Application of protein in prediction of in-vivo influence

Abstract

The invention relates to application of protein in prediction of in-vivo influence. A method of assessing the placement and / or impact of a candidate compound in an in vitro culture and / or suspension to predict the in vivo placement and / or impact of the candidate compound may comprise the steps of: providing a cell culture and / or suspension; exposing the candidate compound to the culture and / or suspension; exposing the culture and / or suspension to a medium providing an in vivo related extracellular environment, such as a medium comprising components (e.g., proteins and / or other components such as lipoproteins, cholic acids, and endogenous compounds such as bilirubin) of physiological concentrations or concentrations having characteristics such as binding characteristics similar to physiological concentrations; wherein any combination of any one of the exposure steps may occur in any order or at the same time; and evaluating the in vitro disposition and / or impact of the compound to predict the in vivo disposition and / or impact of the candidate compound.

Description

[0001] This application is a divisional application of Chinese patent application No. 201580065533.0, filed on October 7, 2015, entitled “Application of protein in prediction of in vivo effects”.

[0002] Citations of Related Applications

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62 / 060,916, filed October 7, 2014, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] The subject matter of the present disclosure relates in some embodiments to assessing the arrangement and / or influence of candidate compounds in vitro culture and / or suspension to predict the method for the in vivo arrangement and / or influence of the candidate compounds. More specifically, the subject matter of the present disclosure relates to assessing the method for arrangement and / influence, which includes but is not limited to the integrated influence of absorption clearance, basolateral eflux clearance, canalicular efflux clearance, intracellular concentration, gallbladder clearance, metabolic clearance and compound kinetics in in vitro culture and / or suspension of candidate compounds to predict the in vivo arrangement and / or influence of candidate compounds. In some embodiments, the method includes exposing culture and / or suspension to a medium providing an extracellular environment relevant in vivo such as a medium comprising a component such as a protein containing a physiological concentration or providing a concentration of a binding feature similar to a physiological concentration. Background Art

[0005] Typically, hepatocytes or related cell lines (suspension, plated, sandwich-cultured or other 3D models, Caco-2, MDCK, Opti-Target TM (Optivia Biotechnology, Menlo Park, California, USA) and under the trade names During the in vitro experiments of those available under (Biopredic International, Saint Grégoire, France), there was no or non-physiological levels of protein during the experiments. Therefore, only unbound drug concentrations were evaluated in all these experiments. This is often for experimental simplicity. In order to translate it into clinical or in vivo situations, a separate in vitro protein binding experiment is performed to determine the fraction of drugs bound to plasma proteins, and this information is used to extrapolate any parameters derived from in vitro experiments to in vivo situations. The impact caused by the "free" compound is then assessed by multiplying by a percentage, which is slightly blindly applied to the results from other experiments. The assumption is that this corrected measurement produces moderately accurate results relevant to clinical or in vivo situations.

[0006] Therefore, there remains a need for methods that do not rely on such calibration measurements and assumptions.There is a need for methods that assess the disposition and / or effects of a candidate compound in in vitro culture and / or suspension to predict the in vivo disposition and / or effects of the candidate compound. Summary of the invention

[0007] In some embodiments, the method comprises 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, such as a medium comprising a component, such as a protein or multiple proteins, at physiological concentrations or at concentrations of proteins that exhibit binding characteristics similar to physiological concentrations; and determining the amount of the candidate compound taken up by the culture and / or suspension to assess the disposition of the compound and / or assess the effect of the compound to predict the in vivo disposition and / or effect of the candidate compound.

[0008] In some embodiments, a method for evaluating the disposition of a candidate compound in an in vitro culture and / or suspension to predict the in vivo disposition of the candidate compound is provided. In some embodiments, the method comprises: 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 the candidate compound absorbed by the culture and / or suspension, thereby evaluating the disposition of the compound to predict the in vivo disposition of the candidate compound. In some embodiments, the cell culture and / or suspension comprises an artificial membrane system suitable for simulating cells. In some embodiments, the artificial membrane system simulates cells in a co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or kupffer cells. In some embodiments, the cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, heart cells, neuronal cells, muscle cells, adipocytes and lung cells. In some embodiments, the cells simulated by the artificial membrane system comprise a cell line, optionally wherein the cell line is selected from the group consisting of In some embodiments, determining the amount of candidate compound taken up by the culture and / or suspension to assess placement comprises: determining intracellular concentration of the candidate compound; determining liver accumulation; determining biliary excretion; and / or determining biliary clearance.

[0009] In some embodiments, a method for screening candidate compounds for sensitivity to bile secretion is provided. In some embodiments, the method comprises: providing a cell culture and / or suspension comprising an artificial membrane system suitable for simulating cells and at least one bile canaliculus; exposing the candidate compound to the cell culture and / or suspension; exposing the cell culture and / or suspension to a medium providing an in vivo relevant extracellular environment; and determining the amount of the candidate compound in at least one bile canaliculus, thereby screening the candidate compound for sensitivity to bile secretion. In some embodiments, determining the amount of the candidate compound in at least one bile canaliculus comprises: simultaneously exposing the cell culture and / or suspension for a period of time sufficient to absorb the candidate compound and a preselected amount of a labeled substrate for a transporter; washing the cell culture and / or suspension; and detecting the amount of the labeled substrate present in at least one bile canaliculus to assess the competition between the candidate compound and the labeled substrate for bile secretion through the transporter, wherein the presence of a reduced amount of the labeled substrate in at least one bile canaliculus compared to the preselected amount of the labeled substrate indicates the sensitivity of the candidate compound to bile secretion through the transporter. In some embodiments, the artificial membrane system simulates cells in co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or Kupffer cells. In some embodiments, the cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells simulated by the artificial membrane system comprise a cell line, optionally wherein the cell line is selected from the group consisting of Cell lines, Caco-2, Opti-Target TM In some embodiments, the labeling substrate comprises a compound selected from the group consisting of a fluorescent compound, a fluorescent compound, a chemiluminescent compound, a colorimetric compound, a radiolabeled compound, and a combination thereof. In some embodiments, the amount of the candidate compound in at least one bile ductule is determined by calculating the bile clearance value of the culture and / or suspension.

[0010] In some embodiments, a method for screening candidate compounds for sensitivity to bile secretion is provided. In some embodiments, the method comprises: establishing a first and a second cell culture and / or suspension and at least one bile canaliculus, the first and the second culture and / or suspension each comprising an artificial membrane system suitable for simulating cells, the first culture and / or suspension having intact bile canaliculi and the second culture and / or suspension having disrupted bile canaliculi; exposing the candidate compound to the first culture and / or suspension and the second culture and / or suspension for a period of time sufficient to allow absorption of the candidate compound; exposing the first culture and / or suspension and the second culture and / or suspension to a medium providing an in vivo relevant extracellular environment; washing and lysing the first culture and / or suspension and the second culture and / or suspension; and determining the amount of the candidate compound present in the lysate obtained from each culture and / or suspension in step (d) and using the amount of the candidate compound in the lysate of each culture and / or suspension to assess the sensitivity of the candidate compound to bile secretion. In some embodiments, the method comprises: exposing the candidate compound to each of the first culture and / or suspension and the second culture and / or suspension for a period of time (T) sufficient to allow absorption of the candidate compound; exposing the first culture and / or suspension and the second culture and / or suspension to a medium that provides an in vivo relevant extracellular environment; washing and lysing the first and second portions of each of the first and second cultures and / or suspensions; measuring the amount of the candidate compound present in the lysate obtained from each of the first and second cultures and / or suspensions in step (iii); calculating the mass in the bile canaliculi as the difference between the amount of the candidate compound present in the lysate from the first culture and / or suspension with intact bile canaliculi and the second culture and / or suspension with disrupted bile canaliculi; and assessing the sensitivity of the candidate compound to bile secretion using the mass calculated in step (iv). In some embodiments, the artificial membrane system mimics cells in a co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or Kupffer cells. In some embodiments, the cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and lung cells. In some embodiments, the cells simulated by the artificial membrane system comprise a cell line, optionally wherein the cell line is selected from the group consisting of Group consisting of cell lines, Caco-2 and MDC.

