Methods and systems for screening candidate compounds for potential systemic or hepatotoxicity

Abstract

A method for screening the compounds for susceptibility to causing systemic or hepatotoxicity. [Solution] A method comprising the steps of providing a compound to be screened, establishing a hepatic cell line (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation, exposing the hepatic cell line (HCS) to a range of concentrations of bile acids to determine the toxicity profile of the bile acids, wherein the bile acid toxicity profile includes toxic potency, exposing the hepatic cell line (HCS) to a range of concentrations of bile acids in the presence of the compound to be screened and determining the toxicity profile of the bile acids, wherein the bile acid toxicity profile includes toxic potency, and comparing the toxic potencies of the bile acids to determine the susceptibility of the compound to causing systemic toxicity and / or hepatotoxicity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims priority to U.S. Provisional Patent Application No. 62 / 395,503, filed September 16, 2016, which is incorporated by reference herein in its entirety. [Technical Field]

[0002] The present invention relates to screening candidate compounds or chemicals for cholestatic hepatotoxicity potential, and more particularly to methods and systems for screening candidate compounds for susceptibility or potential to cause systemic or hepatotoxicity. [Background technology]

[0003] In the development of new therapeutic agents, drugs, and pharmaceutical compounds, it is necessary to screen new chemical entities (NCEs) to determine their potential for cholestatic hepatotoxicity. Drug-induced cholestatic hepatotoxicity is the result of impaired bile acid flow, resulting in the accumulation of toxic concentrations of bile acids in hepatocytes. NCEs that have a high potential for causing drug-induced cholestatic hepatotoxicity may be excluded from consideration in further drug development studies. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for improved in vitro methodologies and systems for assessing the potential of new chemical entities (NCEs), compounds, or drug candidates to cause cholestatic hepatotoxicity in vivo. [Means for solving the problem]

[0005] The above needs are addressed by the present invention. The following lists several embodiments of the present invention, and in many cases, lists variations and permutations of these embodiments. The following are merely exemplary of many different embodiments. Reference to one or more representative features of the embodiments listed herein is likewise exemplary. Such embodiments can generally exist with or without the mentioned features. Likewise, these features may be applied to other embodiments of the present invention, whether or not they are listed herein. To avoid excessive repetition, the following does not list or suggest all possible combinations of such features.

[0006] In some embodiments, a method for screening compounds for their potential to cause systemic and / or hepatotoxicity is provided, comprising the steps of providing a compound to be screened; establishing a hepatic cell line (HCS) capable of bile acid synthesis, bile acid transport, and / or bile acid regulation; exposing the hepatic cell line (HCS) to a range of concentrations of bile acids to determine a toxicity profile of the bile acid, wherein the bile acid toxicity profile comprises a toxic potency; exposing the hepatic cell line (HCS) to a range of concentrations of bile acids in the presence of a compound to be screened and determining a toxicity profile of the bile acid, wherein the bile acid toxicity profile comprises a toxic potency; and comparing the toxic potencies of the bile acids to determine the susceptibility of the compound to causing systemic and / or hepatotoxicity.

[0007] In some embodiments, an in vitro system for predicting the in vivo hepatotoxic potential of a compound is provided, comprising: (i) an in vitro cultured hepatocyte cell line (HCS) capable of bile acid synthesis, bile acid transport, and / or bile acid regulation; (ii) one or more bile acids having established toxic potency in the hepatocyte cell line (HCS); and (iii) an assay for measuring the hepatotoxicity of the compound when exposed to the hepatocyte cell line (HCS) in the presence of one or more bile acids.

[0008] In some embodiments, a method for screening compounds for susceptibility to causing systemic and / or hepatotoxicity is provided, comprising the steps of providing a compound to be screened; establishing a hepatocyte cell line (HCS) capable of bile acid synthesis, bile acid transport, and / or bile acid regulation; exposing the HCS to a range of concentrations of bile acids in the presence of the compound to be screened, and determining the toxicity profile of the bile acid in the presence of the compound to be screened, wherein the toxicity profile of the bile acid is known over the concentration range, and the bile acid toxicity profile of the bile acid comprises the toxic potency of the bile acid; and comparing the toxic potency of the bile acid in the presence and absence of the compound to determine the potential of the compound to cause systemic and / or hepatotoxicity.

[0009] It is therefore an object of the present invention to provide methods and systems for screening compounds for susceptibility to causing systemic and / or hepatotoxicity. This and other objects are achieved in whole or in part by the present invention. Further objects of the present invention, as well as other objects and advantages of the present invention mentioned above, will become apparent to those skilled in the art after studying the following description, figures, and examples. [Brief explanation of the drawings]

[0010] The present invention can be better understood by reference to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention (often diagrammatically). In these figures, like reference numerals designate corresponding parts in the different views. A further understanding of the present invention can be obtained by reference to the embodiments illustrated in the accompanying drawing figures. While the illustrated embodiments are merely exemplary of systems for carrying out the invention, both the organization and method of operation of the present invention, together with further objects and advantages thereof, generally, may be more readily understood by reference to the drawings and their description. These drawings are not intended to limit the scope of the invention, which is particularly set forth in the appended or later amended claims, but are intended merely to clarify and illustrate the invention. For a more complete understanding of the present invention, reference is now made to the following drawings. [Figure 1] Schematic diagram of a hepatocyte showing the normal bile acid homeostasis pathway in vivo (Figure 1A) compared to the compromised bile acid homeostasis (Figure 1B) that can lead to drug-induced cholestatic hepatotoxicity. [Figure 2] FIG. 1 is a schematic diagram of a hepatocyte showing normal bile acid uptake, synthesis and excretion in vivo. [Figure 3] Schematic diagram of a hepatocyte showing the effect of a new chemical entity (NCE) acting as a bile salt export protein (BSEP) inhibitor to initiate a bile acid homeostasis feedback mechanism, leading to the induction of compensatory mechanisms and a decrease in intracellular concentrations of bile acids. [Figure 4] Schematic diagram of hepatocytes showing the effect of a new chemical entity (NCE) acting as a bile salt export protein (BSEP) inhibitor and farnesoid X receptor (FXR) antagonist to prevent activation of the bile acid homeostasis feedback mechanism, resulting in an increase in intracellular bile acid concentrations and leading to bile acid hepatotoxicity. [Figure 5] Schematic of a hepatocyte showing the effect of a new chemical entity (NCE) inhibiting multiple bile acid excretion pathways, resulting in hepatotoxicity. [Figure 6] FIG. 1 is a schematic diagram of hepatocytes showing the effect of a new chemical entity (NCE) inhibiting bile acid uptake, resulting in a decrease in the amount of bile acids presented to the hepatocyte, leading to systemic cholestasis. [Figure 7-8] Graphs showing the effect of increasing bile acids in the presence of a new chemical entity (NCE) on ATP content (FIG. 7) and LDH leakage (FIG. 8). [Figure 9-10] Scatter plots showing the effect of increasing concentrations of the bile acids GCA, GCDCA, and GDCA in sandwich cultures of human hepatocytes (SCHH) on ATP (FIG. 9) and LDH (FIG. 10). [Figure 11-12] 11A-12C are plots showing the effect of glucose concentration (FIG. 11: 5 mM glucose, FIG. 12: 11 mM glucose) on bile acid pool toxicity profiles in the absence and presence of cholestatic agents in sandwich cultures of human hepatocytes (SCHH). [Figure 13] 1 is a plot showing data from a feasibility study evaluating the ability of the disclosed assays, methods, and systems to distinguish between ambrisentan and sitaxsentan. [Figure 14-18] 1 is a plot showing data from a viability study based on exposure to bile acids and bile acid pools. [Figure 19-21] 1 is a plot showing data assessing the effect of free fatty acids on bile acid pool hepatotoxicity in the presence of troglitazone. [Figures 22A-24B] 1 is a histogram showing data from a study assessing the susceptibility of hepatocytes to bile acid toxicity when drug exposure inhibits the bile acid excretion pathway. [Figure 25] 25A and 25B are dot plots showing the results of inhibition of biliary clearance of d8-TCA (FIG. 25A) and biliary excretion of d8-TCA (FIG. 25B) observed in sandwich cultures of human hepatocytes (SCHH) in the presence of troglitazone. [Figure 26] 1 is a histogram showing farnesoid X receptor (FXR) antagonism in sandwich cultures of human hepatocytes (SCHH) following exposure to troglitazone. [Figure 27] Histograms of ATP content and LDH leakage assess cell viability under conditions used to assess farnesoid X receptor (FXR) antagonism. [Figure 28] 1 is a histogram showing the effect of sandwich cultures of human hepatocytes (SCHH) treated with troglitazone, pioglitazone and rosiglitazone under sensitized culture conditions on LDH leakage and reduction of ATP content. BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The present invention is described more fully below. Some, but not all, embodiments of the invention are described therein. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will satisfy applicable legal requirements. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are provided to facilitate the description of the present invention.

