Use of protein in prediction of in-vivo effect

Abstract

To provide a method for 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 a culture medium providing an extracellular environment related to in vivo (the culture medium contains a certain component (e.g., protein or the like) at a concentration having characteristics (e.g., binding characteristics) similar to physiological concentration or physiologic concentration), wherein arbitrary combination of any of the "exposing steps" can 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 disclosure of which is incorporated herein by reference in its entirety. [Technical Field]

[0002] In some embodiments, the present invention relates to a method for assessing 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 assessing the disposition and / or effect, including, but not limited to, the combined effect of uptake clearance, basolateral drainage clearance, canalicular outflow clearance, intracellular concentration, biliary clearance, metabolic clearance, and compound kinetics 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. In some embodiments, the method comprises exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment, e.g., a medium containing components such as proteins at physiological concentrations or concentrations with binding characteristics similar to physiological concentrations. [Background technology]

[0003] Typically, in vitro experiments using hepatocytes or related cell lines (suspended, seeded, sandwich-cultured, or other 3D models, such as Caco-2, MDCK, Opti-Target® (Optivia Biotechnology, Menlo Park, California, USA)), and HepaRG® (Biopredic International, Saint-Gregoire, France) have been performed in which either no protein or only non-physiological levels are present during the experiment. Therefore, in all of these experiments, only unbound drug concentrations are assessed. This is often done for experimental simplicity. To translate this to a clinical or in vivo situation, a separate in vitro protein binding experiment is 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. The effect of the "free" compound is then estimated by multiplying the results of other experiments by this percentage in order to somewhat blindly apply it to the results of other experiments. The premise was that such corrective measures would yield reasonably accurate results relevant to clinical or in vivo situations. Summary of the Invention [Problem to be solved by the invention]

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

[0005] In some embodiments, the methods of the present invention comprise providing a culture and / or suspension of cells, 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 with binding characteristics similar to physiological concentrations), and determining the amount of candidate compound taken up by the culture and / or suspension, thereby assessing 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 comprises an artificial membrane system adapted to mimic cells. In some embodiments, the artificial membrane system mimics cells in co-culture with feeder cells, the feeder cells comprising 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, neuronal cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells mimicked by the artificial membrane system comprise a cell line, optionally selected from the group consisting of the HepaRG® cell line, Caco-2, and MDC. In some embodiments, the step of "determining the amount of candidate compound taken up by the culture and / or suspension, thereby assessing disposition" comprises determining the intracellular concentration, liver accumulation, biliary excretion, and / or biliary clearance of the candidate compound.

[0007] In some embodiments, a method for testing the susceptibility of a candidate compound to bile excretion is provided, comprising providing a culture and / or suspension comprising an artificial membrane system adapted to mimic a cell and at least one bile canaliculus, exposing a candidate compound to the culture and / or suspension, exposing the culture and / or suspension to a medium providing an in vivo relevant extracellular environment, and determining the amount of the candidate compound in the at least one bile canaliculus, thereby testing the susceptibility of the candidate compound to bile excretion. In some embodiments, determining the amount of the candidate compound in at least one bile canaliculus comprises simultaneously exposing the candidate compound and a preselected amount of a labeled substrate for the transport protein to the cell culture and / or suspension for a time sufficient to allow uptake, washing the cell culture and / or suspension, and detecting the amount of labeled substrate present in the at least one bile canaliculus to assess competition between the candidate compound and the labeled substrate for biliary excretion by the transport protein, wherein the presence of a reduced amount of the labeled substrate in the at least one bile canaliculus compared to the preselected amount of the labeled substrate indicates susceptibility of the candidate compound to biliary excretion by the transport protein. In some embodiments, the artificial membrane system mimics cells in co-culture with feeder cells, the feeder cells comprising 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, neuronal cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells mimicked by the artificial membrane system comprise a cell line, optionally selected from the group consisting of HepaRG® cell line, Caco-2, and MDC. In some embodiments, the labeled substrate comprises a compound selected from the group consisting of a fluorogenic compound, a fluorescent compound, a chemiluminescent compound, a colorimetric compound, a radiolabeled compound, and combinations thereof.In some embodiments, the amount of the candidate compound in the at least one bile canaliculus is determined by calculating the bile clearance value of the culture and / or suspension.

[0008] In some embodiments, a method for testing the bile excretion sensitivity of a candidate compound is provided, the method comprising: (a) establishing first and second cell cultures and / or suspensions, each comprising an artificial membrane system adapted to mimic a cell and at least one bile canaliculus, the first culture and / or suspension having intact bile canaliculi and the second culture and / or suspension having collapsed bile canaliculi; (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 that provides an in vivo relevant extracellular environment; (d) washing and lysing the first and second cultures and / or suspensions; and (e) detecting and detecting the bile excretion sensitivity of the candidate compound. determining the amount of the candidate compound present in the lysate obtained from each culture and / or suspension of step (d), and using this amount of the candidate compound in each culture and / or suspension to assess the susceptibility of the candidate compound to bile excretion. In some embodiments, the method comprises the steps of: (i) exposing a candidate compound to the first and second cultures and / or suspensions for a time (T) sufficient to allow uptake of the candidate compound; (ii) exposing the first and second cultures and / or suspensions to a medium that provides an in vivo relevant extracellular environment; (iii) washing and lysing each of the first and second fractions of the first and second cultures and / or suspensions; (iv) determining the amount of candidate compound present in a lysate obtained from each of the first and second cultures and / or suspensions of step (iii); (v) calculating the amount in the bile canaliculi as the difference between the amount of candidate compound present in two lysates obtained from the first culture and / or suspension having intact bile canaliculi and the second culture and / or suspension having disrupted bile canaliculi; and (vi) assessing the susceptibility of the candidate compound to bile excretion using the amount calculated in step (iv). In some embodiments, the artificial membrane system mimics cells in co-culture with feeder cells, where the feeder cells include 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, cardiomyocytes, neurons, muscle cells, adipocytes, and lung cells. In some embodiments, the cells mimicked by the artificial membrane system are a cell line, optionally selected from the group consisting of the HepaRG® cell line, Caco-2, and MDC.

