one or more compounds on cells such as (but not limited to) hepatocytes.

[0051] The term "bile excretion" refers to the biological process by which a substance is removed from a subject's circulatory system by being taken up by hepatocytes and excreted into the bile via bile canals (i.e., uptake and excretion). For example, uptake into hepatocytes is mediated by transport systems intrinsically present in hepatocytes, such as Ntcp, Oatpl, and Oatp2 (but not limited to these). Excretion into bile canals is mediated by efflux transporters, such as Mrp2, Mdr3, Pgp, and Bsep (but not limited to these). Bile canals are structures within liver tissue that receive components excreted from hepatocytes and transport them into the bile ducts via bile to remove them from the subject.

[0052] The method of the present invention may include a step of establishing a sandwich culture of hepatocytes in which at least one hepatocyte layer is formed between two matrix layers. While a sandwich culture-like configuration is preferred for culture, any suitable configuration as will be apparent to those skilled in the art is within the scope of the present invention. For example, clusters, aggregates, or other associations or groups of cells (e.g., hepatocytes, but not limited to them) in cultures and / or suspensions in which at least one bile canal is formed and the functional properties of the cells (e.g., hepatocytes, but not limited to them) are established are within the scope of the present invention. Cells (e.g., hepatocytes, but not limited to them) in co-culture with other cell types, such as Kupffer cells and fibroblasts or other cell types derived from primitive mesenchyme cells, are also within the scope of the present invention. Optionally, the configuration of the culture and / or suspension facilitates the formation of multiple bile canals reflecting in vivo hepatocytes. Also, optionally, the configuration of the culture facilitates the formation of a bile canal network. Furthermore, the composition of this culture optionally facilitates the establishment of cultures of cells (e.g., hepatocytes, but not limited to them) having substantially similar desired metabolic characteristics to in vivo cells (e.g., hepatocytes, but not limited to them). Similarly, the desired transporter expression and function, substantially similar to those of in vivo cells (e.g., hepatocytes, but not limited to them), can optionally be established.

[0053] Furthermore, in sandwich structures, cells (e.g., hepatocytes, but not limited to them) can be cultured in a monolayer or scaffold between two matrix layers. However, these cells (e.g., hepatocytes, but not limited to them) can also be embedded in the matrix, or the matrix can be unevenly extended vertically, horizontally, obliquely, or any combination thereof to form one-dimensional and three-dimensional aggregates. Furthermore, cultures and / or suspensions can be established in bioreactor systems (but not limited to) such as three-dimensional flow-through systems, microenvironments, or three-dimensional scaffolds. See, for example, Griffith and Naughton, (2002) Science 295:1009-1014. Cultures and / or suspensions of cells (e.g., hepatocytes, but not limited to them) can be formed by mixing the cells (e.g., hepatocytes, but not limited to them) with a suitable matrix and inserting this mixture into a suitable culture vessel such as a multiwell plate or culture chamber.

[0054] Collagen is a typical substrate or scaffold for cell cultures and / or suspensions (e.g., hepatocytes, but not limited to these), but any suitable substrate or scaffold, whether natural, synthetic, or a combination thereof, as will be apparent to those skilled in the art, is within the scope of the present invention. For example, other bio-substrates, including basement membranes derived from laminin and biological cell culture substrates, sold under the registered trademark MATRIGEL by Collaborative Biomedical Products, Inc. (Bedford, Massachusetts, USA), are suitable substrate or scaffold materials. Synthetic matrix materials, base materials, or scaffold materials, typically made from various materials such as polymers, also fall within the scope of the present invention. Various component substances having specific matrices for use when culturing cells (e.g., hepatocytes, but not limited to these) are also provided according to the methods of the present invention.

[0055] Any suitable cell source (e.g., hepatocytes, but not limited to those skilled in the art) that would be apparent to those skilled in the art in reviewing this disclosure is also within the scope of the present invention. Exemplary sources include the warm-blooded vertebrates listed above. In particular, exemplary sources include, but are not limited to, humans, rats, mice, monkeys, apes, cattle, dogs, piglets, slaughter pigs, cattle, bulls, sheep, horses, turkeys, chickens, ducks, and geese.