[0012] All technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined below. References to technology employed herein are intended to refer to technology as commonly understood in the art, including variations of those technologies or equivalent technology substitutions that are apparent to those of ordinary skill in the art. While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are provided to facilitate the description of the present invention. In describing the present invention, it will be understood that several techniques and steps are disclosed, each of which has particular advantages and each of which can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Thus, in the following description, for the sake of clarity, we will refrain from repeating every possible combination of the individual steps in an unnecessary manner, but the specification and claims should nevertheless be read with the understanding that such combinations are fully within the scope of the invention and claims.

[0013] Following long-standing patent law convention, the terms "a," "an," and "the" when used in this application, including the claims, refer to "one or more." Thus, for example, a reference to "a cell" includes a plurality of such cells, and so forth. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations and may vary depending upon the desired properties sought to be obtained by the present invention.

[0014] As used herein, when referring to a value or amount of a composition, a dosage, a sequence identity (e.g., when comparing two or more nucleotide or amino acid sequences), a mass, a weight, a temperature, a time, a volume, a concentration, a percentage, and the like, the term "about" is meant to encompass variations from the specified value by, 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 appropriate for practicing the disclosed methods or using the disclosed compositions.

[0015] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. "Comprising" is a term used in claim language to mean that the named elements are present, but other elements may be added and still form a construct or method within the scope of the claim. 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 a clause in the body of a claim rather than immediately following a preamble, it limits only the elements recited in that clause and does not exclude other elements from the claim as a whole. As used herein, the phrase "consisting essentially of" limits the scope of a claim to the materials or steps specified and that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. With respect to the terms "comprising," "consisting of," and "consisting essentially of," when one of these three terms is used herein, the claimed subject matter of this disclosure may include the use of either of the other two terms. As used herein, the term "and / or," when used in the context of listing entities, refers to the entities present either alone or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also any and all combinations and subcombinations of A, B, C, and D.

[0016] As used herein, a hepatocyte cell line (HCS) refers to a hepatocyte cell line capable of bile acid synthesis, bile acid transport, and / or bile acid regulation. In some embodiments, the hepatocyte cell line (HCS) can comprise a two-dimensional culture, such as, but not limited to, a sandwich culture of hepatocytes (SCH), including a sandwich culture of human hepatocytes (SCHH). In some embodiments, the hepatocyte cell line (HCS) can comprise a three-dimensional culture (e.g., but not limited to, a 3D scaffold-based culture, a spheroid culture, etc.). The hepatocyte cell line (HCS) can comprise primary hepatocytes or other related hepatocyte cell lines, such as HepaRG, Huh7, co-culture systems, and / or stem cell-derived hepatocytes. In some embodiments, one or more free fatty acids and / or a preselected concentration of glucose are used in the hepatocyte cell line (HCS), such as in the incubation medium of the hepatocyte cell line (HCS).

[0017] Disclosed herein are methods and systems for screening candidate compounds for susceptibility to, or potential for, causing systemic or hepatotoxicity. As used herein, terms used to refer to the "ability," "susceptibility," "potential," "likelihood," "likelihood," and the like, of a candidate compound are used interchangeably and generally indicate that a compound may affect, cause, or contribute to systemic or hepatic toxicity and related conditions as disclosed and discussed herein.

[0018] In some embodiments, the present invention provides in vitro methodologies and systems for evaluating the potential of new chemical entities (NCEs), compounds, or drug candidates to cause cholestatic hepatotoxicity in vivo. Primary bile acids are synthesized in hepatocytes by one or more hepatic enzymes, including, for example, Cyp7A1, and then rapidly conjugated to both taurine and glycine, and can be further modified by glucuronidation and sulfation. Bile acids and conjugates are excreted into bile by bile salt export protein (BSEP) and MRP2, and into the portal circulation by MRP3 / 4 or OSTα / β. Bile acids are further metabolized in the intestine, absorbed into enterocytes by ASBT, and excreted from enterocytes into the portal vein by OSTα / β. Bile acids are primarily taken up from the portal circulation by NTCP. However, some studies have suggested that OATPs may also be involved to some extent in uptake. Bile acids can act as surfactants, and high intracellular concentrations (ICC) of bile acids have been shown to be hepatotoxic.

[0019] Drug-induced cholestatic hepatotoxicity is generally believed to result from impaired bile acid flow resulting from the inhibition of bile salt export protein (BSEP) alone by new chemical entities (NCEs). However, impaired hepatocellular bile acid homeostasis adaptive responses mediated by farnesoid X receptor (FXR) likely play a pivotal role in drug-induced liver injury. Without being bound by a specific theory or mechanism of action and / or current dogma, it is currently believed that the inhibition of bile salt export protein (BSEP) by new chemical entities (NCEs) is merely the initiating event leading to increased intracellular bile acid concentrations. When intracellular concentrations of bile acids are sufficiently high, bile acids activate farnesoid X receptor (FXR), which initiates a decrease in the expression of CYP7A1, the rate-limiting enzyme in bile acid production. As a result, bile acid synthesis is reduced, leading to the induction of both bile salt export protein (BSEP) and OSTα / β expression. Because new chemical entities (NCEs) inhibit transport through that pathway, this increase in bile salt export protein (BSEP) expression would have minimal effect. However, this induction of OSTα / β significantly increases bile acid clearance from hepatocytes into the portal circulation, thereby reducing the intracellular concentration of bile acids and potentially reducing hepatotoxicity. Therefore, the proposed mechanism for cholestatic hepatotoxicity is that potential cholestatic hepatotoxicants inhibit bile acid export pathways (e.g., bile salt export protein (BSEP) and / or MRP3 / 4) and either 1) prevent (antagonize) the farnesoid X receptor (FXR) from sensing elevated intracellular bile acid concentrations, or 2) inhibit bile acid export via OSTα / β (the bile acid export compensatory pathway), or 3) a combination of both.