[0009] In some embodiments, methods are 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 methods comprise providing a culture and / or suspension of cells, exposing the culture and / or suspension at least once to at least one candidate compound, exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment, 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 methods comprise providing a culture and / or suspension comprising an artificial membrane system adapted to mimic cells and at least one bile canaliculus. In some embodiments, the artificial membrane system mimics cells in co-culture with feeder cells, the feeder cells comprising 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, neuronal cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells mimicked by the artificial membrane system comprise a cell line, optionally selected from the group consisting of HepaRG® cell line, Caco-2, and MDC. In some embodiments, the effect is selected from the group consisting of transduction and other types of studies (metabolism, induction, and toxicity); metabolic studies, including metabolite identification and metabolic stability (parental longevity); gene regulation (induction / repression); P450 and transporter-drug interactions; intracellular accumulation and free or total (bound + free) intracellular concentrations (e.g., nuclear, mitochondrial); 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 mouse, rat, rabbit, human, monkey, ape, cat, dog, piglet, hog, cow, ox, sheep, horse, turkey, chicken, fish, duck, and goose. In some embodiments of the present invention, the culture and / or suspension further comprises a long-term culture and / or suspension. In some embodiments of the present invention, the culture and / or suspension comprises a bile canalicular 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, at least one bile canalicular network within the at least one layer of cells. In some embodiments of the invention, the sandwich culture and / or suspension further comprises a long-term sandwich culture and / or suspension. In some embodiments of the invention, the at least one layer of cells is sandwiched between two layers of matrix. In some embodiments of the invention, the matrix is ​​selected from the group consisting of a biological matrix medium, a synthetic matrix medium, a co-culture medium of a feeder cell type, and combinations thereof. In some embodiments of the invention, the biological matrix medium is selected from the group consisting of collagen, laminin, complexes derived from basement membranes, derivatives thereof, and combinations thereof.

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

[0012] It is an object of the present invention to provide a method for assessing the disposition and / or effect of a candidate compound in in vitro culture and / or suspension in order to predict the in vivo disposition and / or effect of the candidate compound. The objects of the presently disclosed subject matter are achieved in whole or in part by the presently disclosed subject matter, and other objects will become apparent as the description proceeds in connection with the accompanying examples best described herein below. BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The present invention will now be described more fully hereinafter. Some, but not all, embodiments of the invention will be described therein. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0014] In some embodiments of the present invention, plasma proteins and / or other components are generally involved in the binding of drugs, chemicals, and endogenous compounds, so methods are provided for adding extracellular components, such as proteins (often referred to as albumin), at physiological or other relevant levels to mimic the in vivo environment. In particular, a more in vivo-relevant extracellular environment defines in vivo-relevant intracellular concentrations and compound kinetics (time courses). Both these intracellular concentrations and kinetics are factors that guide the assessment of changes in cellular processes and the testing of cellular mechanisms. These processes include compound uptake, compound efflux (basolateral and canalicular in the case of the liver), intracellular concentration of the compound, compound metabolism, induction potential of the compound, and compound toxicity. Experimental results obtained with this approach have yielded surprising results that could not be predicted using conventional methods in the art.

[0015] Typically, when performing in vitro experiments using hepatocytes or related cell lines (suspension, plated, sandwich culture, or other 3D models, such as Caco-2, MDCK, cell lines available under the trademarks Opti-Target® (Optivia Biotechnology, Menlo Park, California, USA)), Hurellux® (Hurel Corporation, North Brunswick, New Jersey, USA), and HepaRG® (Biopredic International, Saint-Gregoire, France), components such as proteins are absent or present at non-physiological levels during the experiments. Therefore, in all of these experiments, only the unbound drug concentration is assessed. 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 drug bound to plasma proteins, and this information is used to extrapolate the parameters obtained from the in vitro experiments to the in vivo situation. As a further example, protein binding experiments, such as those using equilibrium dialysis, provide information on the "free fraction" or proportion of unbound compound (Fu), which can then be applied somewhat blindly to the results of other experiments to estimate the effect of the "free" compound by multiplying the results of the other experiments by this fraction.

[0016] Typically, experiments to assess hepatic uptake and biliary excretion of test compounds are performed in the absence of extracellular protein, which is often done for reasons of experimental simplicity and representativeness, and separately determined fractional release is applied to these results.

[0017] According to the present invention, it is recognized that blind application of free fraction correction factors to hepatocyte measurements (such as uptake, biliary excretion, hepatobiliary clearance, or intracellular concentration) does not provide physiological data to that extent. Indeed, in certain instances, it has been observed that the addition of protein alters (often dramatically) the pharmacokinetics of compound uptake into cells, modifies the kinetics of uptake proteins, and changes thermodynamic binding parameters. When determining the free fraction separately, these parameters are not or cannot be taken into account at all. Finally, 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 herein that the addition of protein does not always have the expected effect, i.e., the observed amount of compound uptake and the intracellular concentration of the compound do not match 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 biliary clearance and hepatic concentrations under conditions approximating the in vivo environment (such as in the presence of protein) cannot be predicted by adjusting the data using free fraction values ​​obtained from separate studies (two-stage method). Rather, assessments are performed under conditions approximating the in vivo environment, such as, but not limited to, in the presence of physiological concentrations of protein, to determine in vivo-relevant values ​​for these parameters. Thus, as shown herein, estimated or calculated (as opposed to observed) biliary clearance and hepatic concentrations can, in some cases, be surprisingly dramatically over- or under-predicted.

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

[0020] In some embodiments, the present invention includes exposing the culture and / or suspension to a candidate compound and exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment. This extracellular environment can be, for example, a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations with properties (e.g., binding properties) that approximate physiological concentrations. Some embodiments of the present invention include simultaneous exposure of the candidate compound and the components in an uptake medium. Indeed, the present invention includes any combination of multiple exposure steps, in any order or simultaneously. In some embodiments, the 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 the protein, followed by evaluation in a medium containing the candidate compound but without the protein. Furthermore, in some embodiments, the protein can be obtained directly from the species of interest, such as albumin, alpha-1-acid glycoprotein at physiological concentrations, or a mixture of proteins at concentrations determined to have binding properties similar to those observed at physiological protein concentrations. Additionally, in some embodiments, the protein may be a mixture of proteins (albumin, alpha-1-acid glycoprotein) at physiological concentrations or at concentrations determined to have binding properties similar to those observed at physiological protein concentrations, including 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 biliary excretion of compounds of interest, such as drug compounds. As disclosed in U.S. Patent No. 6,780,580, the entire contents of which are incorporated herein by reference, screening compounds of interest (e.g., therapeutic compositions) is desirable because such compounds are taken up and excreted extensively through the biliary excretion process, minimizing the likelihood that they will have a therapeutic effect on the subject. Therefore, in the early evaluation process, it is desirable to establish an in vivo test method for the susceptibility of compounds to hepatocyte uptake and biliary excretion so that compounds with undesirably high susceptibility can be easily eliminated from further evaluation as therapeutic agents. Thus, hepatocyte cultures provide a model for screening compounds of interest for susceptibility to biliary excretion because they maintain desired functional properties that reflect in vivo hepatocytes. The following U.S. patent publications are also incorporated by reference herein in their entireties: 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 publication of U.S. Patent Application No. US-2010-0035293-A1.

