Methods and systems for screening candidate compounds for their potential to cause systemic or hepatic toxicity

Abstract

To assess the potential of a new chemical entity (NCE), compound or drug candidate to cause cholestatic hepatotoxicity in vivo.SOLUTION: A method of screening a compound for its potential to cause systemic or hepatic toxicity comprises the steps of: providing a compound to be screened; establishing a hepatic cell system (HCS) having a capacity for bile acid synthesis, bile acid transport and / or bile acid regulation; exposing the hepatic cell system (HCS) to a range of concentrations of a bile acid to determine a toxicity profile of the bile acid, where the bile acid toxicity profile comprises a toxicity potency; exposing the hepatic cell system (HCS) to a range of concentrations of a bile acid in the presence of the compound to be screened and determining a toxicity profile of the bile acid, where the bile acid toxicity profile comprises a toxicity potency; and comparing the toxicity potency of the bile acid to determine the susceptibility of the compound to cause systemic and / or hepatic toxicity.SELECTED DRAWING: None

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. and is incorporated herein by reference in its entirety. [Technical Field]

[0002] The present invention provides a method for screening candidate compounds or chemicals for potential cholestatic hepatotoxicity. More particularly, the present invention relates to the treatment of steroids that cause systemic or hepatotoxicity. To screen candidate compounds for susceptibility to or potential to cause The present invention relates to a method and a system. [Background technology]

[0003] In the development of new therapeutic agents, drugs and pharmaceutical compounds, their cholestatic hepatotoxicity New chemical entities (NCEs) need to be screened to determine their potential. Cholestatic hepatotoxicity induced by benzodiazepine is the result of impaired bile acid flow, resulting in liver damage. Accumulation of toxic concentrations of bile acids in cells, leading to drug-induced cholestatic hepatotoxicity High potential new chemical entities (NCEs) are excluded from consideration for further drug development testing. There is a possibility that this will occur. Summary of the Invention [Problem to be solved by the invention]

[0004] New Chemical Entities (NCEs), compounds, or drug candidates cause cholestatic hepatotoxicity in vivo. Improved in vitro methodologies and systems for assessing the potential for inflammatory bowel disease are needed. are. [Means for solving the problem]

[0005] The above needs are addressed by the present invention. Below, several embodiments of the present invention are listed, and in many cases, Variations and substitutions are listed below. The following are merely exemplary of the many different embodiments. Reference to one or more representative features of the embodiments listed above is likewise exemplary. Such embodiments may generally exist with or without the features recited. Similarly, these features, whether or not listed herein, are also included in other aspects of the invention. To avoid excessive repetition, all such features are described below. It does not list or suggest all possible combinations.

[0006] In some embodiments, the potential for causing systemic and / or hepatotoxicity A method for screening a compound, comprising providing a compound to be screened. A hepatic cell line (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation is provided. Establishing a range of bile acid concentrations to determine the toxicity profile of the bile acid. exposing a hepatic cell line (HCS) to a bile acid, wherein the bile acid toxicity profile comprises toxic potency. In this step, the hepatocyte cell line (HCS) is incubated with a range of bile duct concentrations in the presence of the compound to be screened. and determining a bile acid toxicity profile, The toxic effects of the bile acid and the bile acid are compared to determine systemic toxicity and / or hepatic toxicity. determining the susceptibility of said compound to causing toxicity.