[0020] To accurately predict the effects of bile acid on hepatic excretion in vivo, an integrated system such as that provided herein may be important, if not necessary. In some embodiments, this integrated system includes synthesis, transport (uptake, basolateral excretion, and canalicular excretion), and regulation (transport, metabolism, and synthesis). The sandwich culture of human hepatocytes (SCHH) model is one well-characterized system that can combine all of these pathways.

[0021] The method and system of the present invention combine the use of a sandwich culture of human hepatocytes (SCHH) model to measure bile salt export protein (BSEP) inhibition and bile acid regulation via the farnesoid X receptor (FXR). Furthermore, it was observed that the intracellular concentration of bile acids is the driving force for activation of the farnesoid X receptor (FXR) feedback mechanism. Because this intracellular bile acid concentration is one factor for developing in vitro methodologies to predict cholestatic hepatotoxicity, it was determined that the intracellular concentration (ICC) of bile acids is required to activate the farnesoid X receptor (FXR) feedback mechanism. Furthermore, because different bile acids are known to have different hepatotoxic potential, multiple bile acids and their combinations (bile acid pools) were evaluated for their potential to induce hepatotoxic responses using standard assays for toxicity (e.g., but not limited to, ATP, LDH, caspase 3 / 7, and APOTOX Glo). A mixture of various bile acids, including deoxycholic acid (DCA), was identified as one of the most potent hepatotoxic bile acids. The use of these assays allowed differentiation between cell viability, necrosis, and apoptosis in this system.

[0022] Generally, in some embodiments, the methods of the present invention can include culturing sandwich cultures of human hepatocytes (SCHH) with a range of concentrations of a bile acid (e.g., DCA) or a bile acid pool to identify the toxicity profile of the bile acid (e.g., by measuring ATP and / or LDH; dotted lines in Figures 7 and 8). In parallel studies, sandwich cultures of human hepatocytes (SCHH) can be exposed to a range of concentrations of DCA or a combination of bile acids in the presence of potential cholestatic hepatotoxicants. Three possible outcomes can be observed: 1. No change in bile acid (BA) toxic potency in the presence of new chemical entities (NCE) = no predicted effect. 2. Increased bile acid (BA) toxic potency in the presence of new chemical entities (NCEs) = potential for cholestatic hepatotoxicity (e.g., bile acid (BA) export inhibitors, farnesoid X receptor (FXR) antagonists, or both); or 3. Reduced bile acid (BA) toxic potency in the presence of new chemical entities (NCEs) = may cause cholestasis and result in systemic toxicity (e.g., bile acid (BA) uptake inhibitors).

[0023] Thus, new chemical entities (NCEs) can be classified as those that do not affect bile acid homeostasis (1), or those that have a high potential to cause cholestatic hepatotoxicity, whether or not associated with elevated systemic bile acid concentrations (2), or those that may result in the observation of clinical cholestasis (3) (systemic bile acid concentrations may be elevated, but hepatic cholestasis is unlikely), or a combination of these.

[0024] Thus, in some embodiments, provided herein are methods for screening compounds for their potential to cause systemic and / or hepatotoxicity. In some embodiments, the methods include: (a) providing a compound to be screened; (b) establishing a hepatic cell line (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation; (c) exposing the hepatic cell line (HCS) to a range of bile acids in concentrations to determine a toxicity profile of the bile acid, the bile acid toxicity profile comprising a toxic potency; (d) exposing the hepatic cell line (HCS) to a range of bile acids in the presence of a compound to be screened to determine a toxicity profile of the bile acid, the bile acid toxicity profile comprising a toxic potency; and (e) comparing the toxic potencies of the bile acids to determine the potential of the compound to cause systemic and / or hepatotoxicity.

[0025] In some embodiments, determining the potential for the compound to cause systemic and / or hepatotoxicity further comprises determining whether there is an alteration in the toxic potency of the bile acid in the presence of the compound compared to the absence of the compound, whether there is an increase in the toxic potency of the bile acid in the presence of the compound compared to the absence of the compound, or whether there is a decrease in the toxic potency of the bile acid in the presence of the compound compared to the absence of the compound. Compounds that do not cause an alteration in the toxic potency of bile acids are characterized as not causing systemic or hepatotoxicity.

[0026] However, compounds that cause an increase in the toxic potency of bile acids are characterized as having the potential to cause cholestatic hepatotoxicity. This cholestatic hepatotoxicity can be caused by inhibition of bile acid excretion (in which case the compound is characterized as a bile acid excretion inhibitor), and / or this cholestatic hepatotoxicity is caused by antagonism of the farnesoid X receptor (FXR) (in which case the compound is characterized as a farnesoid X receptor (FXR) antagonist), and / or a combination of both. In some embodiments, inhibition of bile acid excretion comprises inhibition of bile salt export protein (BSEP). Nevertheless, compounds that cause a decrease in the toxic potency of bile acids are characterized as having the potential to cause cholestasis, which leads to systemic toxicity caused by inhibition of bile acid uptake.

[0027] In some embodiments, determining the compound's potential to cause systemic or hepatotoxicity comprises characterizing the compound as a bile acid export inhibitor, a farnesoid X receptor (FXR) antagonist, a bile acid uptake inhibitor, or the absence of the above. In some embodiments, the compound can be a candidate drug, which is characterized as having a low potential to cause systemic and / or hepatotoxicity or as having a high potential to cause systemic and / or hepatotoxicity. In some embodiments, bile acid transport includes bile acid uptake, basolateral excretion, and / or canalicular excretion. The bile acid concentration range includes an intracellular concentration that mimics the fasting and / or postprandial concentrations in vivo. The bile acid concentration range includes an intracellular concentration of bile acid sufficient to activate the farnesoid X receptor (FXR) feedback mechanism.

[0028] In some embodiments, determining the toxicity profile of a bile acid can include measuring the hepatotoxic response using a cytotoxicity assay. Such a cytotoxicity assay can be selected from the group consisting of, but not limited to, an enzyme leakage assay, ATP, APOTOX Glo, and combinations thereof. The enzyme leakage assay can be selected from the group consisting of ALT, AST, and LDH. In some embodiments, the method of screening compounds for their potential to cause systemic and / or hepatotoxicity further comprises exposing a hepatocyte cell line (HCS) to a plurality of bile acids to determine a plurality of toxicity profiles for the plurality of bile acids, wherein the bile acid toxicity profile comprises a plurality of toxic potencies that can be measured in the hepatocyte cell line (HCS) in the absence and presence of the compound to predict the hepatotoxic potential of the compound.

[0029] In some embodiments, the method for screening compounds for their potential to cause systemic toxicity and / or hepatotoxicity can utilize a hepatocyte cell system (HCS), including two-dimensional or three-dimensional cultures, using primary hepatocytes or other hepatocyte cell lines. The two-dimensional culture can include a sandwich culture (SCH) of hepatocytes, where the SCH contains human hepatocytes. The three-dimensional culture can include a three-dimensional (3D) scaffold-based culture or a spheroid culture. Other hepatocyte cell lines include, but are not limited to, HepaRG, Huh7, co-culture systems, stem cell-derived hepatocytes, and combinations thereof. In some embodiments, the compound can be exposed to a hepatocyte cell system (HCS) over a range of concentrations. The bile acid or bile acids can be GCA, GCDCA, GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof. Additionally, one or more free fatty acids and / or predetermined concentrations of glucose can be employed in the hepatocyte cell system (HCS).