[0022] As will be appreciated by those skilled in the art, in order to accurately model in vivo biological processes at a desired level, in vitro cultures and / or suspensions of cells (e.g., but not limited to, hepatocytes) should be structurally and functionally similar to in vivo cells (e.g., but not limited to, hepatocytes). Thus, in some embodiments, the cultures and / or suspensions of the present invention establish structural and functional characteristics that are exhibited in vivo. For example, the present invention establishes a transport system, such as a sinusoidal or bile canalicular transport system, or both a sinusoidal and bile canalicular transport system. In particular, the present invention provides for establishing at least one bile canalicular structure in a culture and / or suspension of cells (e.g., but not limited to, hepatocytes). A single culture and / or suspension may include multiple bile canaliculi. The multiple bile canaliculi may comprise a bile canalicular network. Establishment of at least one bile duct or bile canaliculus network allows cultured cells (e.g., but not limited to, hepatocytes) to excrete bile and bile components into at least one bile canaliculus, similar to bile excretion in vivo.

[0023] In addition to the bile canalicular transport system, specific transporters can be established in in vitro liver or liver-associated cultures. These liver-associated cultures can include complete artificial membrane systems to mimic cells such as vesicles, or cell lines transfected or knocked out for human-specific proteins (e.g., transporters, P450s), such as HepaRG®, Hurellux®, Opti-Target®, Caco-2, and MDCK. Exemplary transporters include, but are not limited to, Ntcp, cMoat, Oatp1, Oatp2, Mrp2, Mrp3, Pgp, Bsep, and Mdr2. The expression and function of these hepatic transporters can be substantially similar to that found in in vivo hepatocytes.

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

[0025] In some embodiments, such methods can include 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 an in vivo relevant extracellular environment, and determining the amount of candidate compound taken up in the culture and / or suspension, thereby assessing the disposition of the candidate compound and predicting the in vivo disposition of the candidate compound. The extracellular environment can be, for example, a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations with properties (e.g., binding properties) that approximate physiological concentrations. Determining the amount of candidate compound taken up in the culture and / or suspension, thereby assessing disposition, can further include determining the intracellular concentration, liver accumulation, biliary excretion, and / or biliary clearance of the candidate compound.

[0026] In some embodiments, a method for testing the bile excretion sensitivity of a candidate compound is provided. Such a method for testing the bile excretion sensitivity of a candidate compound can include 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 canaliculus), exposing a candidate compound to the cell culture and / or suspension, exposing the cell culture and / or suspension to a medium that provides an in vivo relevant extracellular environment, and determining the amount of the candidate compound in the at least one bile canaliculus, thereby testing the bile excretion sensitivity of the candidate compound. This extracellular environment can be, for example, a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations with properties (e.g., binding properties) that approximate physiological concentrations.

[0027] In some embodiments, determining the amount of the candidate compound in the at least one bile canaliculus can include simultaneously exposing the candidate compound and a preselected amount of a labeled substrate for the transport protein to the cell culture and / or suspension for a time sufficient to allow uptake, washing the cell culture and / or suspension, and detecting the amount of labeled substrate present in the at least one bile canaliculus to assess competition between the candidate compound and the labeled substrate for biliary excretion by the transport protein. In some embodiments, a decrease in the amount of the labeled substrate in the at least one bile canaliculus compared to the preselected amount of the labeled substrate indicates susceptibility of the candidate compound to biliary excretion by the transport protein. In some embodiments, the amount of the candidate compound in the at least one bile canaliculus is determined by calculating a bile clearance value for the culture and / or suspension.

[0028] Additionally, in some embodiments, the cell culture and / or suspension in the above methods may comprise an artificial membrane system adapted to mimic cells alone or in co-culture with support 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, neuronal cells, muscle cells, adipocytes, and / or lung cells. Additionally, in some embodiments, the cells mimicked by the artificial membrane system may comprise a cell line. In some embodiments, the cell line is selected from the group consisting of HepaRG® cell line, Hurellux® 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 substrate can comprise a compound selected from the group consisting of a fluorogenic compound, a fluorescent compound, a chemiluminescent compound, a colorimetric compound, a radiolabeled compound, and combinations thereof.

[0030] In some embodiments, a method for testing the bile excretion sensitivity of a candidate compound is provided. Such a method can include establishing first and second cell cultures and / or suspensions and at least one bile canaliculus, where the first culture and / or suspension has intact bile canaliculi and the second culture and / or suspension has disrupted bile canaliculi. Such a method can further include 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, exposing the first and second cultures and / or suspensions to a medium that provides an in vivo relevant extracellular environment, washing and lysing the first and second cultures and / or suspensions, and determining the amount of candidate compound present in the lysate obtained from each culture and / or suspension, and using the amount of candidate compound in each culture and / or suspension to assess the bile excretion sensitivity of the candidate compound. The extracellular environment can be, for example, a culture medium containing components (eg, proteins and / or other components) at physiological concentrations or concentrations with properties (eg, binding characteristics) that approximate physiological concentrations.

[0031] In some embodiments, each of the first and second cultures and / or suspensions may comprise an artificial membrane system adapted to mimic cells. Furthermore, in some embodiments, the cultures and / or suspensions of the above methods may comprise an artificial membrane system adapted to mimic cells alone or in co-culture with support 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, neural cells, muscle cells, adipocytes, and / or lung cells. Furthermore, in some embodiments, the cells mimicked by the artificial membrane system may comprise a cell line. In some embodiments, the cell line is selected from the group consisting of the HepaRG® cell line, Caco-2, and / or MDC.

[0032] In some embodiments, this method using first and second cell cultures and / or suspensions may further include 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; exposing the first and second cultures and / or suspensions to a medium that provides an in vivo relevant extracellular environment; washing and lysing each of the first and second fractions of the first and second cultures and / or suspensions; determining the amount of candidate compound present in a lysate obtained from each of the first and second cultures and / or suspensions; calculating the amount in the bile canaliculi as the difference between the amount of candidate compound present in two lysates obtained from the first culture and / or suspension having intact bile canaliculi and the second culture and / or suspension having disrupted bile canaliculi; and using this calculated amount to assess the susceptibility of the candidate compound to bile excretion. The extracellular environment can be, for example, a culture medium containing components (eg, proteins and / or other components) at physiological concentrations or concentrations with properties (eg, binding characteristics) that approximate physiological concentrations.

[0033] In some embodiments, methods are provided for evaluating the effects of candidate compounds in in vitro cultures and / or suspensions to predict their in vivo effects. These methods may include providing a cell culture and / or suspension, exposing the culture and / or suspension at least once to at least one candidate compound, 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 effects 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 with properties (e.g., binding characteristics) that approximate physiological concentrations. Effects evaluated in these methods may include transduction 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 free or total (bound + free) intracellular concentrations (e.g., nuclear, mitochondrial); and / or toxicological effects.