[0011] In some embodiments, a method is provided for evaluating the effects of a candidate compound in an in vitro culture and / or suspension to predict the in vivo effects of the candidate compound. In some embodiments, the method comprises: providing a cell culture and / or suspension; exposing the cell culture and / or suspension to at least one candidate compound at least once; exposing the cell culture and / or suspension to a medium that provides an in vivo relevant extracellular environment; and evaluating the effects of exposure to at least one candidate compound on the cell culture and / or suspension to predict the in vivo effects of the candidate compound. In some embodiments, the method comprises providing a cell culture and / or suspension comprising an artificial membrane system suitable for simulating cells and at least one bile ductule. In some embodiments, the artificial membrane system simulates cells in a co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or Kupffer cells. In some embodiments, the cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes, and lung cells. In some embodiments, the cells simulated by the artificial membrane system comprise a cell line, optionally wherein the cell line is selected from the group consisting of Cell lines, Caco-2, Opti-Target TM In some embodiments, the effect is selected from the group consisting of: conduction and other types of studies (metabolism, induction and toxicity); metabolic studies, including metabolite ID and metabolic stability (maternal lifespan); gene regulation (induction / inhibition); P450 and transporter drug interactions; subcellular accumulation and free or total (bound + free) intracellular concentration (e.g., nucleus, mitochondria); and toxicological effects. In some embodiments, the culture and / or suspension is exposed to a plurality of candidate compounds. In some embodiments, the culture and / or suspension is repeatedly exposed to one or more candidate compounds.

[0012] In some embodiments of the subject matter of the present disclosure, cells are isolated from a source selected from the group consisting of: mice, rats, rabbits, humans, monkeys, apes, cats, dogs, pigs, hogs, cattle, oxen, sheep, horses, turkeys, chickens, fish, ducks and geese. In some embodiments of the subject matter of the present disclosure, the culture and / or suspension further comprises a long-term culture and / or suspension. In some embodiments of the subject matter of the present disclosure, the culture and / or suspension comprises a microtubule network. In some embodiments of the subject matter of the present disclosure, the culture and / or suspension is characterized by having a structure selected from the group consisting of a cluster, an aggregate, at least one layer of cells and a combination thereof. In some embodiments of the subject matter of the present disclosure, cells are embedded in a matrix. In some embodiments of the subject matter of the present disclosure, the culture and / or suspension further comprises a sandwich culture and / or suspension, and the sandwich culture and / or suspension comprises at least one layer of cells and optionally at least one bile canaliculus within at least one layer of cells. In some embodiments of the disclosed subject matter, sandwich culture and / or suspension further comprise long-term sandwich culture and / or suspension.In some embodiments of the disclosed subject matter, at least one layer of cells is sandwiched between two layers of matrix.In some embodiments of the disclosed subject matter, matrix is ​​selected from the group consisting of biomatrix medium, synthetic matrix medium, co-culture of supporting cell type and their combination.In some embodiments of the disclosed subject matter, biomatrix medium is selected from the group consisting of collagen, laminin, complex derived from basement membrane, their derivatives and their combination.

[0013] In some embodiments of the presently disclosed subject matter, the medium providing an in vivo relevant extracellular environment comprises a medium comprising a component at a physiological concentration or a concentration having characteristics similar to a physiological concentration. In some embodiments of the presently disclosed subject matter, the component is selected from the group comprising albumin, beta-lipoprotein, alpha-1-acid glycoprotein, plasma or serum, bile acid or a mixture of bile acids, bilirubin, and combinations thereof, derived from mouse, rat, rabbit, human, monkey, ape, cat, dog, pig, swine, cattle, bull, sheep, horse, turkey, chicken, fish, duck, or goose.

[0014] In some embodiments of the disclosed subject matter, the method is performed in at least one hole of a multi-well plate. In some embodiments of the disclosed subject matter, the method further comprises screening a plurality of candidate compounds simultaneously. In some embodiments of the disclosed subject matter, the medium comprising a protein of physiological concentration comprises another compound that modulates the properties of the candidate compound. In some embodiments of the disclosed subject matter, any combination of any of the exposure steps can occur in any order or simultaneously.

[0015] It is an object of the presently disclosed subject matter to provide methods of assessing the disposition and / or effects of a candidate compound in in vitro culture and / or suspension to predict the in vivo disposition and / or effects of the candidate compound.

[0016] One object of the subject matter of the present disclosure is set forth above, which is fully or partially achieved by the subject matter of the present disclosure, as described in conjunction with the accompanying embodiments best described below. Other objects will become apparent. DETAILED DESCRIPTION

[0017] The subject matter disclosed herein will be described more fully hereinafter, in which some, but not all, embodiments of the subject matter of the present disclosure are described. Indeed, the subject matter of the present disclosure 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.

[0018] According to some embodiments of the subject matter of the present disclosure, there is provided a method for adding physiological or other relevant levels of components such as protein (often generally referred to as albumin) to approach the in vivo environment outside the cell, because usually plasma proteins and / or other components are related to combined drugs, chemicals and endogenous compounds. Specifically, the extracellular environment that is more relevant in vivo will indicate the intracellular concentration and compound dynamics (changing over time) that are more relevant in vivo. Both intracellular concentration and dynamics are driving factors for assessing the change of cell processes and test cell results. These processes include the absorption of compounds, the outflow (efflux) of compounds (in the case of liver, basolateral (basolateral) and microtubules (canalicular)), the intracellular concentration of compounds, the metabolism of compounds, the induction potential of compounds and the toxicity of compounds. The experimental results obtained by the method are unexpected and produce results that cannot be predicted using the current methods in the art.

[0019] Typically, hepatocytes or related cell lines (suspension, plated, sandwich-cultured or other 3D models, Caco-2, MDCK, Opti-Target TM (Optivia Biotechnology, Menlo Park, California, USA), Hμrelflux TM (Hμrel Corporation, North Brunswick, New Jersey, USA) and under the trade name (Biopredic International, Saint Grégoire, France) during the in vitro experiments available under those, there is no or there is a non-physiological level of components such as proteins during the experiment. Therefore, in all these experiments, only the unbound drug concentration is evaluated. In order to translate it into clinical or in vivo situations, a separate experiment such as an in vitro protein binding experiment is performed to determine, for example, the fraction of the drug that is bound to plasma proteins, and this information is used to extrapolate any parameters derived from the in vitro experiments to the in vivo situation. By other embodiments, protein binding experiments (such as using equilibrium dialysis) will provide a percentage of a "free fraction" or a fraction of unbound (Fu) compounds. The impact caused by the "free" compound is then evaluated by multiplying the percentage, which is applied to the results from other experiments (slightly blindly).

[0020] Typically, experiments evaluating hepatic uptake and biliary excretion of test compounds are evaluated under conditions in which no protein is present outside the cell. This is often for reasons of experimental simplicity, and typically, the solely determined free fraction is applied to these results.

[0021] According to the disclosed subject matter, it is recognized that blindly applying a free fraction correction factor to hepatocyte measurements (such as absorption, biliary excretion, hepatobiliary clearance, or intracellular concentration) does not provide in-scope physiological data. In fact, in specific embodiments, it is observed that the addition of protein can change, often significantly, the pharmacokinetics of compound absorption into cells, change the kinetics of protein absorption, and change thermodynamic binding parameters. These parameters are not or cannot be considered when determining the free fraction alone. Ultimately, it is assumed that the absorption rate is proportional to the unbound drug concentration, which may be wrong in many cases. In fact, it is shown herein that the addition of protein does not always have the expected effect, i.e., the observed absorption and intracellular concentration of the compound in the presence of protein does not match the predicted absorption. This observation is unexpected.

[0022] Indeed, as demonstrated and discussed in the Examples section herein, experimental data unexpectedly demonstrate that for some compounds, in vivo biliary clearance and liver intracellular concentrations cannot be predicted by adjusting the data with free fraction values ​​obtained from separate studies (two-step method) in the presence of conditions close to the in vivo environment, such as the presence of protein. Instead, evaluations performed in the presence of conditions close to the in vivo environment, such as, but not limited to, physiological concentrations of protein, determined in vivo relevant values ​​for these parameters. Thus, as demonstrated herein, in some cases, estimated or calculated (as opposed to observed) biliary clearance and liver intracellular concentrations can be unexpectedly and significantly overestimated or underestimated.

[0023] Thus, in some embodiments, provided herein are methods that employ integrated systems that combine in vivo relevant extracellular environments (e.g., protein and / or other component binding effects) with assessment of intracellular placement and / or effects, such as hepatocyte placement and / or toxicity.