[0030] In some embodiments, an in vitro system for predicting the in vivo hepatotoxic potential of a compound is provided, comprising: (i) an in vitro cultured hepatocyte cell line (HCS) capable of bile acid synthesis, bile acid transport, and / or bile acid regulation; (ii) one or more bile acids having established toxic potency in the hepatocyte cell line (HCS); and (iii) an assay for measuring the hepatotoxicity of the compound when exposed to the hepatocyte cell line (HCS) in the presence of one or more bile acids. In some embodiments, the in vitro cultured hepatocyte cell line (HCS) can comprise an integrated hepatocyte cell line with bile acid synthesis, transport, and bile acid homeostasis feedback mechanisms. The in vitro cultured hepatocyte cell line (HCS) can comprise two-dimensional or three-dimensional cultures using primary hepatocytes or other hepatocyte cell lines. The in vitro cultured hepatocyte cell line (HCS) can comprise a sandwich culture (SCH) of hepatocytes, where the SCH can comprise human hepatocytes. The in vitro cultured hepatocyte cell line (HCS) can comprise a three-dimensional (3D) scaffold-based culture or a spheroid culture. The in vitro cultured hepatocyte cell line (HCS) can comprise HepaRG, Huh7, co-culture, stem cell-derived hepatocytes, and combinations thereof.

[0031] In some embodiments, the bile acid or acids is GCA, GCDCA, GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof. The established toxicity potency of the one or more bile acids can include a toxicity profile of the one or more bile acids based on hepatotoxicity responses using cytotoxicity assays, including, but not limited to, enzyme leakage assays, ATP, APOTOX Glo, and combinations thereof, such as ALT, AST, and LDH.

[0032] In some embodiments, such in vitro systems can be configured to characterize compounds that do not cause a change in the toxic potency of bile acids as compounds that are unlikely to cause systemic or hepatotoxicity. In some embodiments, such in vitro systems can be configured to characterize compounds that cause an increase in the toxic potency of bile acids as compounds that are likely to cause cholestatic hepatotoxicity. In some embodiments, such in vitro systems can be configured to characterize compounds that cause a decrease in the toxic potency of bile acids as compounds that are likely to cause cholestasis leading to systemic toxicity.

[0033] In some embodiments, a method for screening compounds for their potential to cause systemic toxicity and / or hepatotoxicity is provided.In some embodiments, this method comprises: (a) providing the compound to be screened; (b) establishing a hepatocyte cell line (HCS) with the ability of bile acid synthesis, bile acid transport and / or bile acid regulation; (c) exposing the hepatocyte cell line (HCS) to a range of bile acids in the presence of the compound to be screened, and determining the toxicity profile of the bile acids in the presence of the compound to be screened, wherein the toxicity profile of the bile acids is known over the range of concentrations, and the bile acid toxicity profile of the bile acids comprises the toxicity potency of the bile acids; and (d) comparing the toxicity potency of the bile acids in the presence and absence of the compound to determine the sensitivity of the compound to cause systemic toxicity and / or hepatotoxicity.In this method, the compound can be exposed to the hepatocyte cell line (HCS) over a range of concentrations.

[0034] In some embodiments, the bile acid can comprise a combination of bile acids, the toxicity profile of which is known, including toxic potency. The combination of bile acids can comprise bile acids provided in a predetermined ratio. The combination of bile acids can comprise bile acids in concentrations and ratios configured to mimic the concentrations and ratios of bile acids in vivo. In some embodiments, the step of determining the susceptibility of the compound to causing systemic and / or hepatotoxicity can further include the steps of confirming no change in bile acid toxicity potency in the presence of the compound compared to the absence of the compound, confirming an increase in bile acid toxicity in the presence of the compound compared to the absence of the compound, or confirming a decrease in bile acid toxicity in the presence of the compound compared to the absence of the compound.

[0035] Compounds that do not cause changes in the toxic potency of bile acids can be characterized as not causing systemic toxicity or hepatotoxicity. On the other hand, compounds that cause an increase in the toxic potency of bile acids can be characterized as having the potential to cause cholestatic hepatotoxicity. This cholestatic hepatotoxicity can be caused by inhibition of bile acid excretion (in which case the compound is characterized as a bile acid excretion inhibitor), and / or this cholestatic hepatotoxicity can be caused by antagonism of farnesoid X receptor (FXR) (in which case the compound is characterized as a farnesoid X receptor (FXR) antagonist), and / or a combination of both. This inhibition of bile acid excretion can include inhibition of bile salt excretion protein (BSEP). Nevertheless, compounds that cause a decrease in the toxic potency of bile acids can be characterized as having the potential to cause cholestasis leading to systemic toxicity, which can be caused by inhibition of bile acid uptake.

[0036] In some embodiments, determining the compound's potential to cause systemic or hepatotoxicity comprises characterizing the compound as a bile acid export inhibitor, a farnesoid X receptor (FXR) antagonist, a bile acid uptake inhibitor, or the absence of the above. The compound may be a drug candidate, which is characterized as having a low potential to cause systemic and / or hepatotoxicity or a high potential to cause systemic and / or hepatotoxicity. Bile acid transport can include bile acid uptake, basolateral excretion, and / or canalicular excretion. This bile acid concentration range can include an intracellular concentration that mimics the fasting and / or postprandial concentration in vivo. This bile acid concentration range can include an intracellular concentration of bile acid sufficient to activate the farnesoid X receptor (FXR) feedback mechanism. In some embodiments, the bile acid or multiple bile acids can be, but are not limited to, GCA, GCDCA, GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof.

[0037] In some embodiments, determining the toxicity profile of the bile acid can include measuring the hepatotoxic response using a cytotoxicity assay. The cytotoxicity assay can be, for example, an enzyme leakage assay, ATP, APOTOX Glo, or a combination thereof. The enzyme leakage assay can be, for example, ALT, AST, and LDH. In such methods, the hepatocyte cell line (HCS) can comprise a two-dimensional or three-dimensional culture utilizing primary hepatocytes or other hepatocyte cell lines. The two-dimensional culture can comprise a sandwich culture (SCH) of hepatocytes, which can comprise human hepatocytes. Meanwhile, the three-dimensional culture can comprise a three-dimensional (3D) scaffold-based culture or a spheroid culture. Other hepatocyte cell lines can include HepaRG, Huh7, co-culture systems, stem cell-derived hepatocytes, and combinations thereof. Finally, in such methods, one or more free fatty acids and / or a predetermined glucose concentration can be employed in the hepatocyte cell line (HCS). [Example]

[0038] The following examples are included to further illustrate various embodiments of the invention. However, those of skill in the art will recognize that, in light of the present disclosure, many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0039] Example 1 Bile acid homeostasis As demonstrated herein, drug-induced cholestatic hepatotoxicity results from impaired bile acid flow via inhibition of bile salt export protein (BSEP) by new chemical entities (NCEs) and impaired hepatocellular bile acid homeostasis adaptive responses. One possible mechanism of action leading to drug-induced cholestatic hepatotoxicity could consist of cholestatic hepatotoxicants inhibiting bile salt export protein (BSEP) (initial injury) and 1) preventing the farnesoid X receptor (FXR) from sensing elevated intracellular concentrations of bile acids (antagonism), 2) inhibiting all bile acid export pathways (basolateral and canalicular), or 3) a combination of both.