[0034] In some embodiments, such methods further include providing an artificial membrane system adapted to mimic cells and a cell culture and / or suspension comprising at least one bile canaliculus. In some embodiments, the cell culture and / or suspension of the above methods can include an artificial membrane system adapted to mimic cells in co-culture with support cells, e.g., fibroblasts and / or Kupffer cells, or single cells. Cells mimicked by the artificial membrane system can include, for example, vesicles, hepatocytes, liver-derived cells, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neural cells, muscle cells, adipocytes, and / or lung cells. In some embodiments, the cells mimicked by the artificial membrane system can comprise a cell line, such as, but not limited to, a cell line selected from the group consisting of HepaRG® cell line, Hurellux® cell line, Opti-Target® cell line, Caco-2, and / or MDC. In some embodiments, such cells may be isolated from a source selected from the group consisting of mouse, rat, rabbit, human, monkey, ape, cat, dog, piglet, hog, cow, ox, sheep, horse, turkey, chicken, fish, duck, and goose.

[0035] In some embodiments, the culture and / or suspension of this method can further comprise a long-term culture and / or suspension. The culture and / or suspension can comprise a bile canalicular network. The culture and / or suspension can be characterized as having an organization selected from the group consisting of clusters, aggregates, at least one layer of cells, and combinations thereof. Additionally, in some embodiments, the cells can be embedded in a matrix.

[0036] Furthermore, in some embodiments, the culture and / or suspension can further comprise a sandwich culture and / or suspension, the sandwich culture and / or suspension comprising at least one layer of cells and, optionally, at least one bile canaliculus within the at least one layer of cells. The sandwich culture and / or suspension can further comprise a long-term sandwich culture and / or suspension. The at least one layer of cells can be sandwiched between two layers of matrix, which can be selected from the group consisting of a biological matrix medium, a synthetic matrix medium, a co-culture medium of a feeder cell type, and combinations thereof. The biological matrix medium can be selected from the group consisting of collagen, laminin, complexes derived from basement membranes, 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 (e.g., proteins) that can be added at physiological concentrations or concentrations with properties (e.g., binding properties) that approximate physiological concentrations include, but are not limited to, albumin; α-1-acid glycoprotein; β-lipoprotein; bilirubin; bile acid or a mixture of bile acids; and / or plasma or serum from a representative or desired subject, such as a mouse, rat, rabbit, human, monkey, ape, cat, dog, piglet, hog, cow, ox, sheep, horse, turkey, chicken, fish, duck, or goose. Furthermore, in some embodiments, a medium representing an in vivo relevant extracellular environment, such as a medium containing components (e.g., proteins and / or other components) at physiological concentrations or concentrations with properties (e.g., binding properties) that approximate physiological concentrations, can contain other compounds that modulate the properties of the candidate compound. Furthermore, a medium containing a bile acid or a mixture of bile acids; bilirubin; β-lipoprotein; and a protein or protein mixture can contain other compounds that modulate the properties of the candidate compound. Furthermore, in addition to physiological concentrations, concentrations of components (e.g., proteins and / or other components) can also be higher or lower, but have a similar effect. Thus, in some embodiments, non-physiological concentrations that have a similar effect, such as a similar binding effect, are also provided.

[0039] In the methods disclosed herein, such methods can be performed in at least one well of a multi-well plate. In such methods, multiple candidate compounds can be screened simultaneously. Furthermore, in the methods disclosed herein, any combination of these multiple exposing steps can be performed simultaneously or in any order.

[0040] Exemplary calculations that can be used in the present invention include the calculation of the biliary excretion index (BEI) and biliary clearance value. In some embodiments, the biliary excretion index indicates the proportion of a compound that is excreted in bile relative to the compound taken up by hepatocytes. In some embodiments, the biliary clearance value indicates the likelihood that a compound will be excreted in bile and is the best predictor of biliary elimination of a compound in vivo. In some embodiments, the biliary excretion index (BEI) is calculated from the uptake and excretion of the candidate compound as follows: Bile Efflux 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, biliary clearance is calculated as follows: Biliary clearance = ((uptake into intact bile canaliculi in culture) - (uptake into hepatocytes in calcium (Ca2+)-free medium only)) / (incubation time multiplied by the concentration of candidate compound in buffer)

[0041] In some embodiments, the bile clearance value can be calculated as the ratio of the amount of bile canaliculi in the culture medium to the area under the curve (AUC), which 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, this area under the curve (AUC) can be expressed as:

number

[0042] Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are provided to facilitate description of the present invention.

[0043] An "in vivo relevant extracellular environment" refers to an environment that mimics or approximates in vivo conditions relevant to evaluating the disposition and / or effect of a candidate compound in vitro in accordance with the present invention. For example, an 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. The binding properties include the degree of binding as well as the "tightness" of protein binding (as indicated by K (association constant) or K (dissociation constant)).

[0044] The term "disposition and / or effect" includes, but is not limited to, uptake clearance; basolateral outflow clearance; canalicular outflow clearance; metabolic clearance; intracellular concentration; compound kinetics; toxicological effects; metabolite ID and metabolic stability (parent 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" also includes pharmacokinetics (PK), which can be broadly defined as (1) how an organism or system interacts with a compound of interest, and (2) the clearance (uptake, efflux, metabolism) of all additives in culture and / or suspension systems. Indeed, this term "disposition and / or effect" can include any desired evaluation, as would be apparent to one of ordinary skill in the art upon review of this disclosure.

[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 Hank's balanced salt solution (HBSS). As will 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. The use of a calcium-free buffer provides collapsed bile canaliculi according to some embodiments of the present invention.

[0046] The terms "normal metabolic function," "normal metabolic activity," and "desirable metabolic characteristics" are used interchangeably herein and refer to 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 basal conditions in vivo.

[0047] The term "functional property" includes any biological property that confers a specific function involved in the biology of an organism, a cell, or a biochemical reaction. According to the present invention, this functional property may include enzyme activity, enzyme function, enzyme expression, transporter expression and function, and the 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 (exogenously administered or endogenously produced) whose susceptibility to metabolism, toxicity, hepatic uptake, or biliary excretion is desirable. Exemplary compounds, target compounds, or drug compounds include exogenous biological substances such as drugs and other therapeutic agents, carcinogens and environmental pollutants, and endogenous biological substances such as steroids, bile acids, fatty acids, and prostaglandins.

[0049] The subject compounds that are therapeutic agents may be useful in the treatment of warm-blooded vertebrates, and thus the present invention relates to mammals and birds. Provided herein are treatments for mammals such as humans, endangered and therefore important 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), e.g., non-human carnivores (e.g., cats and dogs), pigs (pigs, hogs, wild boars), ruminants (e.g., cows, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also provided herein are treatments for birds, including endangered and zoo-raised birds and poultry, more particularly domesticated birds, i.e., poultry (e.g., turkeys, chickens, ducks, geese, guinea pigs, etc.), because of their economic importance to humans. Accordingly, provided herein are treatments for livestock, including, but not limited to, domesticated pigs (pigs and domestic pigs), ruminants, horses, poultry, etc.