[0024] In some embodiments, the subject matter of the present disclosure includes exposing the candidate compound to the culture and / or suspension; and exposing the culture and / or suspension to a medium that provides an in vivo relevant extracellular environment, such as a medium containing components (such as proteins and / or other components) at physiological concentrations or concentrations having characteristics similar to physiological concentrations, such as binding characteristics. Some embodiments of the subject matter of the present disclosure include exposing the candidate compound and the components to the absorption medium at the same time. In fact, the subject matter of the present disclosure includes any combination of exposure steps in any order or at the same time. In some embodiments, the medium contains proteins and contains another component that can modulate the properties of the candidate compound. Therefore, in some embodiments, in the presence of a medium containing proteins, the culture and / or suspension can be exposed to a putative inducer or inhibitor and then evaluated with a medium containing the candidate compound but not containing proteins. Further, in some embodiments, the protein can be a mixture of proteins (albumin, alpha-1-acid glycoprotein) at physiological concentrations or concentrations determined to have binding characteristics similar to those observed at physiological protein concentrations. Serum or plasma obtained directly from the species of interest is also included.

[0025] In some embodiments of the subject matter of the present disclosure, hepatocyte cultures such as sandwich cultured hepatocytes can be used to evaluate the hepatic absorption rate and bile secretion of compounds of interest such as drug compounds. As disclosed in U.S. Pat. No. 6,780,580 (incorporated herein in its entirety by reference), screening for compounds of interest such as therapeutic compositions is desirable because such compounds can be absorbed and secreted on a large scale through the bile secretory process, whereby they have the least chance of imparting a therapeutic effect to a subject. It is therefore desirable to establish an in vitro test for the sensitivity of compounds to hepatocyte absorption and bile secretion to facilitate the elimination of compounds with undesirable high sensitivity from further evaluation of therapeutic agents as an initial stage of the evaluation process. Accordingly, because cultures of hepatocytes maintain the desired functional properties that reflect hepatocytes in vivo, they provide a model for screening the sensitivity of compounds of interest to bile secretion. The following U.S. patent documents 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 published U.S. patent application No. US-2010-0035293-A1.

[0026] As will be understood by those of ordinary skill in the art, in order to accurately model biological processes in vivo at the desired level, in vitro cultures and / or suspensions of cells (such as, but not limited to, hepatocytes) should be similar in structure and function to cells (such as, but not limited to, hepatocytes) in vivo. Therefore, in some embodiments of cultures and / or suspensions disclosed herein, the structural and functional properties displayed in vivo are established. For example, the establishment of a transport system such as a sinusoidal or canalicular transport system or both a sinusoidal and canalicular transport system is provided according to the subject matter of the present disclosure. In particular, the establishment of at least one bile ductule in a cell (such as, but not limited to, hepatocyte) culture and / or suspension is provided according to the subject matter of the present disclosure. Cultures and / or suspensions may include a variety of bile ductules. A variety of bile ductules may include a microtubule network. The establishment of at least one bile ductule or microtubule network may allow cultured cells (such as, but not limited to, hepatocytes) to secrete bile and bile components to at least one bile ductule, which is similar to bile secretion in vivo.

[0027] In addition to the microtubule transport system, specific transporters in in vitro liver or liver-related cultures have been established. Liver-related can include completely artificial membrane systems designed to mimic cells, such as vesicles, or cell lines such as Cell lines, Hμrelflux TM Cell lines, Opti-Target TMCell lines, Caco-2 and MDCK, which are either transfected or "knocked out" for human specific proteins (e.g., transporters, P450s). Exemplary 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 that seen in hepatocytes in vivo.

[0028] According to the subject matter of the present disclosure, normal metabolic capacity established in cell (such as but not limited to hepatocyte) culture and / or suspension is also provided, including metabolic enzyme expression and activity. Therefore, the culture can contain the metabolic capacity that basically reflects the metabolism of cells (including but not limited to hepatocyte) in vivo. The subject matter of the present disclosure provides, for example, but not limited to, normal expression, function and activity of I phase metabolic enzymes such as multiple P450 isozymes, II phase metabolic enzymes such as UDP-glucuronosyltransferase (UDP-glucuronosyltransferase) (UGT) and other enzymes responsible for primary bile acid (primary bile acid) being bound to taurine and glycine in vitro cell (such as but not limited to hepatocyte) culture and / or suspension.

[0029] Therefore, in some embodiments, provided herein is a method for evaluating the placement of candidate compounds in in vitro cultures and / or suspensions to predict the in vivo placement of candidate compounds. In some embodiments, such methods may include providing cell cultures and / or suspensions; exposing candidate compounds to cultures and / or suspensions; exposing cultures and / or suspensions to a medium providing an in vivo relevant extracellular environment, such as a medium containing components (such as proteins and / or other components) at physiological concentrations or having characteristics similar to physiological concentrations such as concentrations of binding characteristics; and determining the amount of candidate compounds absorbed by cultures and / or suspensions, thereby evaluating the placement of compounds to predict the in vivo placement of candidate compounds. Determining the amount of candidate compounds absorbed in cultures and / or suspensions to evaluate placement may further include determining the intracellular concentration of candidate compounds, determining liver accumulation, determining bile secretion, and / or determining bile clearance.

[0030] In some embodiments, a method for screening candidate compounds for sensitivity to bile secretion is provided. This method for screening candidate compounds for sensitivity to bile secretion may include providing a cell culture and / or suspension (such as but not limited to a suspension containing an artificial membrane system suitable for simulating cells) and at least one bile canaliculus; exposing the candidate compound to the cell culture and / or suspension; exposing the cell culture and / or suspension to a medium providing an in vivo relevant extracellular environment, such as a medium containing a component (such as a protein and / or other component) at a physiological concentration or having a characteristic similar to a physiological concentration, such as a concentration of a binding characteristic; and determining the amount of the candidate compound in at least one bile canaliculus, thereby screening the candidate compound for sensitivity to bile secretion.

[0031] In some embodiments, the step of determining the amount of the candidate compound in at least one bile canaliculus can include simultaneously exposing the cell culture and / or suspension for a period of time sufficient to absorb the candidate compound and a preselected amount of a labeled substrate for a transporter, washing the cell culture and / or suspension, and detecting the amount of the labeled substrate present in at least one bile canaliculus to assess the competition between the candidate compound and the labeled substrate for bile secretion through the transporter. In some embodiments, the presence of a reduced amount of labeled substrate in at least one bile canaliculus compared to a preselected amount of labeled substrate indicates the sensitivity of the candidate compound to bile secretion through the transporter. In some embodiments, the amount of the candidate compound in at least one bile canaliculus is determined by calculating the bile clearance value of the culture and / or suspension.

[0032] In addition, in some aspects, the cell culture and / or suspension in the above method can include an artificial membrane system suitable for simulating cells, cultured alone or in combination with supporting cells such as, for example, fibroblasts and / or kupffer cells. Cells simulated by the artificial membrane system can, for example, include vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and / or lung cells. In addition, in some aspects, the cells simulated by the artificial membrane system can include a cell line. In some embodiments, the cell line is selected from a cell line comprising Cell lines, Hμrelflux TM Cell lines, Opti-Target TM Groups of cell lines, Caco-2 and / or MDC.

[0033] Where a labeled substrate is used in any of the methods described herein, such labeled substrate may 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 / or combinations thereof.

[0034] In some embodiments, a further method for screening candidate compounds for sensitivity to bile secretion is provided. This method may include establishing a first and a second cell culture and / or suspension and at least one bile ductule, the first culture and / or suspension having intact bile ductules and the second culture and / or suspension having damaged bile ductules. This method may further include exposing the candidate compound to the first culture and / or suspension and the second culture and / or suspension for a period of time sufficient to allow absorption of the candidate compound; exposing the first and second cultures and / or suspensions to a medium providing an in vivo relevant extracellular environment, such as a medium containing a component (such as a protein and / or other component) of a physiological concentration or having a characteristic similar to a physiological concentration such as a concentration of a binding characteristic; washing and lysing the first culture and / or suspension and the second culture and / or suspension; and determining the amount of the candidate compound present in the lysate obtained from each culture and / or suspension and using the amount of the candidate compound in each culture and / or suspension lysate to assess the sensitivity of the candidate compound to bile secretion.

[0035] In some embodiments, each of the first and second cultures and / or suspensions may comprise an artificial membrane system suitable for simulating cells. In addition, in some aspects, the cell culture and / or suspension in the above method comprises an artificial membrane system suitable for simulating cells, alone or co-cultured with supporting cells such as, for example, fibroblasts and / or Kupffer cells. Cells simulated by the artificial membrane system may, for example, include alveoli, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and / or lung cells. In addition, in some aspects, the cells simulated by the artificial membrane system may comprise a cell line. In some embodiments, the cell line is selected from a group comprising Groups of cell lines, Caco-2 and / or MDC.