[0040] 1-6 are schematic diagrams of a hepatocyte cell line 10 (in vivo or in vitro) containing multiple hepatocytes 12 with tight junctions 14 and one or more bile ducts 16 between them. The components, enzymes, and transporters within this cell line include: bile acids (BAs); adenosine triphosphate (ATP); farnesoid X receptor (FXR); bile salt efflux protein (BSEP); multidrug resistance-associated protein 2 (MRP2); multidrug resistance-associated protein 3 (MRP3); multidrug resistance-associated protein 4 (MRP4); normal bile acid uptake (NTCP); basolateral efflux transporter (OST); and bile acid synthase (Cyp7A1), which are discussed and defined herein. Figure 1A shows the normal bile acid homeostasis pathway in vivo, while Figure 1B shows compromised bile acid homeostasis potentially resulting in drug-induced cholestatic hepatotoxicity. In Figure 1B, one or more of the farnesoid X receptor (FXR), bile salt export protein (BSEP), and / or basolateral efflux transporter (OST) are blocked or inhibited, causing interference with bile acid homeostasis and transport.

[0041] Thus, in some embodiments, the screening assays, methods and systems of the present invention comprise the following conceptual components. -Bile salt export protein (BSEP) inhibition -Bile acid homeostasis (farnesoid X receptor (FXR)) feedback mechanism -Understanding the need to utilize an integrated hepatocyte cell system that maintains bile acid transport and bile acid homeostasis feedback mechanisms; the sandwich culture of human hepatocytes (SCHH) system satisfies the first two items above. -Recognition that the sandwich culture of human hepatocytes (SCHH) system requires additional bile acids to behave more in vivo-like (e.g., fasting and postprandial); the system uses bile acid (BA) doses to model fasting and postprandial states in the presence and absence of new chemical entities (NCEs). -Identifying the physiologically relevant bile acid or bile acid pool (i.e., DCA) that elicits the toxic response; and / or -Relate cholestasis to toxicity in initial in vivo assays.

[0042] Example 2 Bile acid homeostasis feedback mechanism FIG. 2 shows normal bile acid (BA) uptake (NTCP), synthesis (CYP7A1), and excretion (bile salt export protein (BSEP)) in vivo. Figure 3 shows the effect of new chemical entities (NCEs) acting as bile salt export protein (BSEP) inhibitors to initiate bile acid homeostasis feedback mechanisms (e.g., activation of farnesoid X receptor (FXR)), resulting in the induction of compensatory mechanisms (e.g., basolateral efflux transporters OSTα / β) and a decrease in intracellular bile acid concentrations. Initiating the bile acid feedback mechanism prevents bile acid hepatotoxicity (also known as cholestatic hepatotoxicity). Figure 4 shows the effect of a new chemical entity (NCE) acting as a bile salt export protein (BSEP) inhibitor and farnesoid X receptor (FXR) antagonist to prevent activation of the bile acid homeostasis feedback mechanism, resulting in an increase in intracellular bile acid concentrations and leading to bile acid hepatotoxicity (also known as cholestatic hepatotoxicity). Figure 5 shows the effect of new chemical entities (NCEs) inhibiting multiple bile acid export pathways, resulting in hepatotoxicity. While bile acid feedback mechanisms remain intact, compensatory mechanisms (e.g., OSTα / β basolateral export) and other bile acid export mechanisms are inhibited, resulting in increased intracellular bile acid concentrations and hepatotoxicity (i.e., cholestatic hepatotoxicity). FIG. 6 shows the effect of new chemical entities (NCEs) on inhibiting bile acid uptake, resulting in a decrease in the amount of bile acids presented to hepatocytes and causing systemic cholestasis.

[0043] Example 3 Development of a cholestatic hepatotoxicity screening assay Provided herein are assays, methods, and systems for identifying new chemical entities (NCEs) that may interfere with the bile acid processing ability of hepatocytes. In some embodiments, such assays, methods, and systems are configured to link cholestasis to hepatotoxicity based on toxicity assays (e.g., ATP / LDH / caspase 3 / 7 / 8) and / or by monitoring LDH relative to clinical measurements (AST / ALT) typically used to monitor liver function. In some embodiments, such assays, methods, and systems are configured to classify new chemical entities (NCEs) as (i) cholestatic, (ii) ineffective, or (iii) causing cholestatic hepatotoxicity.

[0044] The possible results (bile acids (BA) in the presence of new chemical entities (NCE)) can be represented in some embodiments as shown in Figures 7 and 8. In Figures 7 and 8, dotted, dashed, and solid lines indicate the following: -dotted line: Bile acid (BA) toxic potency (TC) in the absence of new chemical entities (NCE) 50 Bile acid (BA) dose response to determine - System ID normal response calibration - Demonstrate how hepatocytes respond to a wide range of bile acid (BA) concentrations No change in potency of bile acid (BA) toxicity in the presence of new chemical entities (NCE) = no predicted effect -Dash line: Increased bile acid (BA) toxic potency in the presence of new chemical entities (NCEs) = potential for cholestatic hepatotoxicity Bile acid (BA) excretion inhibitors, farnesoid X receptor (FXR) antagonists, or both -solid line: In the presence of new chemical entities (NCEs), the decreased potency of bile acid (BA) toxicity is equivalent to the potential for cholestasis, which can lead to systemic toxicity. Bile acid (BA) uptake inhibitors

[0045] The bile acids GCA, GCDCA, and GDCA were assayed at increasing concentrations in sandwich cultures of human hepatocytes (SCHH) over a 24-hour period. ATP (Figure 9) and LDH (Figure 10) contents were measured. As shown in Figures 9 and 10, the results of these assays indicate that sandwich cultures of human hepatocytes (SCHH) are fairly resistant to bile acid (BA) toxicity for up to 24 hours of exposure. More specifically, GCA was not cytotoxic, GCDCA was comparable to GDCA in the ATP assay, and GDCA was greater than GCDCA in the LDH assay.

[0046] In sandwich cultures of human hepatocytes (SCHH), glucose concentrations were assessed in the absence and presence of cholestatic agents (e.g., troglitazone and sitaxsentan) to examine their effect on (i.e., shift in) bile acid pool toxicity profiles. Using a method similar to that described in the previous section, ApoTox-Glo TM Hepatotoxicity was evaluated using a triplex assay, and the results are shown in Figures 11 and 12. Low glucose conditions were necessary to enhance the sensitivity of the sandwich culture of human hepatocytes (SCHH) to the effects of troglitazone on cell viability and bile acid potency (Figures 11-12). Low glucose conditions improved the dynamic range of the cell viability assay, likely by improving its dependence on hepatic mitochondrial function, thereby increasing ATP production and promoting mitochondrial function. The addition of sitaxsentan did not alter the hepatotoxicity of the bile acid pool.