[0056] Cultured cells (e.g., hepatocytes, but not limited to them) can be cultured as “long-term cultures and / or suspensions.” “Long-term cultures and / or suspensions” means cells (e.g., hepatocytes, but not limited to them) that have been cultured for at least about 12 hours. Optionally, “long-term cultures and / or suspensions” means cells (e.g., hepatocytes, but not limited to them) that have been cultured for at least about 24 hours, at least about 48 hours, or at least about 72 hours. Also optionally, “long-term cultures and / or suspensions” means cells (e.g., hepatocytes, but not limited to them) that have been cultured for at least about 96 hours, at least about 1 week, or at least about 28 days. Long-term cultures promote the formation of bile canaliculi and the establishment of functional properties such as metabolic pathways in the cultures and / or suspensions.

[0057] While hepatocyte cultures and / or suspensions are described herein as representative cultures, the present invention provides for culturing or using any cells or cell types of interest, either alone or in any combination of cell types and supporting cells, that can modulate the function of the disclosed cell types. Accordingly, by referring to this disclosure, those skilled in the art can adapt the above descriptions and approaches for use of any desired cell culture and / or suspension. Representative cell cultures and / or suspensions include, but are not limited to, cell cultures and / or suspensions containing cells selected from the group consisting of hepatocytes, renal cells, gastrointestinal cells, pancreatic cells, muscle cells, cardiac cells, nerve cells, and lung cells. According to some embodiments of the present invention, co-cultures of one or more cell types with other cells that provide a supporting matrix (e.g., fibroblasts, but not limited to them) or function (e.g., Kupffer cells, but not limited to them) are provided.

[0058] In accordance with the long-standing patent law treaties, the terms “a” and “an,” when used in this application including claims, mean “one or more.” Unless otherwise indicated, all figures used herein and in the claims, representing quantities of components, reaction conditions, etc., should be understood in all cases to be modified by the term "approximately." Accordingly, unless otherwise indicated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties to be obtained by the present invention. As used herein, the term “about” when referring to a value, or mass, weight, time, volume, concentration, or percentage, means ±20%, ±10%, ±5%, ±1%, ±0.5%, and ±0.1% of a specified amount, in some embodiments, such that the variation is applicable to the practice of the disclosed method.

[0059] As used herein, "and / or" refers to entities that exist individually or in combination when used in a context listing entities. Therefore, for example, "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any one of A, B, C, and D, as well as all combinations and subcombinations thereof. "Comprising" is synonymous with "including," "containing," or "characterized by," but is comprehensive or open-ended and does not exclude additional, unlisted elements and / or steps of method. "Comprising" is a term that means named elements and / or steps exist, but other elements and / or steps added may still fall within the scope of the claim. As used herein, the term "consisting of" means excluding any element, process, or component not specifically listed. When the term "consisting of" is used in the body of a claim without following a preamble, it is limited to the elements described therein, and all other elements are excluded from the claim as a whole. "Consisting essentially of" limits the claims to the specified materials or steps, and further limits any materials or steps that do not substantially affect the fundamental and novel features of the claimed invention. With respect to "comprising," "consisting of," and "consisting essentially of," when one of these three terms is used herein, the present invention may include the use of any of the other two terms.

[0060] As used herein, “significance / strikingness” or “significant / significant” refers to the statistical analysis of the probability that a non-random association exists between two or more entities. To determine whether the relationship is “significant / striking,” statistical manipulation of the data can be performed, and the probability can be calculated and expressed as a “p-value.” These p-values ​​below a user-defined cutoff point are considered “significant / striking.” In some embodiments, p-values ​​less than or equal to 0.05, less than 0.01, less than 0.005, and less than 0.001 are considered “significant / striking.” Therefore, p-values ​​greater than or equal to 0.05 are not considered “significant / striking.” [Examples]

[0061] The results of several exemplary experiments comparing the hepatic bile dispositions of multiple compounds in the absence and presence of different types of proteins are described below. The following examples are given to illustrate representative aspects of the present invention. By referring to this disclosure, those skilled in the art will understand that the following examples are representative only and that many changes, modifications and alterations can be adopted without departing from the spirit and scope of the invention.

[0062] Example 1 Evaluation of the free percentage of selected compounds First, using equilibrium dialysis, we identified several compounds with a certain range of free proportions (also called unbound proportions). This free proportion represents the amount of the compound that is thermodynamically free in solution, compared to the amount that is thought to be thermodynamically bound to the albumin protein. For example, a free proportion of 0.428 indicates that approximately 43% of the compound is unbound and free in solution under these conditions. These values ​​are shown in Table 1.