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

[0008] In some embodiments, the susceptibility to causing systemic toxicity and / or hepatotoxicity A method for screening a compound, comprising providing a compound to be screened. A hepatic cell line (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation is provided. Establishing a step of inducing hepatocytes to a range of bile acids in the presence of the compound to be screened. Exposing the HCS to the toxicity profile of bile acids in the presence of the compounds being screened determining the toxicity profile of the bile acid over the concentration range; wherein the bile acid toxicity profile of the bile acid comprises the toxic potency of the bile acid; and To compare the toxic potency of bile acids in the presence and absence of determining the potential of the compound to cause

[0009] It is therefore an object of the present invention to evaluate compounds for their susceptibility to causing systemic and / or hepatotoxicity. The present invention provides a method and system for screening substances. The other objects of the present invention are achieved in whole or in part by the present invention. The above and other objects and advantages of the present invention will become apparent to those skilled in the art after studying the following description, drawings and examples. It will be clear to those who [Brief explanation of the drawings]

[0010] The present invention can be better understood by reference to the following figures, in which: The components are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. In these drawings, like reference numbers refer to different Corresponding parts are shown in the figures. Reference is made to the embodiments illustrated in the figures of the accompanying drawings, in which: A further understanding of the present invention can be obtained by the following. Although the present invention is merely exemplary of a system for implementing the present invention, both the structure and method of operation of the present invention are not intended to be limiting. The present invention, together with further objects and advantages thereof, will be more readily understood by reference to the drawings and accompanying description. These drawings are intended to be illustrative and not restrictive of the principles set forth in the appended or later amended claims. It is not intended to limit the scope of the invention as described, but merely to clarify the invention. It is intended to illustrate and illustrate the principles of 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 more fully described below, in which some, but not all, embodiments of the present invention are described. Indeed, the present invention can be embodied in many different forms, It should not be construed as being limited to the embodiments set forth herein. 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. It is not intended to limit 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 for purposes of describing the present invention. It is shown for ease of understanding.

[0012] All technical and scientific terms used herein are defined as follows unless otherwise defined below: , are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed in this document are intended to refer to techniques as commonly understood in the art. The following are examples of the present invention, including variations or equivalent technical substitutions of those techniques that are apparent to those skilled in the art. Although the terms are believed to be well understood by those skilled in the art, the following definitions are provided to facilitate the description of the present invention. This is intended to In describing the present invention, it will be understood that several techniques and steps are disclosed. Each of these has its own advantages, and each also provides one of the techniques disclosed in the others. It can be used in conjunction with one or more, or possibly all. Therefore, in the following description, for the sake of clarity, all possible combinations of the individual steps will be described. Nevertheless, the specification and claims will refrain from repeating the same in an unnecessary manner. The scope of the claims is understood to be such that such combinations are fully within the scope of the present invention and claims. It should be read with understanding.

[0013] In accordance with long-standing patent law practice, the terms "a," "an," and "the" are used in patent applications. As used in this application, including the claims, it refers to "one or more." For example, a reference to "a cell" includes a plurality of such cells, and so forth. Unless otherwise specified, the amounts of ingredients, reaction conditions, etc. used in the specification and claims are All numbers in the figures should be understood to be modified in all instances by the term "about." Therefore, unless otherwise indicated, the numbers set forth in this specification and claims The value parameters are approximations and do not necessarily represent the desired properties sought to be obtained by the present invention. It may change depending on the situation.

[0014] As used herein, a value or amount of a composition, a dose, a sequence identity (e.g., 2 or more) (when comparing more than 100 nucleotide or amino acid sequences), mass, weight, temperature, time, When referring to a product, concentration, percentage, and the like, the term "about" refers to a range of values from the stated value to an extent that the range is within a certain embodiment. In some embodiments, ±20%, in some embodiments, ±10%, in some embodiments, ±5%, In some embodiments, ±1%, in some embodiments, ±0.5%, in some embodiments, ±0 It is intended to include a 0.1% variation, however, that such variations are within the scope of the disclosed method. or be suitable for use in the disclosed compositions.