[0047] Example 4 Potential of cholestatic hepatotoxicity screening assays A feasibility study was also conducted to evaluate the ability of the disclosed assays, methods, and systems to distinguish between ambrisentan and sitaxsentan, the results of which are shown below and in Figure 13. Characteristics of sitaxsentan and ambrisentan are listed below. Sitaxentan (2-200 μM) -Reduced toxic potential of bile acids (BAs) -In vitro assays demonstrated potent / effective normal bile acid uptake (NTCP) inhibitors. -In vitro assays showed no bile salt export protein (BSEP) effect. -Cmax (maximum (or peak) concentration achieved after administration) = 23.5 μM Toxicity in clinic after 5 months; liver likely not the primary site of toxicity. No toxicity at 3 months Ambrisentan (2-200 μM) -No change in efficacy of bile acid (BA) toxicity -Cmax = 3 μM - Well tolerated and non-toxic - In vitro assays showed no effect on normal bile acid uptake (NTCP) / bile salt export protein (BSEP).

[0048] The potential of individual bile acids and bile acid mixtures (bile acid pools) to modify the hepatotoxicity of troglitazone (100 μM) and sitaxenten (60 μM) was assessed in sandwich-cultured human hepatocytes after 24 h of exposure in the presence of 5 mM glucose. The effects of test compounds were assessed in the presence of individual bile acids and in the presence of the bile acid pool. These effects were compared with the effects of bile acid treatment alone. DMSO treatment alone was used as a control for hepatotoxicity. Hepatotoxicity was assessed using ApoTox-Glo, a method that assesses viability, cytotoxicity, and apoptosis. TM Evaluation was performed using a triplex assay.

[0049] [Table 1]

[0050] A bile acid mixture was evaluated, consisting of four different bile acids maintained in physiologically relevant ratios of each bile acid as shown in Table 1. Individual bile acids were administered at the concentrations they represent in the mixture. A left-shift of troglitazone was observed only in the presence of the bile acid pool, indicating that fewer bile acids were required to reduce cell viability (Figure 14). Exposure to individual bile acids did not result in a left-shift in cell viability (Figures 15-18). These data suggest that the bile acid pool is a necessary component that, in some embodiments, allows the system to differentiate between the effects of compounds on bile acid homeostasis. Troglitazone interferes with the ability of hepatocytes to respond to increased intracellular bile acid concentrations, thereby increasing the hepatotoxicity of bile acids.

[0051] Example 5 Effect of free fatty acids on the hepatotoxicity of test compounds. Using the same methodology as in the previous section, the effects of troglitazone (100 μM) and sitaxenten (60 μM) on the hepatotoxicity of bile acid pools in the presence of two free fatty acid ratios (oleic acid:palmitic acid = 2:1 and 0:3) were evaluated. A treatment group without free fatty acids served as a control. Hepatotoxicity was assessed using ApoTox-Glo, which assesses viability, cytotoxicity, and apoptosis. TM Evaluation was performed using a triplex assay. In the absence of free fatty acids, the hepatotoxicity of bile acids was increased in the presence of troglitazone (Figure 19). 50 The bile acid concentration at which hepatocytes were 50% toxic was reduced to 44% of the control (from 1.48 μM to 0.65 μM) (Table 2).

[0052] [Table 2]

[0053] Addition of free fatty acids at both oleic acid:palmitic acid ratios further altered the hepatotoxicity of the bile acid pool in the presence of troglitazone. See Figure 20. TC for bile acids in the presence of troglitazone and oleic and palmitic acids (2:1 oleic:palmitic ratio). 50 was reduced to 9.5% of the control (from 1.32 μM to 0.13 μM) (Table 3).

[0054] [Table 3]

[0055] A similar effect was observed in the presence of an oleic acid:palmitic acid ratio of 0:3 (Figure 21). TC of bile acids in the presence of troglitazone and oleic and palmitic acids (oleic acid:palmitic acid ratio of 0:3). 50 was reduced to 17% of the control (from 1.28 μM to 0.22 μM) (Table 4).

[0056] [Table 4]

[0057] The addition of free fatty acids alone at any ratio of oleic acid:palmitic acid had little effect on the hepatotoxicity profile of the pooled bile acids. 50 was reduced to only 89% and 86% of the control when either ratio of free fatty acids (2:1 or 0:3 oleic acid:palmitic acid) was added, respectively. See Table 5 below.

[0058] [Table 5]

[0059] This unexpected effect of free fatty acids on bile acid hepatotoxicity in the presence of troglitazone suggests that the addition of free fatty acids may be a factor in the evaluation of test compounds for their potential to alter bile acid excretion and predict hepatotoxicity.

[0060] Example 6 Applications of cholestatic hepatotoxicity screening assays As mentioned above, drug exposure that inhibits bile acid export pathways (e.g., bile salt export protein (BSEP) and / or MRP3 / 4) and thus 1) prevents (antagonizes) the farnesoid X receptor (FXR) from sensing elevated intracellular concentrations of bile acids, 2) inhibits bile acid efflux via basolateral efflux transporter (OST) α / β, or 3) both, renders hepatocytes susceptible to bile acid toxicity (e.g., cholestatic hepatotoxicity). To further illustrate this finding, sandwich cultures of human hepatocytes (SCHH) were cultured in low glucose (about 2 mM to about 10 mM, or about 5 mM) standard medium or sensitized medium containing low glucose (about 2 mM to about 10 mM, or about 5 mM), or in some embodiments, other suitable energy sources (e.g., galactose), free fatty acids (about 100 μM to about 2 mM, or about 250 μM to about 1.5 mM, or about 500 μM to about 1 mM, or about 1 mM), and bile acid pool (about 5 μM to about 1 mM, or about 50 μM to about 500 μM, or about 100 μM to about 250 μM, or about 250 μM) with a bile salt export protein (BSEP) inhibitor (e.g., cyclosporine A (CsA); Ansede et al., 2010), or a compound with properties of both a bile salt export protein (BSEP) inhibitor and a farnesoid X receptor (FXR) antagonist (e.g., troglitazone; Marion et al., 2007 and Kaimal et al. 2009) for 24 hours.

[0061] Briefly, hepatocytes were established in quadruplicate in a sandwich culture configuration. On day 4 of culture, sandwich cultures of human hepatocytes (SCHH) were exposed to cyclosporine A (CsA) (10 μM; 13×Cmax) or troglitazone (100 μM; 16×Cmax) for 24 hours and cultured in standard or sensitization medium. The concentrations of CsA and troglitazone were based on the Cmax values ​​reported in Dawson et al. (2012). To account for potential convergence in the portal vein after oral administration of the test compounds, drug exposure ranged from approximately 8-fold to approximately 16-fold Cmax. After 24 hours, LDH leakage and ATP content were assessed using commercially available assays. Under standard culture conditions, no significant increase in LDH secretion or significant decrease in ATP content was observed in any of the three SCHH donors treated with either cyclosporine A (CsA) (10 μM; 13×Cmax) or troglitazone (100 μM; 16×Cmax) (Figures 22A and 22B). However, under sensitized culture conditions, exposure to troglitazone, but not CsA, increased LDH secretion to over 490% of control (Tukey's sigma; p<0.05) and decreased ATP content to below 0.9% of control (Tukey's sigma; p<0.05) in the three SCHH donors (Figures 22A and 22B).