[0063] [Table 1]

[0064] Example 2 Evaluation of endogenous bile clearance measurements in rat hepatocytes The hepatic uptake, efflux, intracellular concentration, and bile clearance of the nine compounds listed in Table 1 were evaluated in the presence and absence of physiologically regulated protein (4% bovine serum albumin, BSA) and serum obtained from Wistar rats. These data were measured for each compound in sandwich-cultured rat hepatocytes at a concentration of 1 μM and an exposure time of 10 minutes in either a protein-free buffer, a buffer containing 4% BSA, or rat serum. Specifically, the following parameters were determined: total accumulation (reflecting uptake under +Ca conditions); cellular accumulation (reflecting intracellular concentration under -Ca (calcium-free) conditions); bile efflux index (BEI: indicating the proportion of compounds effluxed into bile out of compounds taken up by hepatocytes); and endogenous bile clearance (indicating the likelihood of a compound being effluxed into bile and being the best predictor of compound removal into bile in vivo). The results for endogenous bile clearance, considering both compound uptake and efflux, are shown in Table 2. In summary, these data clearly demonstrate that the addition of extracellular proteins can yield very different results compared to traditional measurements in buffer solutions.

[0065] [Table 2]

[0066] Example 3 Comparison of measured (observed) and predicted (calculated) bile clearance values ​​in rat liver cells. Next, the measured bile clearance of these compounds in rat hepatocytes in the presence of extracellular proteins was compared to the predicted endogenous bile clearance, which was measured in the absence of proteins and adjusted using appropriate free percentage values ​​shown in Table 1. The latter method is the current industry standard. Table 3 summarizes these results and shows the effect of using a one-step integrated system to measure bile clearance compared to a two-step process based on separate experiments. If the two-step process is equivalent to this one-step integrated system, these values ​​should match. However, this was not the case with pitavastatin and rosuvastatin, where the predicted bile clearance was underestimated compared to the measured value, which is thought to be due to unexpected changes in the presence of albumin. Note also that the predicted bile clearance of DPDPE (1 μM) was overestimated compared to the measured value. In this case, the possibility of overestimation or underestimation of clearance, or the fact that these values ​​are not fundamentally equivalent, indicates that the interaction is of an unexpected nature and that it is desirable to use an integrated method when evaluating cellular disposition.

[0067] [Table 3]

[0068] Typically, no significant difference is expected in these bile clearance values ​​(as this value takes into account the degree of protein binding). As can be seen in Table 2, this is true for many of the compounds evaluated, including methotrexate, valsartan, DPDPE, pravastatin, digoxin, and taurocholate. However, for the two compounds pitavastatin and rosuvastatin, the endogenous bile clearance values ​​predicted in the presence of protein are much greater than those observed in the absence of protein. The effects observed in serum are similar, though not identical, suggesting that protein composition also influences the prediction of compound clearance.

[0069] Example 4 Comparison of measured (observed) and predicted (calculated) intracellular concentrations in rat liver cells. The intracellular concentration (ICC) of compounds, which reflects the balance of cellular uptake, metabolism, and efflux, can also be determined in the absence and presence of protein. Using a method similar to that of Example 3 above, a one-step method using protein within the system can be compared with data from a two-step method in which intracellular concentration (ICC) was determined and adjusted using the free percentage values ​​in Table 1. Table 4 shows the results of comparing intracellular concentrations observed using a one-step integrated system for various compounds with predicted values ​​obtained by first conducting the experiment in the absence of protein and then adjusting using protein-binding data from another experiment. If these methods are equivalent, these values ​​should closely match.

[0070] [Table 4]

[0071] For several compounds, the protein-binding information from intracellular concentration data obtained from separate experiments in the absence of protein (ICC predictions) was consistent with the observed intracellular concentrations (ICC measurements). However, for four compounds (valsartan, pravastatin, pitavastatin, and rosuvastatin), the observed intracellular concentrations differed from the predicted values, with the predicted values ​​being either more than 50% overestimated (valsartan) or less than 50% underestimated (pravastatin, pitavastatin, and rosuvastatin). Without measurements using an integrated system, it would be impossible to predict how much of a compound was taken up by cells and the resulting intracellular concentrations.

[0072] For several compounds, these data—namely, in vivo bile clearance and intrahepatic concentrations in the presence of protein (physiological conditions)—cannot be predicted by adjusting the data using free percentage values ​​obtained from separate studies (two-step method). The in vivo related values ​​of these parameters can only be determined by conducting experiments in the presence of physiological concentrations of protein. This effect cannot be predicted based on protein binding of the compounds, as valsartan, pitavastatin, and rosuvastatin all bind to a similar extent to BSA. The effect of protein addition on valsartan's bile clearance could be easily predicted from protein binding data obtained from other studies. However, the protein effect of pitavastatin and rosuvastatin on bile clearance was unexpected and could not be predicted using protein binding data from separate experiments. Accurate prediction of the bile clearance parameters of pitavastatin and rosuvastatin was only obtained by conducting experiments in the presence of physiological concentrations of protein.