[0036] In some embodiments, the method using a first and a second cell culture and / or suspension can further include exposing the candidate compound to each of the first culture and / or suspension and the second culture and / or suspension for a time (T) sufficient to allow absorption of the candidate compound; exposing the first and second cultures and / or suspensions to a medium that provides an in vivo relevant extracellular environment, such as a medium containing components (such as proteins and / or other components) at physiological concentrations or concentrations having characteristics similar to physiological concentrations, such as binding characteristics; washing and lysing the first and second portions of each of the first culture and / or suspension and the second culture and / or suspension; measuring the amount of the candidate compound present in the lysate obtained from each of the first culture and / or suspension and the second culture and / or suspension; calculating the mass in the bile canaliculi as the difference between the amount of the candidate compound present in the lysate from the first culture and / or suspension having intact bile canaliculi and the second culture and / or suspension having disrupted bile canaliculi; and using the calculated mass to assess the sensitivity of the candidate compound to biliary secretion.

[0037] In some embodiments, methods for evaluating the effects of candidate compounds in in vitro cultures and / or suspensions to predict the in vivo effects of candidate compounds are provided. In some embodiments, these methods may include providing cell cultures and / or suspensions; exposing cell cultures and / or suspensions to at least one candidate compound at least once; exposing cell cultures and / or suspensions to a medium providing an in vivo relevant extracellular environment, such as a medium containing components (such as proteins and / or other components) at physiological concentrations or having characteristics similar to physiological concentrations such as binding characteristics; and evaluating the effects of exposure to at least one candidate compound on cell cultures and / or suspensions to predict the in vivo effects of candidate compounds. In some embodiments, the effects evaluated in the above methods may include conduction and other types of studies (metabolism, induction, and toxicity); metabolic studies, including metabolite ID and metabolic stability (maternal lifespan); gene regulation (induction / inhibition); P450 and transporter drug interactions; subcellular accumulation and free or total (bound + free) intracellular concentrations (e.g., nucleus, mitochondria); and / or toxicological effects.

[0038] In addition, in some aspects, the method can further include providing a cell culture and / or suspension comprising an artificial membrane system suitable for simulating cells and at least one bile ductule. In some aspects, the cell culture and / or suspension in the above method can include an artificial membrane system suitable for simulating cells, cultured alone or in combination with supporting cells such as, for example, fibroblasts and / or Kupffer cells. Cells simulated by the artificial membrane system can, for example, include vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, heart cells, neuronal cells, muscle cells, adipocytes and / or lung cells. In addition, in some aspects, the cells simulated by the artificial membrane system can include cell lines, such as, but not limited to, selected from cells comprising Cell lines, Hμrelflux TM Cell lines, Opti-Target TM In some embodiments, the cell may be isolated from a source selected from the group consisting of mice, rats, rabbits, humans, monkeys, apes, cats, dogs, pigs, hogs, cattle, oxen, sheep, horses, turkeys, chickens, fish, ducks, and geese.

[0039] In some aspects, the culture and / or suspension in the disclosed method may further include long-term culture and / or suspension. The culture and / or suspension may contain a microtubule network. The culture and / or suspension may be characterized by having a structure selected from the group consisting of a cluster, an aggregate, at least one layer of cells, and a combination thereof. In some embodiments, the cells may be embedded in a matrix.

[0040] In addition, in some aspects, the culture and / or suspension in the disclosed method may further include sandwich culture and / or suspension, and sandwich culture and / or suspension include at least one layer of cells and at least one bile duct in at least one layer of cells optionally. Sandwich culture and / or suspension may further include long-term sandwich culture and / or suspension. At least one layer of cells may be sandwiched between two layers of matrix, wherein the matrix may be selected from the group consisting of a biomatrix medium, a synthetic matrix medium, a co-culture of a supporting cell type and a combination thereof. The biomatrix medium may be selected from the group consisting of collagen, laminin, a complex derived from basement membrane (basement membrane-derived complex), their derivatives and a combination thereof.

[0041] In any of the methods disclosed herein, in some embodiments, 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.

[0042] By way of example but not limitation, in the methods disclosed herein, components (such as proteins) that can be added at physiological concentrations or concentrations having characteristics similar to physiological concentrations such as binding characteristics can include albumin, alpha-1-acid glycoprotein, beta-lipoprotein, bilirubin, bile acid or a mixture of bile acid, and / or plasma or serum from representative or desired subjects such as mice, rats, rabbits, humans, monkeys, apes, cats, dogs, pigs, edible pigs, cattle, bulls, sheep, horses, turkeys, chickens, fish, ducks or geese. In addition, in some aspects, a medium providing an in vivo relevant extracellular environment, such as a medium containing components (such as proteins and / or other components) at physiological concentrations or having characteristics similar to physiological concentrations such as binding characteristics, can contain another compound that regulates the properties of the candidate compound. In addition, a medium containing bile acid or a mixture of bile acid, bilirubin, beta-lipoprotein, and a protein or protein mixture can contain another compound that regulates the properties of the candidate compound. Further, the concentration of components (such as proteins and / or other components) other than physiological concentrations also includes concentrations that may be higher or lower but have similar effects. Thus, in some embodiments, non-physiological concentrations having similar effects, such as similar binding effects, are also provided.

[0043] In the methods described herein, this method can be carried out in at least one hole of a multi-well plate. In this method, multiple candidate compounds can be screened simultaneously. In addition, in the disclosed methods, any combination of any of the exposure steps can occur in any order or simultaneously.

[0044] Representative calculations that can be employed in accordance with the subject matter of the present disclosure include calculating a bile secretion index and a bile clearance value. In some embodiments, the bile secretion index (BEI) represents the percentage of a compound absorbed into hepatocytes (i.e., secreted into bile), and the bile clearance value represents the likelihood of a compound being cleared into bile and is the best predictor of the clearance of a compound into bile in vivo. In some embodiments, the BEI is a calculation of the absorption and secretion of a candidate compound as follows: 100% X {(absorption in cultures with intact bile ductules - absorption only in hepatocytes in Ca2+-free cultures) / (absorption in cultures with intact bile ductules)}. In some embodiments, the bile clearance calculation is performed as follows: (absorption in cultures with intact bile ductules - absorption only in cultures without Ca2+)} 2+=(absorption in hepatocytes in culture) / (incubation time x concentration of candidate compound in buffer medium). In some embodiments, the bile clearance value can be calculated as the ratio of the mass in the bile canaliculi in the culture medium and the area under the curve (AUC), where AUC represents the integral of the candidate compound in the medium from time 0 to time T (time can be measured in any desired units, and is typically measured in minutes). In practice, the term AUC can be related to the following equation:

[0045] where C is the concentration in the medium.

[0046] This equation is set forth in Pharmacokinetics, Second Edition (Marcel Dekker, Inc. 1982), by Gibaldiand Perrier, (pp. 13-14).

[0047] While it is believed that the following terms are fully understood by those of ordinary skill in the art, the following definitions are set forth to aid in explaining the subject matter of the present disclosure.

[0048] An "in vivo relevant extracellular environment" is an environment that simulates or approximates in vivo conditions relevant to the in vitro disposition and / or effects of candidate compounds for evaluation according to the disclosed subject matter. For example, an in vivo relevant extracellular environment can provide components (such as proteins and / or other components) at physiological concentrations or concentrations having characteristics similar to physiological concentrations, such as binding characteristics, including the degree of binding (determined by the association constant, K) in addition to the "tightness" of protein binding. a Or dissociation constant K d express).

[0049] The combination of the terms "disposition and / or effect" includes, but is not limited to, the following: absorption clearance; basolateral efflux clearance; canalicular efflux clearance; metabolic clearance; intracellular concentration; compound kinetics; toxicological effects; metabolite ID and metabolic stability (maternal lifespan); gene regulation (induction / inhibition); P450 and transporter drug interactions; subcellular accumulation and free or total (bound + free) intracellular concentration (e.g., nucleus, mitochondria) and overall bile clearance. The combination of the terms "disposition and / or effect" also includes pharmacokinetics (PK), which can be broadly defined as (1) how an organism or system responds to a compound of interest and (2) all additive clearances (absorption, efflux, metabolism) in a culture and / or suspension system. Indeed, combinations of the terms "arrange and / or influence" may include any desired assessment, as would be apparent to one of ordinary skill in the art upon reading this disclosure.

[0050] The term "calcium-free buffer" is intended to refer to any buffer that is substantially free of calcium. A non-limiting example of a calcium-free buffer is a calcium-free Hank's balanced salt solution. As will be appreciated by those of ordinary skill in the art, any suitable buffer that is substantially free of calcium falls within the scope of the subject matter of the present disclosure. The use of a calcium-free buffer provides bile ductules for destruction according to some embodiments of the subject matter of the present disclosure.