[0062] The sensitization medium contains a physiological mixture of free fatty acids and the above-mentioned primary and secondary bile acids (GCDCA, GCA, DCA, and GDCA) at an appropriate concentration (250 μM) to minimize toxicity while challenging hepatocyte bile acid homeostasis mechanisms. After 24 h of exposure to the sensitization medium, LDH secretion did not increase significantly (Tukey's p value <0.05) in two of three human hepatocyte sandwich culture (SCHH) preparations (Figure 23A). In the same donor, ATP content decreased by 24.8% or less in the sensitization medium (Figure 23B). These results indicated that the sensitization medium was well tolerated across multiple donors.

[0063] Under standard culture conditions, in the presence of the cholestatic hepatotoxicant troglitazone, hepatocytes lack sufficient concentrations of bile acids or the appropriate mixtures to induce toxicity. This was demonstrated by the lack of effects on LDH and ATP levels in all three donors examined when treated with troglitazone under standard culture conditions (Figures 24A and 24B). To fully assess the function of the bile acid homeostasis mechanism, hepatocytes require a bile acid load. More than 95% of bile acids secreted after a meal are reabsorbed in vivo from the ileum into the portal vein (Chiang, 2009). Accordingly, in vivo bile acid portal vein concentrations are dynamic, reported to increase more than threefold between fasting and postprandial states, represented by standard and primed media, respectively (Angelin B et al., 1982).

[0064] The primary toxicities associated with cyclosporine A (CsA) therapy in humans are nephrotoxicity and neurotoxicity, not hepatotoxicity, and the incidence is relatively low compared to other treatments (LiverTox Database, Magnasco et al., 2008). In contrast, clinical studies with troglitazone suggest that the incidence of liver injury may be as high as 1:1000 patients, clearly indicating that troglitazone causes liver injury (LiverTox Database). The clear difference in the incidence of clinical liver injury between these two potent bile salt export protein (BSEP) inhibitors suggests that other processes, such as interference with bile acid homeostasis mechanisms (e.g., farnesoid X receptor (FXR) antagonism) may be involved in addition to BSEP inhibition. This clinical liver injury incidence was in complete agreement with the results of the in vivo assays for cyclosporine A (CsA) and troglitazone, suggesting that the cholestatic hepatotoxicity assay can distinguish between cyclosporine A (CsA), a non-drug-induced liver injury (DILI) agent, and troglitazone, a cholestatic drug-induced liver injury (DILI) agent.

[0065] Titration of troglitazone (a thiazolidinedione compound) in three different sandwich culture cultures of human hepatocytes (SCHH) preparations at 50 μM (8×Cmax), 75 μM (12×Cmax), and 100 μM (16×Cmax) established a dose-dependent toxicity manifested only in sensitized media (Figures 24A and 24B). In all three donors treated with troglitazone at concentrations ranging from 50 to 75 μM (8×Cmax to 12×Cmax), LDH leakage of >590% of control and loss of ATP content of >95% of control were observed (Figure 24B). These results demonstrated that the hepatotoxic pathway is not idiosyncratic and that dose-dependent toxicity is conserved across donors. At all concentrations evaluated, no significant increases in LDH leakage or ATP loss were observed in sandwich cultures of human hepatocytes (SCHH) exposed to troglitazone under standard media conditions. These results demonstrated that sandwich cultures of human hepatocytes (SCHH) require specific culture conditions (e.g., sensitized medium) to identify cholestatic DILI agents. These results suggested that under conditions requiring hepatocytes to process large amounts of bile acids (e.g., sensitized conditions), troglitazone disrupts the bile acid homeostasis mechanism, resulting in bile acid or cholestatic hepatotoxicity.

[0066] This conclusion was further supported by the inhibition of biliary clearance of d8-TCA (Figure 25) and farnesoid X receptor (FXR) antagonism (Figure 26) observed in sandwich cultures of human hepatocytes (SCHH) in the presence of troglitazone. The biliary clearance of this model bile acid (d8-TCA) was significantly decreased in a dose-dependent manner in sandwich cultures of human hepatocytes (SCHH) treated with troglitazone (Figure 25A). The biliary excretion of d8-TCA (e.g., biliary excretion index) was also significantly decreased in a dose-dependent manner in sandwich cultures of human hepatocytes (SCHH) exposed to troglitazone (Figure 25B). These results suggest that bile acid excretion across the canalicular domain was reduced in the presence of troglitazone, consistent with a previous report (Marion et al., 2007). Troglitazone (75–100 μM) exposure also prevented the synergistic induction of OSTβ mRNA content in sandwich cultures of human hepatocytes (SCHH) after co-treatment with CDCA and cyclosporine A (CsA) (Figures 26A and 26B). A similar effect was observed in sandwich cultures of human hepatocytes (SCHH) treated with a combination of CDCA, CsA, and DY268 (a novel and potent farnesoid X receptor (FXR) antagonist) (Yu et al., 2014). Alternatively, the decrease in OSTβ mRNA content could be explained by cytotoxicity under the conditions evaluated. However, no significant decrease in ATP content or increase in LDH leakage was observed under the conditions examined, suggesting that the decrease in OSTβ mRNA content was due to antagonism of farnesoid X receptor (FXR) activation (Figure 27).

[0067] We conducted a preliminary evaluation of the predictive accuracy of screening for cholestatic hepatotoxicity using other thiazolidinedione compounds, including pioglitazone and rosiglitazone. Troglitazone, a thiazolidinedione compound and a well-established drug-induced liver injury (DILI) agent, had a clinical liver injury incidence of 1:1000 patients and was removed from the market due to liver injury (LiverTox Database). In contrast, despite widespread use, fewer than 12 cases of liver injury have been reported for either pioglitazone or rosiglitazone (LiverTox Database). In large-scale clinical trials, ALT elevations of more than three times the upper limit of normal were not different from placebo recipients for either pioglitazone or rosiglitazone. This suggests that the potential for liver injury is low for either of these thiazolidinedione compounds. All three thiazolidinedione compounds were evaluated in a cholestatic hepatotoxicity screening assay over a wide range of concentrations (1, 5, 10, 25, 50, and 100 μM), including the Cmax of troglitazone (Cmax: 6.4 μM; Dawson et al. 2012), pioglitazone (Cmax: 2.9 μM; Dawson et al. 2012), and rosiglitazone (Cmax: 1.0 μM; Dawson et al. 2012). In sandwich cultures of human hepatocytes (SCHH) treated with troglitazone (100 μM), a significant increase in LDH leakage (863% of control) and a significant loss of ATP content (>99% of control) were observed only under sensitized culture conditions (Figures 28A and 28B), consistent with previous results. No significant increase in LDH leakage or decrease in ATP content was observed in sandwich cultures of human hepatocytes (SCHH) treated with either pioglitazone or rosiglitazone at any concentration under any culture condition examined (Figures 28A and 28B). These in vitro results were generally consistent with the clinical liver injury incidence of thiazolidinedione compounds, demonstrating the predictive accuracy of the disclosed cholestatic hepatotoxicity screening assay.