[0015] Synonymous with "including," "containing," or "characterized by" The term "comprising" is inclusive or open-ended and is not intended to be a citation. It does not exclude additional elements or method steps. "Comprising" is used in claim language. It is a term of art, and although the named elements are present, other elements may be added and still be to form a structure or method within the scope of the claims. As used herein, the phrase "consisting of" shall mean any material that is The phrase "consisting of" excludes any element, step, or ingredient that is not included in the preceding sentence. If it appears in a clause in the body of a claim rather than immediately afterwards, it is The invention is limited to only those elements which are listed, and does not exclude other elements from the scope of the entire claim. As used herein, the phrase "consisting essentially of" extends a claim to: The materials or steps specified and the basic and novel feature(s) of the claimed subject matter ) and will be limited to those that do not have a substantial impact on the With respect to the terms "comprising," "consisting of," and "consisting essentially of," When one of the terms is used herein, the claimed subject matter of this disclosure includes the other. It can involve the use of either of the two terms As used herein, when used in the context of listing entities, the term "and / or" refers to The terms "A, B, C, The phrase "and / or D" includes A, B, C, and D individually, but also A, B, C, and D.

[0016] As used herein, a hepatocyte cell line (HCS) is a cell line that is involved in bile acid synthesis, bile acid transport, and / or refers to a hepatic cell line that has the ability to regulate bile acids. In some embodiments, this hepatic cell line (HCS) is a sandwich culture of hepatocytes, including sandwich cultures of human hepatocytes (SCHH). These include, but are not limited to, two-dimensional cultures such as spheroids (SCH). In some embodiments, the hepatic cell line (HCS) is grown in three-dimensional culture (e.g., in a 3D scaffold). These may include, but are not limited to, cultures based on spheroids, spheroid cultures, etc. This hepatocyte cell line (HCS) can be cultured using primary hepatocytes or other related hepatocyte cell lines, such as HepaRG, Huh 7, co-culture systems, and / or stem cell-derived hepatocytes. In hepatocyte cell lines (HCS), such as in the incubation medium of hepatocyte cell lines (HCS), In this case, one or more free fatty acids and / or preselected concentrations of glucose are used. do.

[0017] As used herein, the term "susceptibility to causing systemic or hepatotoxicity" refers to a condition in which a compound or compound(s) is or are present in a compound or compound(s) that ... and a system for screening candidate compounds for their potential to cause As used herein, the terms "ability," "susceptibility," and "potential" of a candidate compound are used interchangeably. Terms used to refer to "likelihood," "probability," "likelihood," etc. are used interchangeably; Generally, a compound may be used to treat a variety of conditions, including but not limited to, system or liver toxicity, as disclosed and discussed herein. and conditions related thereto, which may affect, cause, or contribute to such conditions. It suggests that there is.

[0018] The present invention provides, in some embodiments, a new chemical entity (NCE), compound, or drug candidate. In vitro methodology for assessing the potential to cause cholestatic hepatotoxicity in vivo The primary bile acid may be one or more of the following: It is synthesized in liver cells by liver enzymes and then rapidly binds to both taurine and glycine, and can be further modified by glucuronidation and sulfation. Bile Acids and Conjugations The body excretes them into the bile via bile salt export protein (BSEP) and MRP2, and MRP Bile acids are excreted into the portal circulation by serotonin 3 / 4 or OSTα / β. Bile acids are further metabolized in the intestine. It is absorbed into enterocytes by ASBT and excreted from enterocytes into the portal vein by OSTα / β. Acid is taken up from the portal circulation mainly by NTCP. However, some studies have shown that OA It has also been suggested that TP may be involved to some extent in the uptake. They can act as surfactants, and high intracellular concentrations (ICC) of bile acids are hepatotoxic. It has been shown that