[0051] The phrases "normal metabolic function," "normal metabolic activity," and "desirable metabolic characteristics" are used interchangeably herein and are intended to 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 in vivo basal conditions.

[0052] The term "functional property" includes any biological property that confers a specified function in the biology of an organism, a cell, or a biochemical reaction. According to the subject matter of the present disclosure, functional properties can include enzyme activity, enzyme function, enzyme expression, transporter expression and transporter function, and the regulatory pathways responsible for enzyme and transporter expression.

[0053] The terms "compound," "candidate compound," "compound of interest," or "drug compound" are used interchangeably herein and are intended to refer to any compound (exogenously administered or endogenously produced) in which the characteristics of the compound's metabolism, toxicity, hepatic uptake, or sensitivity to biliary secretion are desirable. Exemplary compounds, compounds of interest, or drug compounds include xenobiotics such as drugs and other therapeutic agents, carcinogens, and environmental pollutants, as well as endobiotics such as steroids, bile acids, fatty acids, and prostaglandins.

[0054] Compounds of interest as therapeutic agents are useful for treating warm-blooded vertebrates. Thus, the disclosed subject matter relates to mammals and birds.

[0055] Treatments of mammals are provided, such as humans, and those mammals that are important because they are endangered (such as Siberian tigers), are of economic importance (animals raised on farms for human consumption), and / or are of social importance to humans (as pets or animals in zoos), such as carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Treatments of birds are also provided, including those species of birds that are endangered, those kept in zoos, and fowl, and more specifically domesticated fowl, i.e., poultry such as turkeys, chickens, ducks, geese, guinea fowls, etc., because they are also of economic importance to humans. Thus, treatments of livestock are provided, including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, etc.

[0056] The phrase "evaluating toxicological effect" is intended to refer to any suitable method of quantitatively and / or qualitatively measuring one or more toxic effects of a compound on cells, such as but not limited to hepatocytes.

[0057] The term "biliary excretion" is intended to refer to a biological process in which a substance is removed from a subject's circulatory system by being taken up by hepatocytes and secreted via bile canaliculi, i.e., absorption and efflux. For example, absorption into hepatocytes is mediated by an endogenous transport system of hepatocytes, including but not limited to Ntcp, Oatp1, and Oatp2. Secretion into the bile canaliculi is mediated by efflux transporters, including but not limited to Mrp2, Mdr3, Pgp, and Bsep. The bile canaliculi are structures within liver tissue that receive secreted components from hepatocytes and transport bile to the bile duct for removal from a subject.

[0058] The method of the present disclosure can include setting up a sandwich culture of hepatocytes, wherein at least one hepatocyte layer is formed between two layers of matrix. Although the structure as a sandwich culture is a preferred structure for the culture, any suitable structure obvious to those of ordinary skill in the art is within the scope of the subject matter of the present disclosure. For example, clusters, aggregates or other associations (associations) or groupings (groupings) of cells (such as but not limited to hepatocytes) in cultures and / or suspensions fall within the scope of the subject matter of the present disclosure, wherein at least one bile ductule is formed, and wherein the functional properties of cells (such as but not limited to hepatocytes) are established. In addition, cells (such as but not limited to hepatocytes) co-cultured 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 subject matter of the present disclosure. Optionally, the culture and / or suspension structure contributes to the formation of a variety of bile ductules reflecting hepatocytes in vivo. Also optionally, the culture structure contributes to the formation of a microtubule network. Further, the culture configuration optionally helps to establish a culture of cells (such as but not limited to hepatocytes) with desired metabolic characteristics that are substantially similar to those of in vivo cells (such as but not limited to hepatocytes). Likewise, the desired transporter expression and function are optionally established so as to be substantially similar to those of in vivo cells (such as but not limited to hepatocytes).

[0059] In addition, in sandwich construction, cells (such as but not limited to hepatocytes) can be cultured in a monolayer between two layers of matrix or support. However, cells (such as but not limited to hepatocytes) can also be embedded in the matrix or it can be extended non-uniformly by matrix vertically, horizontally, diagonally or any combination thereof, thereby forming one-dimensional, two-dimensional and three-dimensional aggregates. In addition, cultures and / or suspensions can be set up in bioreactor systems, microenvironments or three-dimensional support such as but not limited to three-dimensional flow-through systems. See, for example, Griffith and Naughton, (2002) Science 295: 1009-1014. Therefore, cells (such as but not limited to hepatocytes) can be mixed with suitable matrix and the mixture can be inserted into suitable culture containers such as multi-well plates or culture chambers to form cell (such as but not limited to hepatocyte) cultures and / or suspensions.

[0060] Although collagen is a representative substrate or scaffold for culture and / or suspension of cells (such as but not limited to hepatocytes), any suitable substrate or scaffold, whether natural, synthetic, or a combination thereof, that would be apparent to one of ordinary skill in the art is within the scope of the disclosed subject matter. For example, other biological substrates, including but not limited to laminin and The biological cell culture substrate derived from the basement membrane sold by Collaborative Biomedical Products, Inc. (Bedford, Massachusetts, the U.S.) under the present disclosure comprises a suitable substrate or scaffold material. Typically, the matrix material, substrate material or scaffold material made of various materials such as polymers also fall within the scope of the subject matter of the present disclosure. The method according to the subject matter of the present disclosure also provides a change in the component material (having a specific matrix) for culturing cells (such as but not limited to hepatocytes).

[0061] Any suitable source of cells (such as but not limited to hepatocytes) that will be apparent to one of ordinary skill in the art upon reading this disclosure is within the scope of the subject matter of this disclosure. Exemplary sources include the warm-blooded vertebrates listed above. Specifically, exemplary sources include, but are not limited to, humans, rats, mice, monkeys, apes, cats, dogs, pigs, hogs, cattle, bulls, sheep, horses, turkeys, chickens, ducks, and geese.

[0062] The cultured cells (such as but not limited to hepatocytes) can be cultured as "long-term cultures and / or suspensions". "Long-term cultures and / or suspensions" are intended to refer to cells (such as but not limited to hepatocytes) that have been cultured for at least about 12 hours. Alternatively, "long-term cultures and / or suspensions" are intended to refer to cells (such as 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. Also optionally, "long-term cultures and / or suspensions" are intended to refer to cells (such as but not limited to hepatocytes) that have been cultured for at least about 96 hours, at least about one week, or at least about 28 days. Long-term culture contributes to the formation of bile canaliculi in the culture and / or suspension and the establishment of functional properties such as metabolic pathways.

[0063] Although hepatocyte cultures and / or suspensions are described above as representative cultures, the subject matter of the present disclosure provides the culture and use of any cell of interest or any combination of cell types of supporting cells that can regulate the functions of the disclosed cell types alone or in combination. Therefore, after reading this disclosure, one of ordinary skill in the art can rewrite the description and methods provided above for the 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, kidney cells, gastrointestinal cells, pancreatic cells, muscle cells, cardiac cells, neuronal cells, and lung cells. According to some embodiments of the subject matter of the present disclosure, there is provided a single or multiple cell type and a co-culture of other cells that provide a supporting matrix (such as, but not limited to, fibroblasts) or a function (such as, but not limited to, Kupffer cells).

[0064] Following long-standing patent law convention, when used in this application, including the claims, the terms "a," "an," and "an" mean "one or more."

[0065] 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 modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0066] As used herein, the term "about" when referring to a value or an amount of mass, weight, time, volume, concentration, or percentage, is intended to include variations from the specified amount, 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%, as such variations are suitable for performing the disclosed methods.

[0067] As used herein, the term "and / or," when used in the context of listing entities, refers to the entities as being present either individually or in combination. Thus, for example, the phrase "A, B, C and / or D" includes A, B, C, and D individually, and includes any and all combinations and subcombinations of A, B, C, and D.

[0068] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive and open-ended and does not exclude additional, unrecited elements or method steps. "Comprising" is a term of art used in claim language that means the named elements are provided, but other elements may be added and still form a construction or method within the scope of the claim.

[0069] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When the phrase "consisting of" appears in the body of a claim rather than immediately following a preamble, it limits only the elements set forth in that statement; other elements are not excluded from the claim as a whole.

[0070] As used herein, the phrase "consisting essentially of limits the scope of a claim to the specified materials or steps plus those that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0071] With respect to the terms "comprising," "consisting of," and "consisting essentially of," where one of these three terms is used in the specification, the presently disclosed and claimed subject matter may include the use of either of the other two terms.