[0050] The phrase "assessing toxicological effects" refers to any suitable method for quantitatively and / or qualitatively measuring one or more toxic effects of a compound on cells, such as, but not limited to, hepatocytes.

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

[0052] The methods of the present invention can include establishing a sandwich culture of hepatocytes, in which at least one layer of hepatocytes is formed between two matrix layers. While sandwich-like configurations are preferred for culture, any suitable configuration, as would be apparent to one skilled in the art, is within the scope of the present invention. For example, clusters, aggregates, or other associations or populations of cells (e.g., but not limited to, hepatocytes) in culture and / or suspension, in which at least one bile canaliculus is formed and functional characteristics of the cells (e.g., but not limited to, hepatocytes) are established, are within the scope of the present invention. Cells (e.g., but not limited to, hepatocytes) in co-culture with other cell types, such as Kupffer cells and fibroblasts or other cell types derived from primitive mesenchyme, are also within the scope of the present invention. Optionally, the culture and / or suspension configuration facilitates the formation of multiple bile canaliculi, which mirror hepatocytes in vivo. Optionally, the culture configuration also facilitates the formation of a bile canalicular network. Furthermore, this culture configuration optionally facilitates the establishment of cultures of cells (e.g., but not limited to, hepatocytes) that have desired metabolic properties substantially similar to those of in vivo cells (e.g., but not limited to, hepatocytes), as well as the establishment of desired transporter expression and function substantially similar to those of in vivo cells (e.g., but not limited to, hepatocytes).

[0053] Furthermore, in a sandwich structure, cells (e.g., but not limited to, hepatocytes) can be cultured in a monolayer or scaffold between two matrix layers. However, the cells (e.g., but not limited to, hepatocytes) can also be embedded in the matrix, or the matrix can be stretched unevenly vertically, horizontally, diagonally, or any combination thereof to form one- and three-dimensional aggregates. Furthermore, the culture and / or suspension can be established in a bioreactor system, such as, but not limited to, a three-dimensional flow-through system, a microenvironment, or a three-dimensional scaffold. See, for example, Griffith and Naughton (2002) Science 295:1009-1014. The culture and / or suspension of cells (e.g., but not limited to, hepatocytes) can be formed by mixing the cells (e.g., but not limited to, hepatocytes) with an appropriate matrix and inserting the mixture into an appropriate culture vessel, such as a multi-well plate or culture chamber.

[0054] While collagen is an exemplary substrate or scaffold for cell (e.g., but not limited to, hepatocyte) cultures and / or suspensions, any suitable substrate or scaffold, whether natural, synthetic, or a combination thereof, as would be apparent to one of skill in the art, is within the scope of the present invention. For example, other suitable substrate or scaffold materials include basement membranes derived from laminin and biological cell culture substrates sold under the registered trademark MATRIGEL by Collaborative Biomedical Products, Inc. (Bedford, Massachusetts, USA). Synthetic matrix, substrate, or scaffold materials, typically made from a variety of materials such as polymers, are also within the scope of the present invention. Various component materials with specific matrices for use in culturing cells (e.g., but not limited to, hepatocytes) are also provided in accordance with the methods of the present invention.

[0055] Any suitable cell source (e.g., but not limited to, hepatocytes) as would be apparent to one of skill in the art upon review of this disclosure is 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, cats, dogs, piglets, hogs, cows, oxen, sheep, horses, turkeys, chickens, ducks, and geese.

[0056] Cultured cells (e.g., but not limited to, hepatocytes) can be cultured as "long-term cultures and / or suspensions." "Long-term cultures and / or suspensions" refer to cells (e.g., but not limited to, hepatocytes) that have been cultured for at least about 12 hours. Optionally, "long-term cultures and / or suspensions" refer to cells (e.g., but not limited to, hepatocytes) that have been cultured for at least about 24 hours, at least about 48 hours, or at least about 72 hours. Optionally, "long-term cultures and / or suspensions" refer to cells (e.g., but not limited to, hepatocytes) 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 liver cell cultures and / or suspensions are described herein as exemplary cultures, the present invention provides for the culture or use of any cell or cell type of interest, alone or in any combination of cell types and support cells, that can modulate the function of the disclosed cell types. Thus, with reference to this disclosure, one of skill in the art can adapt the above descriptions and approaches for use with any desired cell culture and / or suspension. Exemplary cell cultures and / or suspensions include, but are not limited to, cell cultures and / or suspensions comprising cells selected from the group consisting of hepatocytes, kidney cells, gastrointestinal cells, pancreatic cells, muscle cells, cardiac cells, neural cells, and lung cells. According to some embodiments of the present invention, co-cultures of single or multiple cell types with other cells that provide a support matrix (e.g., but are not limited to, fibroblasts) or function (e.g., but are not limited to, Kupffer cells) are provided.

[0058] Following long-standing patent law convention, the terms "a" and "an" mean "one or more" when used in this application, including the claims. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. As used herein, the term "about" when referring to a value, or to a mass, weight, time, volume, concentration, or percentage, is in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% of the specified amount, such variation being applicable to the practice of the disclosed methods.

[0059] As used herein, "and / or," when used in the context of listing entities, refers to those entities present either alone or in combination. Thus, for example, "A, B, C, and / or D" includes A, B, C, and D individually, but also any and all combinations and subcombinations of A, B, C, and D. "Comprising" is synonymous with "including," "containing," or "characterized by," but is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a term that implies the presence of the named elements and / or steps, but other elements and / or steps could be added and still fall within the scope of the claim. As used herein, the term "consisting of" means excluding any element, step, or ingredient not specifically recited. When the term "consisting of" appears in the body of a claim without following a preamble, it limits the claim to only those elements recited therein and excludes other elements from the entire claim. "Consisting essentially of" limits the claim to the specified materials or steps and further limits materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter. With respect to "comprising," "consisting of," and "consisting essentially of," when one of these three terms is used herein, the invention can include the use of either of the other two terms.

[0060] As used herein, "significance" or "significant" refers to a statistical analysis of the probability that a non-random association exists between two or more entities. To determine whether the relationship is "significant," statistical manipulations of the data are performed and a probability can be calculated and expressed as a "p-value." Those p-values ​​that fall below a user-defined cutoff point are considered "significant." In some embodiments, a p-value of 0.05 or less, in some embodiments less than 0.01, in some embodiments less than 0.005, and in some embodiments less than 0.001 are considered "significant." Thus, a p-value of 0.05 or greater is not considered "significant." [Example]

[0061] The results of several exemplary experiments comparing the hepatobiliary disposition of several compounds in the absence and presence of different types of proteins are described below. The following examples are provided to illustrate representative aspects of the present invention. In light of this disclosure, those skilled in the art will recognize that the following examples are merely representative, and that many variations, modifications, and variations can be employed without departing from the spirit and scope of the present invention.