[0073] Accurate estimation of bile clearance is beneficial when attempting to predict in vivo clearance in human studies. More accurate predictions of true in vivo clearance can lead to better clinical study designs, a reduced need for human experiments, and shorter clinical development times.

[0074] Intracellular concentrations of a drug are a driving force for any process that occurs within a hepatocyte. Changes in intracellular concentrations can affect all types of interactions that occur within hepatocytes. These may include, but are not limited to, transporter-based drug interactions; the degree of a compound's metabolism; metabolic interactions; the induced potential of the compound (metabolism or transport); and the toxicity produced by the compound or its metabolites.

[0075] Observed effects of proteins on intracellular concentrations can lead to overestimation or underestimation of the compound's effects, potentially dramatically altering expected clinical outcomes. For example, we observed this in the case of telmisartan. While previously considered a potential human cholestasis based on its transporter inhibition profile, data showed that, in the presence of albumin protein, intracellular concentrations in human hepatocytes were higher than predicted values ​​adjusted for a 100-fold free ratio using data from studies conducted in the absence of the protein. However, these intracellular concentrations never reached levels high enough to produce hepatotoxic effects. In vivo data support these conclusions, as telmisartan has no known toxicity associated with its use. Since the effects of proteins are unexpected and unpredictable, it is desirable to expose cells to the protein during experiments.

[0076] A further example involves liver accumulation experiments in and without physiologically charged BSA to understand the differences in in vivo toxicity between two compounds. In this experiment, in vitro toxicity tests (conducted in the absence of the protein) and other pharmacological tests showed that the two closely related compounds had the same potential for in vivo toxicity. However, in vivo tests revealed that the two compounds had significantly different toxicity (liver) profiles in rodents. The researchers were unable to explain this difference based on systemic (blood) exposure. The non-toxic compound (AMG-A) had six times higher Cmax concentrations and area under the curve (AUC) values ​​than the toxic compound (AMG-B). Therefore, the researchers measured intracellular liver concentrations and found that the toxic compound (AMG-B) accumulated more in the liver and had approximately 15 times higher intracellular liver concentrations than AMG-A. This difference in intracellular concentration explained the difference in toxicity (Hamadeh et. al Chem. Res. Toxicol., 2010, 23 (6), pp 1025-1033).

[0077] Example 5 Evaluation of hepatic uptake and intracellular concentrations of non-toxic compounds (AMG-A) and toxic compounds (AMG-B) in the presence and absence of physiologically charged BSA. Experiments to determine the hepatic uptake and intracellular concentrations of AMG-A and AMG-B were conducted in sandwich-cultured rat hepatocytes in the absence and presence of physiologically toxic protein (4% BSA) according to the present invention. Significant differences were observed in hepatic accumulation and intracellular concentrations of the two compounds between the absence and presence of physiologically toxic protein. In experiments conducted in the absence of physiologically toxic protein, intracellular concentrations of the non-toxic compound (AMG-A) at doses of 3 and 10 μM were higher than those of the more toxic compound (AMG-B) (Table 5). In vivo intracellular concentrations were achieved only when experiments were conducted in the presence of physiologically active protein (4% BSA), and the intracellular concentration of the more toxic compound (AMG-B) was significantly higher than that of the non-toxic compound (AMG-A) (Table 6). These results indicate that the presence of physiologically toxic protein is necessary to accurately predict the in vivo effect. Since the binding parameters of the two compounds were the same, the differences in liver accumulation and intracellular concentrations of the two compounds in the presence / absence of protein could not be predicted from the protein binding data.

[0078] [Table 5]

[0079] [Table 6]

[0080] The present invention provides the ability to use proteins to evaluate the effects of hepatic bile disposition and / or compounds to predict in vivo-related bile clearance and intracellular concentration in the liver system. Furthermore, the present invention provides the use of proteins in transmission and other types of studies (metabolism, induction, and toxicity) that can yield more predictive results in vivo. Accordingly, in some embodiments, metabolic studies are provided that include metabolite identification and metabolic stability (parent lifespan) (Kilford et 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 et al. Drug Metab Dispos 41 :1949-1956, November 2013) and free or total (bound + free) intracellular concentration (e.g., nucleus, mitochondria). As will be apparent to those skilled in the art reviewing this disclosure, all of the above can be used to reach in vivo-associated intracellular concentrations of metabolic, inhibitory, induced, modulated, and toxicity-controlling factors.