[0072] As used herein, "significant" or "significant" refers to a statistical analysis of the probability that a non-random association exists between two or more entities. In order to determine whether a relationship is "significant" or has "significance", statistical manipulations of the data can be performed to calculate a probability expressed as a "p-value". Those p-values ​​that fall below a user-defined cutoff point are considered significant. In some embodiments, p-values ​​less than or equal to 0.05, 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. Accordingly, p-values ​​greater than or equal to 0.05 are considered insignificant.

[0073] Example

[0074] The following discussion compares the results of multiple exemplary experiments of hepatobiliary deployment of various compounds in the presence and absence of different types of proteins. The following examples are included to illustrate representative modes of the subject matter of the present disclosure. In light of the present disclosure, one of ordinary skill in the art will appreciate that the following examples are intended to be representative only, and that various changes, modifications, and variations may be employed without departing from the spirit and scope of the subject matter of the present disclosure.

[0075] Example 1

[0076] Evaluation of free fraction values ​​for selected compounds

[0077] Initially, equilibrium dialysis was first used to identify a number of compounds that had a free fraction (also called fraction unbound) within a certain range. The free fraction is the amount of compound that is considered thermodynamically free in solution / the amount that is considered thermodynamically bound to albumin. For example, a free fraction of 0.428 means that approximately 43% of the compound is unbound and free in solution under these conditions. These values ​​are shown in Table 1.

[0078] Table 1. Free fraction values ​​of selected compounds

[0079]

[0080] Example 2

[0081] Evaluation of intrinsic biliary clearance measured in rat hepatocytes

[0082] The compounds listed in Table 1 were used to evaluate hepatic absorption, efflux, intracellular concentration, and biliary clearance of nine compounds in the presence or absence of physiological concentrations of protein (4% bovine serum albumin, BSA) and serum obtained from Wistar rats. Data for each compound were determined in sandwich cultured rat hepatocytes at a concentration of 1 μM and an exposure time of 10 minutes in buffer (no protein), in buffer with 4% BSA, or in rat serum. Specifically, the parameters determined were: total accumulation (reflecting absorption, determined under +Ca conditions), cellular accumulation (determined under -Ca conditions, reflecting intracellular concentration), bile secretion index (BEI; representing the percentage of compound absorbed into hepatocytes and secreted into bile), and intrinsic biliary clearance (representing the probability of a compound being eliminated into bile and being the best predictor of elimination of a compound into bile in vivo). Table 2 shows the intrinsic biliary clearance results, which take into account both absorption and efflux of the compound. Taken together, these data clearly show that the addition of extracellular protein can produce very different results compared to traditional results measured in buffer.

[0083] Table 2. Intrinsic bile clearance in rat hepatocytes under different extracellular conditions

[0084]

[0085] Example 3

[0086] Comparison of measured (observed) and calculated (predicted) intrinsic biliary clearance in rat hepatocytes

[0087] Next, experiments were performed to compare the biliary clearance of these same compounds in rat hepatocytes in the presence of extracellular protein with the intrinsic biliary clearance measured in the absence of protein, then adjusted using the appropriate free fraction values ​​(predicted biliary clearance) in Table 1. The latter approach is the current industry standard. Table 3 summarizes these results and shows the effect of measuring biliary clearance using a one-step integrated system compared to a two-step process based on separate experiments. If the two-step methods are equivalent, then the values ​​are compared. However, there are many exceptions, including pitavastatin and rosuvastatin, where the predicted biliary clearance is substantially underestimated, probably due to unexpected changes in absorption when albumin is present. It was also noted that the predicted biliary clearance of DPDPE at 1 μM was overestimated. The observation that clearance can be overestimated or underestimated in this case or that these values ​​are not substantially equivalent indicates unexpected interoperability properties and the desire to use an integrated approach when evaluating cell placement.

[0088] Table 3. Comparison of measured (observed) and calculated (predicted) intrinsic biliary clearance in rat hepatocytes

[0089]

[0090] Normally, there is no difference in the value of the expected gallbladder clearance rate (due to the degree of protein binding being taken into account in this value). From Table 2, it can be observed that this is correct for many compounds evaluated, such as methotrexate (methrotrexate), valsartan (valsartan), DPDPE, pravastatin (pravastatin), digoxin (digoxin) and taurocholate (taurocholate). However, for two compounds, pitavastatin and rosuvastatin, the intrinsic gallbladder clearance rate value predicted in the presence of protein is much larger than the value observed in the absence of protein. Although inconsistent, the effect observed with serum is similar, showing that protein composition can have an impact on the clearance rate prediction of compound.

[0091] Example 4

[0092] Comparison of measured (observed) and calculated (predicted) intracellular concentrations in rat hepatocytes

[0093] The intracellular concentration (ICC) of the compound in the liver can also be determined in the absence or presence of protein, which reflects the balance of absorption, metabolism and efflux to the extracellular space. In a manner similar to Example 3 above, the comparison of the one-step method using protein in the system can be compared with the data from the two-step method, in which the ICC is determined and adjusted using the free fraction values ​​in Table 1. Table 4 provides the results of comparing the intracellular concentrations observed using the one-step integrated method of multiple compounds with the values ​​predicted when the experiment was first performed in the absence of protein and then adjusted using protein binding data from separate experiments. If these methods are equivalent, then these values ​​should match closely.

[0094] Table 4. Comparison of measured (observed) and calculated (predicted) intracellular concentrations in rat hepatocytes

[0095]

[0096] For some compounds, the integration of protein binding information from separate experiments with intracellular concentration data obtained in the absence of protein (predicted ICC) showed agreement with the observed values ​​for intracellular concentration (observed ICC). However, for four compounds (valsartan, pravastatin, pitavastatin and rosuvastatin), the intracellular concentration could not be predicted or was overestimated (valsartan) or underestimated (pravastatin, pitavastatin and rosuvastatin) by more than 50%. Without using an integrated system to measure, it would not be possible to predict how much compound is absorbed into the cell and the resulting intracellular concentration.

[0097] These data show that for some compounds, the in vivo gallbladder clearance and liver intracellular concentration in the presence of protein (physiological conditions) cannot be predicted by adjusting the free fraction value obtained by a separate study (two-step method). Only by conducting experiments in the presence of physiological concentrations of protein, the in vivo relevant values ​​of these parameters can be determined. The protein binding based on the compound cannot predict this effect because valsartan, pitavastatin and rosuvastatin are all bound to BSA to a similar extent. The effect of adding protein on the gallbladder clearance of valsartan cannot be easily predicted by the protein binding data obtained in the separate interest. However, the effect of protein on the gallbladder clearance of pitavastatin and rosuvastatin is unexpected and cannot be predicted using the protein binding data from a separate experiment. Only when experiments are conducted in the presence of physiological concentrations of protein, accurate estimates of the gallbladder clearance parameters of pitavastatin and rosuvastatin can be obtained.

[0098] An accurate estimate of biliary clearance is valuable when attempting to predict in vivo clearance prior to first human studies. A more accurate assessment of true in vivo clearance can lead to better clinical study designs and reduce the need for subsequent experiments in humans, as well as reduce the time to clinical development.

[0099] Intracellular concentrations of a compound are the driving force for any process that occurs within the hepatocyte. Changes in intracellular concentrations can affect any type of interaction that occurs within the hepatocyte. These can include, but are not limited to: transporter-type drug interactions; the extent of metabolism of the compound; metabolic interactions; the induction potential of the compound (metabolism or transport); and toxicity produced by the compound or its metabolites.

[0100] The observed effect of protein on intracellular concentration can lead to over- or under-predictions of compound effects, greatly changing the expected clinical results. For example, we have observed it in the case of telmisartan, and believe that telmisartan has human cholestasis potential based on its transporter inhibition curve (profile); however, the data show that in the presence of albumin, the intracellular concentration in human hepatocytes in the presence of protein is greater than the case predicted by the data predicted by the study conducted in the absence of protein and adjusted by 100X using the free fraction. However, a sufficiently high level of intracellular concentration has never been achieved to cause hepatotoxic effects. In vivo data support these conclusions because telmisartan does not have known toxicity associated with its use. Since the effect of protein is unexpected and unpredictable, it is expected that cells will be exposed to protein during the experiment.

[0101] Other embodiments include liver accumulation experiments in the presence or absence of physiological concentrations of BSA to understand the difference in the in vivo toxicity of the two compounds, where in vitro toxicity tests (performed in the absence of protein) and other pharmacological tests indicate that two closely related compounds should have the same possibility of in vivo toxicity. However, when tested in vivo, the two compounds have significantly different rodent toxicity (liver) curves. Based on systemic (blood) exposure, the researchers cannot explain the difference. The non-toxic compound (AMG-A) has a Cmax concentration and area under the curve (AUC) value, which is 6 times higher than the toxic compound (AMG-B). The researchers then measured the intracellular liver concentration, and the toxic compound (AMG-B) accumulated in the liver to a greater extent and had an intracellular liver concentration about 15 times higher than AMG-A. The difference in intracellular concentration can explain the toxicity difference. (Hamadeh et.al., Chem. Res. Toxicol., 2010, 23 (6), pp1025-1033).