[0062] Example 1 Evaluation of free fraction values ​​for selected compounds Initially, equilibrium dialysis was used to identify several compounds with a range of free fractions (also called unbound fractions). This free fraction is the amount of compound that would be thermodynamically free in solution compared to the amount that would be thermodynamically bound to the albumin protein. For example, a free fraction 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 measurements of endogenous bile clearance in rat hepatocytes. The hepatic uptake, efflux, intracellular concentration, and biliary clearance of nine compounds listed in Table 1 were evaluated in the presence and absence of physiological concentrations of protein (4% bovine serum albumin, BSA) and serum from Wistar rats. These data were measured in sandwich-cultured rat hepatocytes at a concentration of 1 μM for each compound and for a 10-minute exposure time in buffer (protein-free), 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); biliary excretion index (BEI: the percentage of compounds excreted in bile relative to compounds taken up by hepatocytes); and intrinsic biliary clearance (indicating the potential for compound excretion in bile and the best predictor of biliary elimination in vivo). The intrinsic biliary clearance results, which consider both compound uptake and efflux, are shown in Table 2. Taken together, these data clearly demonstrate that the addition of extracellular proteins can produce very different results compared to traditional results measured in buffer.

[0065] [Table 2]

[0066] Example 3 Comparison of measured (observed) and predicted (calculated) bile clearance in rat hepatocytes. Next, the measured biliary clearance of these compounds in rat hepatocytes in the presence of extracellular protein was compared to the predicted endogenous biliary clearance measured in the absence of protein and adjusted using the appropriate free fraction values ​​shown in Table 1. The latter method is the current industry standard. Table 3 summarizes these results, demonstrating the effectiveness of using a one-step integrated system to measure biliary clearance compared to a two-step process based on separate experiments. If the two-step process were equivalent to the one-step integrated system, these values ​​should agree. However, this was not the case with pitavastatin and rosuvastatin, where the predicted biliary clearance was underestimated compared to the measured value, likely due to unexpected changes in the presence of albumin. Note also that the predicted biliary clearance of DPDPE (1 μM) overestimated compared to the measured value. The possibility of over- or underestimation of clearance in this case, or essentially the inequality of these values, indicates the unexpected nature of the interaction and the desirability of using an integrated method when assessing cellular disposition.

[0067] [Table 3]

[0068] Generally, one would expect no difference between these values ​​for biliary clearance (because the values ​​take into account the extent 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 two compounds, pitavastatin and rosuvastatin, the predicted intrinsic biliary clearance in the presence of protein is much greater than that observed in the absence of protein. The effects observed in serum are similar, if not identical, suggesting that protein composition also influences compound clearance predictions.

[0069] Example 4 Comparison of measured (observed) and predicted (calculated) intracellular concentrations in rat hepatocytes The intrahepatic concentration (ICC) of the compound, which reflects the balance between cellular uptake, metabolism, and efflux, can also be determined in the absence and presence of protein. In a manner similar to Example 3 above, a one-stage method using protein in the system can be compared to data from a two-stage method in which the intrahepatic concentration (ICC) is determined and adjusted using the fraction free values ​​in Table 1. Table 4 shows the results of comparing the intrahepatic concentrations observed using the one-stage integrated system for various compounds with predicted values, first performed in the absence of protein and then adjusted using protein binding data from a separate experiment. If the methods are comparable, these values ​​should closely match.

[0070] [Table 4]

[0071] For several compounds, the protein binding information combined with intracellular concentration data from separate experiments obtained in the absence of protein (ICC predictions) agreed 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, being overestimated (valsartan) or underestimated (pravastatin, pitavastatin, and rosuvastatin) by more than 50%. Without measurements using the integrated system, it would be impossible to predict how much compound was taken up by cells and the resulting intracellular concentrations.

[0072] For some compounds, these data, i.e., in vivo biliary clearance and hepatic intracellular concentration in the presence of protein (physiological conditions), indicate that they cannot be predicted by adjusting the data with free fraction values ​​obtained from separate studies (the two-stage method). In vivo-relevant values ​​for these parameters can only be determined by performing experiments in the presence of physiological concentrations of protein. This effect cannot be predicted based on the protein binding of the compounds, since valsartan, pitavastatin, and rosuvastatin all bind to BSA to a similar extent. The effect of protein addition on valsartan biliary clearance was easily predicted from protein binding data obtained in a separate study. However, the protein effect on the biliary clearance of pitavastatin and rosuvastatin was unexpected and could not be predicted using protein binding data from separate experiments. Accurate prediction of the biliary clearance parameters of pitavastatin and rosuvastatin was only possible by performing experiments in the presence of physiological concentrations of protein.

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

[0074] Hepatic intracellular concentration is the driving force for any process occurring within the hepatocyte. Changes in intracellular concentration can affect all types of interactions occurring within the hepatocyte, including, but not limited to, transporter-based drug interactions; the extent of metabolism of the compound; metabolic interactions; the compound's induction potential (metabolism or transport); and toxicity produced by the compound or its metabolites.

[0075] The observed effect of proteins on intracellular concentrations can lead to over- or under-prediction of the compound's effect, dramatically altering expected clinical outcomes. For example, we observed this with telmisartan. Previously, it was thought to have potential for human cholestatic effects based on its transporter inhibition profile, but data showed that in the presence of albumin protein, the intracellular concentration in human hepatocytes was greater than predicted using data predicted from studies conducted in the absence of the protein, adjusted for a 100-fold free fraction. However, the intracellular concentration never reached a level high enough to cause hepatotoxic effects. In vivo data support these conclusions, as telmisartan has no known toxicity associated with its use. Because the effects of proteins are unexpected and unpredictable, it is desirable to expose cells to the protein during experiments.