[0081] For non-hepatic systems (e.g., cell lines such as Caco-2 and MDCK, and organ-specific cell lines of the kidney, gastrointestinal tract, pancreas, heart, neuron, and lung), the present invention provides the use of relevant protein levels to mimic each physiological situation and derive a protected methodology for measuring intracellular concentrations of compounds. Knowledge of transporters and the ability to measure intracellular volume, by using an integrated method, enables the prediction of intracellular concentrations and the disposition and / or effects (e.g., exposure, efflux, etc.) of compounds.

[0082] Example 6 IC of P450 drug-metabolizing enzymes 50 Evaluation of the effect of protein addition on the determination Sandwich cultures (SCH) were prepared using newly isolated or cryopreserved hepatocytes. Newly isolated hepatocytes were placed on 24-well cell culture plates, rinsed with appropriate species-specific culture media (QualGro®) from Qualyst Transporter Solutions (QTS, Durham, North Carolina, USA), and cultured. The cells were then maintained in appropriate species-specific media until consumed in the study.

[0083] Cryopreserved hepatocytes were thawed according to the manufacturer's thawing instructions. Subsequently, the cryopreserved hepatocytes were suspended in QTS hepatocyte medium (QualGro™ culture medium) on 24-well cell culture plates at a density of 0.7–0.8 million viable cells / mL. After plating, the cells were allowed to adhere for 2–4 hours, rinsed, and cultured in warmed (37°C) seeding medium. After 18–24 hours, the cells were supplied with appropriate species-specific QTS suitable medium (QualGro™) supplemented with extracellular matrix (ECM) and Matrigel™ (0.25 mg / mL) and covered. The cells were maintained in QualGro™ hepatocyte culture medium until consumed in the study.

[0084] Cells were cultured as described above until day 6. On day 7, the used culture medium was aspirated and replaced with HBSS culture medium containing or without 4% bovine serum albumin (BSA). A culture medium containing a P450 marker substrate and either fluconazole or ketoconazole was added directly to SCHH to bring the total incubation volume to 0.5 mL. In situ incubation was performed in a cell culture incubator (37°C; 25% CO2; 100% humidity) with shaking at 120 rpm for 20-30 minutes. After incubation, the culture medium was collected and stored at -80°C until use for bioanalysis.

[0085] In situ incubation was analyzed for the detection of P450-mediated metabolite formation from midazolam to hydroxymidazolam and from ibuprofen to hydroxyibuprofen. Briefly, 300 μl of internal standard solution (methanol solution containing 25 nM triazolam and d3-ibuprofen) and 100 μl of HBSS or HBSS (+ 4% BSA) were added to a protein precipitation plate (Millipore MDRPNP4; EDM Millipore, Billerica, Massachusetts, USA) stacked in a 96-deep well block. Before centrifugation, the plate was shaken for 1-2 minutes, and the filtered supernatant was collected. This sample filtrate was evaporated to dryness, and the sample was reconstituted in 200 μL of sample diluent (40 / 60 methanol / 10 mM ammonium acetate) and mixed on a plate shaker for at least 20 minutes. The reconstituted samples were transferred to Millipore 0.45 μm filter plates (Millipore MSHVN45), filtered into Costar 3957 plates by centrifugation before LC-MS / MS analysis, and sealed with silicone cap mats.

[0086] The direct inhibitory effects of fluconazole (CYP2C9) and ketoconazole (CYP3A4), inhibitors of the enzyme activity of CYP2C9 (3-hydroxybutyrophene) and CYP3A4 (hydroxymidazolam), on SCHH were evaluated in the presence and absence of 4% BSA. Fluconazole (CYP2C9) and ketoconazole (CYP3A4) reduced the enzyme activity of CYP2C9 and CYP3A4 to 24.4–45.1% and 32.0–71.1% of the control, respectively. Positive control inhibitors of CYP2C9 and CYP3A4 reduced enzyme activity in a dose-dependent manner, as expected. The difference in the effect of adding protein (4% BSA) to the culture mixture with fluconazole (CYP2C9) was the putative IC of fluconazole (CYP2C9). 50 The estimated IC50 for ketoconazole (CYP3A4) decreased from 56.2 μM to 27.1 μM (Tables 7 and 8), but the estimated IC50 for ketoconazole (CYP3A4) 50However, the concentration increased from 0.0455 μM to 0.117 μM (Tables 9 and 10). This strongly suggests that the protein has a specific effect on the hepatic uptake and intracellular concentration of various compounds (in this case, either the probe inhibitor or the probe substrate midazolam and ibuprofen).