[0102] Example 5

[0103] Hepatic uptake and intracellular concentrations of AMG-A and AMG-B in the presence / absence of physiological concentrations of BSA Degree Assessment

[0104] According to the subject matter of the present disclosure, experiments to determine the liver absorption rate and intracellular concentration of AMG-A and AMG-B were performed in sandwich cultured rat hepatocytes in the absence or presence of physiological concentrations of proteins (4% BSA). Significant differences in the liver accumulation and intracellular concentrations of the two compounds were observed between the absence or presence of physiological concentrations of proteins. In experiments conducted in the absence of physiological concentrations of proteins (Table 5), the intracellular concentration of AMG-A (non-toxic) at 3 and 10 μM was higher than that of the more toxic compound (AMG-B). Only when the experiment was conducted in the presence of physiological concentrations of proteins (4% BSA), the in vivo relevant intracellular concentrations (Table 6) were achieved, wherein the intracellular concentration of the more toxic compound AMG-B was significantly higher than that of the non-toxic compound (AMG-A). These results indicate that in order to correctly predict the in vivo effect, the presence of physiological concentrations of proteins is required. The differences in liver accumulation and intracellular concentrations of the two compounds in the absence and presence of proteins cannot be predicted by protein binding data because the binding parameters are the same for the two compounds.

[0105] Table 5. Concentration and time-dependent absorbance of AMG-A and AMG-B evaluated in the absence of protein (4% BSA)

[0106]

[0107] Table 6. Concentration and time-dependent absorbance of AMG-A and AMG-B evaluated in the presence of protein (4% BSA)

[0108]

[0109] For the liver system, the disclosed subject matter provides the ability to use proteins in assessing the hepatobiliary disposition and / or effects of compounds to predict relevant biliary clearance and intracellular concentrations in vivo. In addition, the disclosed subject matter provides the ability to use proteins in conduction and other types of studies (metabolism, induction, and toxicity) that can generate more predictive results in vivo. Thus, in some embodiments, metabolic studies are provided, including metabolite ID and metabolic stability (maternal lifespan) (Kilford et al., Drug Metab Dispos, 36 (7): 1194-1197, July 2008); gene regulation (induction / inhibition) (Jackson et al. Chemico-Biological Interactions, 179, 263-272, 2009); P450 and transporter drug interactions (including herbal drug interactions); subcellular accumulation (Pfeifer et al. Drug Metab Dispos 41: 1949-1956, November 2013) and free or total (bound + free) intracellular concentration (e.g., nucleus, mitochondria). It will be apparent to those of ordinary skill in the art after reading this disclosure that all of the above can be used to achieve in vivo relevant intracellular concentrations, which are control factors for metabolism, inhibition, induction, regulation, and toxicity.

[0110] For non-liver systems, (e.g., cell lines such as Caco-2, MDCK, and organ-specific cell lines from: kidney, gastrointestinal, pancreatic, cardiac, neuronal, lung), the disclosed subject matter provides a protective method for using relevant protein levels to mimic various physiological situations and derive for measuring compound intracellular concentrations. The knowledge of transporters and the ability to measure intracellular volume coupled with the use of an integrated approach allows prediction of intracellular concentrations and compound disposition and / or effects (e.g., exposure, efflux, etc.).

[0111] Example 6

[0112] IC of added protein on P450 drug metabolizing enzymes 50 Determined impact assessment

[0113] Sandwich-Cultured Hepatocytes (SCH) were prepared using freshly isolated hepatocytes or frozen hepatocytes. Freshly isolated hepatocytes were plated on 24-well cell culture plates, rinsed and incubated with appropriate species-specific Qualyst transporter solution (QTS, Durham, North Carolina, USA) and appropriate culture medium (QualGro TM Maintain cells in appropriate species-specific medium until consumed in the species of interest.

[0114] Thaw the frozen hepatocytes according to the manufacturer's thawing instructions. In a 24-well cell culture plate, frozen hepatocytes were suspended in QTS suitable hepatocyte seeding medium (QualGro TM After plating, cells were allowed to attach for 2-4 hours, rinsed and fed with warm (37°C) inoculum. Eighteen to 24 hours later, cells were fed and supplemented with extracellular matrix (ECM), (0.25 mg / mL) in the appropriate species-specific QTS suitable culture medium (QualGro TM ) to cover. Keep the cells in QualGro TM Hepatocytes are cultured in medium until the cells of interest are consumed.

[0115] Cells were cultured as described above through day 6 of culture. On day 7 of culture, spent culture medium was aspirated and replaced with an HBSS incubation solution containing or not containing 4% bovine serum albumin (BSA). An incubation solution containing either a P450 marker substrate and fluconazole or ketoconazole was added directly to SCHH, with a total incubation volume of 0.5 mL. In situ incubation was performed in a cell culture incubator (37° C.; 5% CO 2 ; 100% humidity) with shaking at 120 rpm for 20-30 minutes. After the incubation period, the incubated solution was collected and stored at -80° C. until processed for bioanalysis.

[0116] In situ incubation was analyzed for the formation of P450-mediated metabolites of hydroxymidazolam and hydroxyibuprofen from ibuprofen. Briefly, 300 μL of internal standard solution (methanol containing 25 nM triazolam and d3-ibuprofen) and 100 μL of HBSS or HBSS plus 4% BSA were added to protein precipitation plates stacked on 96 deep-well blocks (Millipore MDRPNP4; EDM Millipore; Billerica, Massachusetts, the United States). Before centrifugation to collect the filtered supernatant, the plate was allowed to shake for 1 to 2 minutes. 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 on a plate shaker for at least 20 min. The reconstituted sample was transferred to a Millipore 0.45 μm filter plate (Millipore MSHVN45) and filtered by centrifugation into a Costar 3957 plate and sealed with a silicone capmat prior to LC-MS / MS analysis.

[0117] The inhibitors fluconazole (CYP2C9) and ketoconazole (CYP3A4) were evaluated for direct inhibition of CYP2C9 (3-hydroxyibuprofen) and CYP3A4 (hydroxymidazolam) enzyme activities in SCHH in the presence and absence of 4% BSA. Fluconazole (CYP2C9) and ketoconazole (CYP3A4) reduced CYP2C9 and CYP3A4 enzyme activities to 24.4-45.1% and 32.0-71.1% of the control, respectively. Positive control inhibitors of CYP2C9 and CYP3A4 reduced enzyme activities in the expected dose-dependent manner. The difference in the effect of adding protein (4% BSA) in the incubation mixture with fluconazole resulted in a decrease in the IC50 estimated for CYP2C9 from 56.2 μM to 27.1 μM (Tables 7 and 8), while the mixture with ketoconazole increased the IC50 estimated for CYP3A4 from 0.0455 μM to 0.117 μM (Tables 9 and 10). This emphasizes that in the case of either the probe inhibitor or the probe substrate midazolam and ibuprofen, proteins have different effects on the liver absorption rate and intracellular concentration of various compounds.

[0118] Table 7 (pmol / min*million cells)

[0119]

[0120] Table 8

[0121]

[0122]

[0123] (pmol / min*million cells)

[0124] Table 9

[0125]

[0126] (pmol / min*million cells)

[0127] Table 10

[0128]

[0129] (pmol / min*million cells)

[0130] References

[0131] References cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide background or teach the methods, techniques and / or compositions used herein. 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

[0132] Pfeifer et al., (2013) Drug Metab. Dispos., 41:1949-1956

[0133] U.S. Patent No. 6,780,580

[0134] U.S. Patent No. 7,601,494

[0135] U.S. Patent No. 7,604,934

[0136] U.S. Patent No. 7,682,781

[0137] U.S. Patent No. 8,367,630

[0138] U.S. Patent Application Publication No. US-2010-0035293-A1

[0139] It will be appreciated that numerous details of the subject matter of the present disclosure may be varied without departing from the scope of the subject matter of the present disclosure. Furthermore, the above description is for the purpose of illustration only and not for the purpose of limitation.