[0076] A further example is a liver accumulation experiment conducted in the presence and absence of physiological concentrations of BSA to understand the differences in in vivo toxicity of the two compounds. In this experiment, in vitro toxicity tests (conducted in the absence of protein) and other pharmacological studies showed that two closely related compounds had similar potential for in vivo toxicity. However, when in vivo testing was performed, the two compounds had significantly different toxicity (liver) profiles in rodents. The researchers could not explain the differences based on systemic (blood) exposure. The non-toxic compound (AMG-A) had a Cmax concentration and area under the curve (AUC) value six times higher than the toxic compound (AMG-B). Therefore, the researchers measured intracellular liver concentrations and determined that the toxic compound (AMG-B) accumulated more in the liver and had an intracellular liver concentration approximately 15 times higher than AMG-A. This difference in intracellular concentration could explain 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 a non-toxic compound (AMG-A) and a toxic compound (AMG-B) in the presence / absence of physiological concentrations of BSA Experiments to determine the hepatic uptake and intracellular concentrations of AMG-A and AMG-B were conducted in sandwich-cultured rat hepatocytes in accordance with the present invention, in the absence and presence of physiological concentrations of protein (4% BSA). Significant differences were observed in the liver accumulation and intracellular concentrations of the two compounds between the absence and presence of physiological concentrations of protein. In experiments conducted in the absence of physiological concentrations of protein, the intracellular concentrations of the non-toxic compound (AMG-A) at 3 and 10 μM doses 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 concentrations of protein (4% BSA), and the intracellular concentrations of the more toxic compound (AMG-B) were significantly higher than those of the non-toxic compound (AMG-A) (Table 6). These results demonstrate that the presence of physiological concentrations of protein is necessary to accurately predict in vivo effects. As the binding parameters of the two compounds were similar, 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 assess hepatobiliary disposition and / or the effects of compounds to predict in vivo-relevant biliary clearance and intracellular concentrations in the liver system. Furthermore, the present invention provides for the use of proteins in transduction and other types of studies (metabolism, induction, and toxicity) that can yield more predictive results in vivo. Thus, in some embodiments, metabolic studies are provided, including metabolite identification and metabolic stability (parental longevity) (Kilford et al., Drug Metab Dispos, 36(7): 1194-1197, 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 concentrations (e.g., nuclear, mitochondrial). As will be apparent to one of skill in the art upon review of this disclosure, all of the above can be used to achieve in vivo relevant intracellular concentrations of metabolic, inhibitory, inducible, modulating and toxic regulators.

[0081] For non-hepatic systems (e.g., cell lines such as Caco-2, MDCK, and organ-specific cell lines for kidney, gastrointestinal, pancreatic, cardiac, neuronal, and lung), the present invention provides the use of relevant protein levels to mimic each physiological situation and derive a robust methodology for measuring intracellular concentrations of compounds. Knowledge of transporters and the ability to measure intracellular volumes allows prediction of intracellular concentrations and compound disposition and / or effects (e.g., exposure, efflux, etc.) using integrated methods.

[0082] Example 6 IC of P450 drug-metabolizing enzymes 50 Assessment of the effect of protein addition on the determination of Sandwich culture hepatocytes (SCH) were prepared using freshly isolated or cryopreserved hepatocytes. Freshly isolated hepatocytes were plated onto 24-well cell culture plates, rinsed, and cultured in the appropriate species-specific medium (QualGro™) from Qualyst Transporter Solutions (QTS, Durham, North Carolina, USA). Cells were then maintained in the appropriate species-specific medium 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, cells were allowed to attach for 2–4 hours, rinsed, and cultured in warmed (37°C) seeding medium. After 18–24 hours, cells were fed and covered with the appropriate species-specific QTS-compatible medium (QualGro™) supplemented with extracellular matrix (ECM), Matrigel® (0.25 mg / mL). Cells were maintained in QualGro™ hepatocyte culture medium until consumed for the study.

[0084] Cells were cultured as described above until day 6. On day 7, spent medium was aspirated and replaced with HBSS medium containing or without 4% bovine serum albumin (BSA). Medium containing P450 marker substrates and either fluconazole or ketoconazole was added directly to the SCHH to a total incubation volume of 0.5 mL. In situ incubation was performed in a cell culture incubator (37°C; 5% CO2; 100% humidity) with shaking at 120 rpm for 20–30 min. After incubation, the medium was collected and stored at -80°C until use in bioanalysis.

[0085] In situ incubations were analyzed for P450-mediated metabolite formation of midazolam to hydroxymidazolam and ibuprofen to hydroxyibuprofen. Briefly, 300 μl of internal standard solution (25 nM triazolam and d3-ibuprofen in methanol) and 100 μl of HBSS or HBSS (+ 4% BSA) were added to a 96-deep-well stacked protein precipitation plate (Millipore MDRPNP4; EDM Millipore, Billerica, Massachusetts, USA). The plate was shaken for 1–2 min before centrifugation, and the filtered supernatant was collected. The 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 for at least 20 min on a plate shaker. The reconstituted samples were transferred to Millipore 0.45 μm filter plates (Millipore MSHVN45) and filtered by centrifugation onto Costar 3957 plates and sealed with a silicone cap mat before LC-MS / MS analysis.

[0086] The direct inhibitory effects of fluconazole (CYP2C9) and ketoconazole (CYP3A4), inhibitors of CYP2C9 (3-hydroxybutyrophene) and CYP3A4 (hydroxymidazolam) enzyme activity in SCHH were evaluated in the presence and absence of 4% BSA. Fluconazole (CYP2C9) and ketoconazole (CYP3A4) reduced CYP2C9 and CYP3A4 enzyme activity 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 incubation mixture with fluconazole (CYP2C9) was related to the estimated IC value of fluconazole (CYP2C9). 50 The estimated IC of ketoconazole (CYP3A4) decreased from 56.2 μM to 27.1 μM (Tables 7 and 8). 50increased from 0.0455 μM to 0.117 μM (Tables 9 and 10), strongly indicating that the protein has a differential effect on the hepatic uptake and intracellular concentrations of various compounds (in this case either the probe inhibitors or the probe substrates midazolam and ibuprofen).

[0087] [Table 7]

[0088] [Table 8]

[0089] [Table 9]

[0090] [Table 10]

[0091] References The references cited herein are incorporated herein to the extent that they supplement, explain, provide background 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 may be changed without departing from the scope of the invention. Further, the foregoing description is by way of example only and not by way of limitation.

Claims

1. 1. A method for assessing the disposition of a candidate compound in in vitro culture and / or suspension to predict the in vivo disposition of the candidate compound, 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; and (d) determining the amount of candidate compound taken up in the culture and / or suspension, thereby assessing the disposition of the candidate compound and predicting the in vivo disposition of the candidate compound; A method consisting of:

2. The method of claim 1, wherein the culture and / or suspension comprises an artificial membrane system adapted to mimic a cell.

3. The method of claim 2, wherein the artificial membrane system mimics cells in a co-culture with feeder cells, the feeder cells comprising fibroblasts and / or Kupffer cells.

4. 3. The method of claim 2, wherein 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.

5. 3. The method of claim 2, wherein the cells mimicked by the artificial membrane system consist of a cell line, optionally selected from the group consisting of HepaRG (registered trademark) cell line, Caco-2, and MDC.