[0068] References All references, including, but not limited to, patents, patent applications and publications, scientific journal articles, and database entries (e.g., GENBANK® database entries and all annotations available therein), to the extent that they supplement, describe, provide background to, or teach the methodologies, techniques, and / or compositions used herein, are incorporated herein by reference in their entirety. ○ Ansede JH, Smith WR, Perry CH, St. Claire III RL, and Brouwer KR (2010) An in vitro assay to assess transporter-based cholestatic hepatotoxicity using sandwich-cultured rat hepatocytes. Drug Metab and Dispos 38:276-280. ○ Marion TL, Leslie EM and Brouwer KLR (2007) Use of sandwich-cultured hepatocytes to evaluate impaired bile acid transport as a mechanism of drug-induced hepatotoxicity. Mol Pharmaceutics 4(6): 911-918. ○ Kaimal R, Song X, Yan B, King R, and Deng R (2009) Differential modulation of farnesoid X receptor signaling pathway by the thiazolidinediones. J Pharmacol Exp Ther 330: 125-134. ○ Dawson S, Stahl S, Paul N, Barber J, and Kenna JG (2012) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab and Dispos 40: 130-138. ○ Angelin B, Bjorkhem I, Einarsson K, and Ewerth S (1982) Hepatic uptake of bile acids in man. Fasting and postprandial concentrations of individual bile acids in portal venous and systemic blood serum. J Clin Invest. 70: 724-731. ○ Chiang JYL (2009) Bile acids: regulation of synthesis. J Lipid Res 50: 1955-1966. ○ Magnasco A, Rossi A, Catarsi P, Gusmano R, Ginevri F, Perfumo F, Ghiggeri GM (2008) Cyclosporin and organ specific toxicity: clinical aspects, pharmacogenetics and perspectives. Curr Clin Pharmacol. 3(3):166-73. ○ Yu DD, Lin W, Forman BM, and Chen T (2014) Identification of trisubstituted-pyrazol carboxamide analogs as novel and potent antagonists of farnesoid X receptor. Bioorg Med Chem 22: 2919-2938. ○ Dawson S, Stahl S, Paul N, Barber J, and Kenna JG (2012) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab and Dispos 40: 130-138. ○ Hartman et al. Can J. Physiol. Pharmacol. 2010. 88:682-691. It will be understood that various details of the invention may be changed without departing from the scope of the invention. Further, the above description is by way of example only and not by way of limitation. [Explanation of symbols]

[0069] 10 Hepatocyte system 12 Hepatocytes 14 Tight Junctions 16 Bile duct

Claims

1. 1. An in vitro system for predicting the in vivo cholestatic hepatotoxic potential of a compound, comprising: (i) an in vitro cultured hepatocyte cell line (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation; (ii) one or more bile acids with established toxic potency in the hepatocyte cell line (HCS), and (iii) a means for an assay for measuring the toxic potency of said one or more bile acids in the absence or presence of said compound, wherein said HCS is exposed to a range of concentrations of said one or more bile acids in the absence or presence of said compound, and a toxic profile of said one or more bile acids is determined, said toxic profile comprising the toxic potency of said one or more bile acids; Including, an increase in the toxic potency of the one or more bile acids in the presence of the compound compared to the toxic potency of the one or more bile acids in the absence of the compound in the assay indicates the compound has the potential to cause cholestatic hepatotoxicity; A system wherein a decrease in the toxic potency of the one or more bile acids in the presence of the compound compared to the toxic potency of the one or more bile acids in the absence of the compound in the assay indicates the potential for the compound to cause cholestasis resulting in systemic toxicity.

2. 2. The in vitro system of claim 1, wherein the in vitro cultured hepatocyte cell line (HCS) comprises an integrated hepatocyte cell line with bile acid synthesis, transport, and bile acid homeostasis feedback mechanisms.

3. 3. The in vitro system of claim 1 or 2, wherein the in vitro cultured hepatocyte cell line (HCS) comprises a two-dimensional or three-dimensional culture utilizing primary hepatocytes or other hepatocyte cell lines.

4. 4. The in vitro system of claim 1, wherein the in vitro cultured hepatocyte cell line (HCS) comprises a sandwich culture (SCH) of hepatocytes, the SCH comprising human hepatocytes.

5. 5. The in vitro system of any one of claims 1 to 4, wherein the in vitro cultured hepatic cell line (HCS) comprises a three-dimensional (3D) scaffold-based culture or a spheroid culture.

6. 6. The in vitro system of any one of claims 1 to 5, wherein the in vitro cultured hepatocyte cell line (HCS) comprises HepaRG, Huh7, co-culture system, stem cell-derived hepatocytes, and combinations thereof.

7. 7. The in vitro system of claim 1, wherein the one or more bile acids are GCA, GCDCA, GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof.

8. 8. The in vitro system of any one of claims 1 to 7, wherein the established toxic potency of the one or more bile acids is based on hepatotoxicity responses using a means for cytotoxicity assay.

9. 9. The in vitro system of claim 8, wherein the means for cytotoxicity assay is selected from the group consisting of means for measuring enzyme leakage, ATP, APOTOX Glo, and combinations thereof.

10. 10. The in vitro system of claim 9, wherein the enzyme leakage is selected from the group consisting of ALT leakage, AST leakage, and LDH leakage.

11. 11. An in vitro system according to any one of claims 1 to 10, configured to characterize compounds that do not cause a change in the toxic potency of bile acids as compounds that are unlikely to cause systemic or hepatotoxicity.

12. 12. An in vitro system according to any one of claims 1 to 11, configured to characterize compounds that cause an increase in the toxic potency of bile acids as compounds that may cause cholestatic hepatotoxicity.

13. 13. An in vitro system according to any one of claims 1 to 12, configured to characterize compounds that cause a reduction in the toxic potency of bile acids as compounds that may cause cholestasis leading to systemic toxicity.

14. 14. The in vitro system according to any one of claims 1 to 13, wherein the HCS comprises one or more free fatty acids and / or glucose at a predetermined concentration.

15. 15. The in vitro system of claim 14, wherein the HCS comprises one or more free fatty acids.

16. 1. A method for screening compounds for their potential to cause systemic and / or hepatotoxicity, comprising: (a) providing a compound to be screened; (b) establishing a hepatocyte cell line (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation; (c) exposing the hepatic cell line (HCS) to a range of concentrations of bile acids to determine a toxicity profile of the bile acids, wherein the toxicity profile of the bile acids comprises a toxic potency; (d) exposing the hepatic cell line (HCS) to a range of concentrations of bile acids in the presence of the compound to be screened and determining the toxicity profile of the bile acids, wherein the toxicity profile of the bile acids comprises toxic potency; and (e) comparing the toxic potency of the bile acid between step (c) and step (d) to determine the potential of the compound to cause systemic and / or hepatotoxicity. Including, A method wherein the HCS contains one or more free fatty acids and / or glucose at a predetermined concentration, and ATP content and / or LDH leakage are measured.

17. 1. A method for screening compounds for their potential to cause systemic and / or hepatotoxicity, comprising: (a) providing a compound to be screened; (b) establishing a hepatocyte cell line (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation; (c) exposing a hepatic cell line (HCS) to a range of concentrations of bile acids in the absence or presence of a compound to be screened, and determining a toxicity profile of the bile acids in the absence or presence of the compound to be screened, wherein the toxicity profile of the bile acids in the absence of the compound to be screened is first determined over the concentration range, and the toxicity profile of the bile acids comprises the toxic potency of the bile acids; and (d) comparing the toxic potency of bile acids in the presence and absence of the compound to determine the susceptibility of the compound to causing systemic and / or hepatotoxicity. Including, A method wherein the HCS contains one or more free fatty acids and / or glucose at a predetermined concentration, and ATP content and / or LDH leakage are measured.

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