Claims

1. A method for evaluating the disposition of a candidate compound in in vitro culture and / or suspension to predict the in vivo disposition of the candidate compound, the method comprising: (a) providing a cell culture and / or suspension; (b) exposing a candidate compound to the culture and / or suspension; (c) exposing the culture and / or suspension to a medium providing an in vivo relevant extracellular environment; as well as (d) determining the amount of said candidate compound taken up by said culture and / or suspension, thereby assessing the disposition of said compound to predict the in vivo disposition of said candidate compound.

2. The method according to claim 1, wherein: The cell culture and / or suspension comprises an artificial membrane system suitable for mimicking cells.

3. The method according to claim 2, wherein: The artificial membrane system simulates cells in co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or Kupffer cells.

4. The method according to claim 2, wherein: The cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and lung cells.

5. The method according to claim 2, wherein: The cells simulated by the artificial membrane system include cell lines, optionally wherein the cell lines are selected from Group consisting of cell lines, Caco-2 and MDC.

6. The method according to any one of claims 1 to 5, wherein: Determining the amount of the candidate compound taken up by the culture and / or suspension thereby evaluating the arrangement comprises: (a) determining the intracellular concentration of the candidate compound; (b) determination of liver accumulation; (c) determining bile secretion; and / or (d) Determination of bile clearance.

7. A method for screening a candidate compound for sensitivity to bile secretion, the method comprising: (a) providing a cell culture and / or suspension comprising an artificial membrane system suitable for mimicking cells and at least one bile canaliculus; (b) exposing a candidate compound to the cell culture and / or suspension; (c) exposing the cell culture and / or suspension to a medium providing an in vivo relevant extracellular environment; as well as (d) determining the amount of the candidate compound in the at least one bile canaliculus, thereby screening the candidate compound for sensitivity to bile secretion.

8. The method according to claim 7, wherein: Determining the amount of the candidate compound in the at least one bile canaliculus comprises: (a) simultaneously exposing the cell culture and / or suspension for a period of time sufficient for uptake of the candidate compound and a preselected amount of a labeled substrate for the transporter; (b) washing the cell culture and / or suspension; and (c) detecting the amount of the labeled substrate present in the at least one bile canaliculus to assess competition between the candidate compound and the labeled substrate for bile secretion through the transporter, 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 sensitivity of the candidate compound to bile secretion through the transporter.

9. The method according to claim 7, wherein: The artificial membrane system simulates cells in co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or Kupffer cells.

10. The method according to claim 7, wherein: The cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and lung cells.

11. The method according to claim 7, wherein: The cells simulated by the artificial membrane system include cell lines, optionally wherein the cell lines are selected from Cell lines, Caco-2, Opti-Target TM Group consisting of cell lines and MDCs.

12. The method according to claim 7, wherein: The labeled substrate comprises a compound selected from the group consisting of a fluorescence generating compound, a fluorescent compound, a chemiluminescent compound, a colorimetric compound, a radiolabeled compound, and combinations thereof.

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

14. A method for screening candidate compounds for sensitivity to bile secretion, the method comprising: (a) establishing a first cell culture and / or suspension and a second cell culture and / or suspension and at least one bile canaliculus, each of the first culture and / or suspension and the second culture and / or suspension comprising an artificial membrane system suitable for simulating cells, the first culture and / or suspension having intact bile canaliculus and the second culture and / or suspension having destroyed bile canaliculus; (b) exposing a candidate compound to said first culture and / or suspension and to said second culture and / or suspension for a period of time sufficient to allow for uptake of said candidate compound; (c) exposing said first culture and / or suspension and said second culture and / or suspension to a medium providing an in vivo relevant extracellular environment; (d) washing and lysing the first culture and / or suspension and the second culture and / or suspension; as well as (e) determining the amount of the candidate compound present in the lysate obtained from each culture and / or suspension in step (d) and using the amount of the candidate compound in the lysate from each culture and / or suspension to assess the sensitivity of the candidate compound to biliary secretion.

15. The method according to claim 14, comprising: (i) exposing a candidate compound to each of said first culture and / or suspension and said second culture and / or suspension for a time (T) sufficient to allow uptake of said candidate compound; (ii) exposing said first culture and / or suspension and said second culture and / or suspension to a medium providing an in vivo relevant extracellular environment; (iii) washing and lysing the first part and the second part of each of the first culture and / or suspension and the second culture and / or suspension; (iv) measuring the amount of the candidate compound present in a lysate obtained from each of the first culture and / or suspension and the second culture and / or suspension in step (iii); (v) calculating the mass in the bile canaliculi as the difference in the amount of the candidate compound present in lysates from the first culture and / or suspension with intact bile canaliculi and the second culture and / or suspension with disrupted bile canaliculi; as well as (vi) evaluating the sensitivity of the candidate compound to biliary secretion using the mass calculated in step (iv).

16. The method according to claim 14, wherein: The artificial membrane system simulates cells in co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or Kupffer cells.

17. The method according to claim 14, wherein: The cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and lung cells.

18. The method according to claim 14, wherein: The cells simulated by the artificial membrane system include cell lines, optionally wherein the cell lines are selected from Group consisting of cell lines, Caco-2 and MDC.

19. A method of evaluating the effect of a candidate compound in in vitro culture and / or suspension to predict the in vivo effect of the candidate compound; the method comprising: (a) providing a cell culture and / or suspension; (b) exposing the cell culture and / or suspension to at least one candidate compound at least once; (c) exposing the cell culture and / or suspension to a medium providing an in vivo relevant extracellular environment; as well as (d) evaluating the effect of exposure of said at least one candidate compound to said cell culture and / or suspension to predict the in vivo effect of said candidate compound.

20. The method according to claim 19, comprising providing a cell culture and / or suspension comprising an artificial membrane system suitable for simulating cells and at least one bile canaliculus.

21. The method according to claim 20, wherein: The artificial membrane system simulates cells in co-culture with supporting cells, wherein the supporting cells include fibroblasts and / or Kupffer cells.

22. The method according to claim 20, wherein: The cells simulated by the artificial membrane system are selected from the group consisting of vesicles, hepatocytes, cells derived from the liver, kidney cells, gastrointestinal cells, pancreatic cells, cardiac cells, neuronal cells, muscle cells, adipocytes and lung cells.

23. The method according to claim 20, wherein: The cells simulated by the artificial membrane system include cell lines, optionally wherein the cell lines are selected from Cell lines, Caco-2, Opti-Target TM Group consisting of cell lines and MDCs.

24. The method according to claim 19 or 20, wherein: The effects are selected from the group consisting of conduction and other types of studies (metabolism, induction and toxicity); metabolism studies including metabolite ID and metabolic stability (maternal lifespan); gene regulation (induction / inhibition); P450 and transporter drug interactions; subcellular accumulation and free or total (bound + free) intracellular concentrations (e.g., nucleus, mitochondria); and toxicological effects.

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

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

27. The method according to 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, pig, swine, cow, bull, sheep, horse, turkey, chicken, fish, duck, and goose.

28. The method according to any one of claims 2 to 27, wherein: The culture and / or suspension further includes long-term culture and / or suspension.

29. The method according to any one of claims 2 to 28, wherein: The culture and / or suspension comprises a microtubule network.

30. The method according to any one of claims 2 to 29, wherein: The culture and / or suspension is characterized as having a structure selected from the group consisting of clusters, aggregates, at least one layer of cells, and combinations thereof.

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

32. The method according to any one of claims 2 to 31, wherein: The culture and / or suspension further comprises a 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.

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

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

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 with a supporting cell type, and combinations thereof.

36. The method of claim 35, wherein: The biomatrix medium is selected from the group consisting of collagen, laminin, basement membrane-derived complexes, derivatives thereof, and combinations thereof.

37. The method according to any one of claims 1 to 36, wherein: Such media that provide an in vivo relevant extracellular environment include media that contain components at physiological concentrations or concentrations having characteristics similar to physiological concentrations.

38. The method according to any one of claims 1 to 37, wherein: The component is selected from the group comprising albumin, β-lipoprotein, α-1-acid glycoprotein, plasma or serum derived from mouse, rat, rabbit, human, monkey, ape, cat, dog, pig, swine, cattle, bull, sheep, horse, turkey, chicken, fish, duck or goose, bile acid or a mixture of bile acids, bilirubin and combinations thereof.

39. The method according to any one of claims 1 to 38, wherein: The method is performed in at least one well of a multiwell plate.

40. The method of any one of claims 1-39, further comprising screening a plurality of candidate compounds simultaneously.

41. The method according to any one of claims 1 to 40, wherein: The medium containing the protein at physiological concentrations contains another compound that modulates a property of the candidate compound.

42. The method according to any one of claims 1 to 41, wherein: Any combination of any of the exposing steps may occur in any order or simultaneously.

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