6. The step of "determining the amount of the candidate compound taken up into the culture and / or suspension, thereby evaluating the disposition" (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) providing a culture and / or suspension of cells comprising an artificial membrane system adapted to mimic a cell and at least one bile canaliculus; (b) exposing a candidate compound to the culture and / or suspension of cells; (c) exposing this culture and / or suspension of cells to a medium that provides an in vivo relevant extracellular environment; and (d) determining the amount of the candidate compound in said at least one bile canaliculus, thereby testing the susceptibility of the candidate compound to biliary excretion. A method consisting of:

8. (d) determining the amount of the candidate compound in at least one bile canaliculus, (a) exposing the candidate compound and a labeled preselected amount of substrate for the transport protein simultaneously to the cell culture and / or suspension for a time sufficient 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 assessing competition between the candidate compound and the labeled substrate for biliary excretion by the transport protein, wherein the presence of a decreased amount of the labeled substrate in the at least one bile canaliculus compared to a preselected amount of the labeled substrate indicates susceptibility of the candidate compound to biliary excretion by the transport protein. The method of claim 7, comprising:

9. The method of claim 7, wherein the artificial membrane system mimics cells in a co-culture with feeder cells, the feeder cells consisting of fibroblasts and / or Kupffer cells.

10. 8. The method of claim 7, wherein 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.

11. 8. The method of claim 7, wherein the cells mimicked by the artificial membrane system consist of a cell line, optionally selected from the group consisting of HepaRG (registered trademark) cell line, Caco-2, Opti-Target (registered trademark) cell line, and MDC.

12. 8. The method of claim 7, wherein the labeled substrate comprises a compound selected from the group consisting of a fluorogenic compound, a fluorescent compound, a chemiluminescent compound, a colorimetric compound, a radiolabeled compound, and combinations thereof.

13. 8. The method of claim 7, wherein the amount of the candidate compound in the at least one bile canaliculus is determined by calculating a biliary clearance value of the culture and / or suspension.

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, each comprising an artificial membrane system adapted to mimic cells and at least one bile canaliculus, the first culture and / or suspension having intact bile canaliculi and the second culture and / or suspension having disrupted bile canaliculi; (b) exposing a candidate compound to said first culture and / or suspension and said 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 that provides an in vivo relevant extracellular environment; (d) washing and lysing the first and second cultures and / or suspensions; and (e) determining the amount of candidate compound present in the lysate obtained from each culture and / or suspension of step (d), and using the amount of candidate compound in each culture and / or suspension to assess the susceptibility of the candidate compound to biliary excretion; A method consisting of:

15. 15. The method of claim 14, comprising: (i) exposing a candidate compound to said first and said second cultures and / or suspensions for a time (T) sufficient to allow uptake of the candidate compound; (ii) exposing the first and second cultures and / or suspensions to a medium that provides an in vivo relevant extracellular environment; (iii) washing and lysing the first and second fractions of the first and second cultures and / or suspensions, respectively; (iv) determining the amount of candidate compound present in a lysate obtained from each of the first and second cultures and / or suspensions of step (iii); (v) calculating the amount in the bile canaliculi as the difference between the amounts 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 disrupted bile canaliculi; and (vi) assessing the biliary excretion susceptibility of the candidate compound using the amount calculated in step (iv).

16. The method of claim 14, wherein the artificial membrane system mimics cells in co-culture with feeder cells, the feeder cells comprising fibroblasts and / or Kupffer cells.

17. 15. The method of claim 14, wherein 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 myocytes, nerve cells, muscle cells, adipocytes, and lung cells.

18. 15. The method of claim 14, wherein the cells mimicked by the artificial membrane system consist of one cell line, optionally selected from the group consisting of HepaRG (registered trademark) cell line, Caco-2, and MDC.

19. 1. A method for assessing the effect of a candidate compound in in vitro culture and / or suspension to predict the in vivo effect of the candidate compound, comprising: (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; (c) exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment; and (d) assessing 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. A method consisting of:

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

21. 21. The method of claim 20, wherein the artificial membrane system mimics cells in co-culture with feeder cells, the feeder cells consisting of fibroblasts and / or Kupffer cells.

22. 21. The method of claim 20, wherein 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, neuronal cells, muscle cells, adipocytes and lung cells.

23. 21. The method of claim 20, wherein the cells mimicked by the artificial membrane system comprise a cell line, optionally selected from the group consisting of HepaRG® cell line, Caco-2, Opti-Target® cell line, and MDC.

24. The method of claim 19 or 20, wherein the effect is selected from the group consisting of transduction and other types of studies (metabolism, induction and toxicity); metabolic studies including metabolite identification and metabolic stability (parental longevity); gene regulation (induction / repression); P450 and transporter drug interactions; intracellular accumulation and free or total (bound + free) intracellular concentrations (e.g., nuclear, mitochondrial); and toxicological effects.

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

26. The method of any one of claims 19 to 25, wherein the culture and / or suspension is repeatedly exposed to one or more candidate compounds.

27. 27. The method of any one of claims 2 to 26, wherein the cells are isolated from a source selected from the group consisting of mouse, rat, rabbit, human, monkey, ape, cat, dog, piglet, hog, cow, ox, sheep, horse, turkey, chicken, fish, duck and goose.

28. 28. The method of any one of claims 2 to 27, wherein the cultures and / or suspensions further comprise long-term cultures and / or suspensions.

29. 29. The method of any one of claims 2 to 28, wherein the culture and / or suspension comprises a bile canaliculus network.

30. 30. The method of any one of claims 2 to 29, wherein the culture and / or suspension has a configuration selected from the group consisting of clusters, aggregates, at least one layer of cells, and combinations thereof.

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

32. 32. The method of any one of claims 2 to 31, wherein the culture and / or suspension further comprises a sandwich culture and / or suspension, said sandwich culture and / or suspension comprising at least one layer of cells and optionally at least one bile canaliculus within said at least one layer of cells.

33. 33. The method of claim 32, wherein the sandwich cultures and / or suspensions further comprise long-term sandwich cultures and / or suspensions.

34. 34. The method of any one of claims 2 to 33, wherein at least one layer of cells is sandwiched between two layers of matrix.

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

36. 36. The method of claim 35, wherein the biological matrix medium is selected from the group consisting of collagen, laminin, complexes derived from basement membranes, derivatives thereof, and combinations thereof.

37. The method according to any one of claims 1 to 36, wherein the medium providing an in vivo relevant extracellular environment consists of a medium containing components at physiological concentrations or concentrations having properties similar to physiological concentrations.

38. 38. The method of any one of claims 1 to 37, wherein the component is selected from the group consisting of albumin; beta-lipoprotein; alpha-1-acid glycoprotein; plasma or serum from a mouse, rat, rabbit, human, monkey, ape, cat, dog, piglet, hog, cow, ox, sheep, horse, turkey, chicken, fish, duck, or goose; bile acid or a mixture of bile acids; bilirubin; and combinations thereof.

39. The method of any one of claims 1 to 38, which is carried out in at least one well in a multi-well plate.

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

41. 41. The method according to any one of claims 1 to 40, wherein the medium containing the protein at a physiological concentration contains other compounds that modulate the properties of the candidate compound.

42. 42. The method of any one of claims 1 to 41, wherein any combination of said exposing steps may be performed simultaneously or in any order.

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