[0087] [Table 7]

[0088] [Table 8]

[0089] [Table 9]

[0090] [Table 10]

[0091] References References cited herein are incorporated herein to the extent that they are cited to supplement, explain, provide background art to, or teach the methods, techniques and / or compositions employed herein.

[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] It will be understood that various details of the invention disclosed herein can be modified without departing from the scope of the invention. Furthermore, the foregoing description is for illustrative purposes only and not intended to limit.

Claims

1. A method for evaluating the disposition of a candidate compound in order to predict the in vivo disposition of the candidate compound, (a) A step of providing a sandwich culture of hepatocytes, wherein the cell culture comprises an artificial membrane system containing cells in which transporters have been genetically modified or knocked out. (b) Exposing the culture to the candidate compound, (c) Simultaneously providing the relevant extracellular environment of hepatocytes in the liver in vivo, and thereby mimicking the in vivo environment of hepatocytes in the liver while evaluating the disposition of the candidate compound in the cell culture, the step of exposing the culture and the candidate compound to a medium containing BSA or serum at a concentration having similar properties to the physiological concentration of the extracellular environment of hepatocytes in the liver in vivo or the physiological concentration of the extracellular environment of hepatocytes in the liver, and (d) Determining the amount of the candidate compound incorporated into the culture and thereby evaluating the disposition of the candidate compound. A method comprising the evaluation of the disposition, wherein the evaluation of the disposition is an evaluation of the intracellular concentration of the candidate compound.

2. The aforementioned "determining the amount of candidate compound incorporated into this culture and thereby evaluating the disposition of this candidate compound" is (e) A step of determining the intracellular concentration of the candidate compound. The method according to claim 1, including the method described in claim 1.

3. A method for evaluating the effect of a candidate compound in order to predict the in vivo effect of the candidate compound, (a) A step of providing a sandwich culture of hepatocytes, wherein the cell culture comprises cells in which transporters have been genetically modified or knocked out, and comprises an artificial membrane system, (b) Exposing the culture to at least one candidate compound at least once, (c) Simultaneously providing the relevant extracellular environment of hepatocytes in the liver, and thereby mimicking the in vivo environment of hepatocytes in the liver while evaluating the disposition of the candidate compound in the cell culture, the step of exposing the culture and the candidate compound to a medium containing BSA or serum at a concentration having similar properties to the physiological concentration of the extracellular environment of hepatocytes in the liver or the physiological concentration of the extracellular environment of hepatocytes in the liver, and (d) A step to evaluate the effect on the culture of exposure to at least one candidate compound. A method that includes this.

4. The method according to claim 3, wherein the artificial membrane system comprises supporting cells and cells in which a transporter in the co-culture medium has been genetically modified or knocked out, and the supporting cells comprise fibroblasts and / or Kupffer cells.

5. The method according to claim 4, wherein the cells included in the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, and liver-derived cells.

6. The method according to claim 4, wherein the cells included in the artificial membrane system include a cell line, and optionally, this cell line is selected from the group consisting of HepaRG® cell line, Caco-2, Opti-Target® cell line, and MDCK.

7. The method according to claim 3, wherein the effect is selected from the group consisting of transmission and other types of studies (metabolism, induction and toxicity); metabolic studies including metabolite identification and metabolic stability (parent lifespan); gene regulation (induction / repression); P450 and transporter drug interactions; intracellular accumulation and release or all (bound + free) intracellular concentrations (e.g., nucleus, mitochondria); and toxicological effects.

8. A method for evaluating the disposition of a candidate compound in order to predict the in vivo disposition of the candidate compound, (a) A step of providing a sandwich culture of hepatocytes, wherein the cell culture comprises an artificial membrane system containing cells in which transporters have been genetically modified or knocked out. (b) Exposing the culture to the candidate compound, (c) The step of simultaneously exposing the culture and candidate compound to a culture medium containing BSA or serum at physiological concentrations to approximate the in vivo environment of hepatocytes in the liver, and (d) Determining the amount of the candidate compound incorporated into the culture and thereby evaluating the disposition of the candidate compound. A method comprising the evaluation of the disposition, wherein the evaluation of the disposition is an evaluation of the intracellular concentration of the candidate compound.

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