[0019] Drug-induced cholestatic hepatotoxicity is generally caused by the inhibition of bile salt excretion by new chemical entities (NCEs). This is thought to be the result of impaired bile acid flow resulting from the inhibition of only the BSEP. However, hepatocellular bile acid homeostasis mediated by the farnesoid X receptor (FXR) is Impairment of the osteoadaptive response may play a pivotal role in drug-induced liver injury. Highly reliable. Without being bound by any particular theory or mechanism of action and / or current dogma, Inhibition of bile salt export protein (BSEP) by chemicals (NCE) increases intracellular bile acid concentrations. When the intracellular concentration of bile acids is high enough, In this case, bile acids activate the farnesoid X receptor (FXR), which inhibits the expression of CYP7A1. The rate at which bile acid synthesis begins to decrease is limiting for the enzymes involved in bile acid production. This results in the induction of both bile salt export protein (BSEP) and OSTα / β expression. Since NCE inhibits transport along this pathway, this bile salt export protein (BSE) Increased expression of OSTα / β would have minimal effect. Induction significantly increased bile acid clearance from hepatocytes into the portal circulation, thereby increasing hepatic The cells reduce the intracellular concentration of bile acids and reduce the potential for hepatotoxicity. The proposed mechanism for cholestatic hepatotoxicity is potential cholestatic hepatotoxicity. The substance is involved in the bile acid export pathway (e.g., bile salt export protein (BSEP) and / or MRP3 / 4) and 1) inhibit the farnesoid X receptor (FXR) from increasing intracellular bile acid concentrations. or 2) by preventing (antagonizing) biliary secretion via OSTα / β (the bile acid compensatory pathway). 3) inhibition of cerebrospinal fluid secretion, or 3) a combination of both.

[0020] Unless necessary, to accurately predict the effects on hepatic excretion of bile acids in vivo. An integrated system such as that provided herein can be important. In some embodiments, This integrated system involves synthesis, transport (uptake, basolateral excretion, and canalicular excretion), and The sandwich culture of human hepatocytes (SCHH) model includes regulation (transport, metabolism, and synthesis). , a single distinctive system that can combine all of these pathways.

[0021] The methods and systems of the present invention are directed to measuring bile salt export protein (BSEP) inhibition. Using a sandwich culture of human hepatocytes (SCHH) model and the farnesoid X receptor (FXR) R) and bile acid regulation. It was observed that the FXR feedback mechanism is the driving force behind activation This intracellular bile acid concentration is an in vitro methodology for predicting cholestatic hepatotoxicity. The intracellular concentration (ICC) of bile acids is a factor in the development of farnesoid X receptors. It was determined that FXR is required to activate the FXR feedback mechanism. Different bile acids are known to have different potential for hepatotoxicity, so multiple Bile acids and their combinations (bile acid pool) were assayed using standard assays for toxicity. The potential for inducing hepatotoxic responses was assessed (e.g., ATP, LDH, caspase 3 / 7 and APOTOX Glo, but are not limited to these. A mixture of various bile acids, including dihydrochloride (DCA), has been identified as one of the most potent hepatotoxic bile acids. The use of these assays allows for the determination of the relationship between cell viability, necrosis, and apoptosis in this system. This made it possible to distinguish between

[0022] Generally, the methods of the present invention, in some embodiments, involve determining the toxicity profile of bile acids. To identify the bile acids, sandwich cultures of human hepatocytes (SCHH) were incubated with a range of bile acids (e.g., This may include incubation with a bile acid pool (e.g., ATP and / or DCA). (Figures 7 and 8, dotted lines). Sandwich cultures of human hepatocytes (SCHH) were cultured in the presence of various cholestatic hepatotoxic substances. Exposure to a range of DCA concentrations or a combination of bile acids can result in three possible outcomes: The results can be observed. 1. No change in bile acid (BA) toxic potency in the presence of new chemical entities (NCE) = predicted effect none. 2. Increased bile acid (BA) toxic potency in the presence of new chemical entities (NCEs) = cholestatic hepatotoxicity May cause side effects (e.g., bile acid (BA) efflux inhibitors, farnesoid X receptor (FXR) antagonists, or both). 3. The toxic potency of bile acids (BAs) is reduced in the presence of new chemical entities (NCEs) = causing cholestasis However, they may result in systemic toxicity (e.g., bile acid (BA) uptake inhibitors).

[0023] Therefore, new chemical entities (NCEs) do not affect bile acid homeostasis (1). or cholestatic hepatotoxicity, whether associated with elevated systemic bile acid concentrations (2) or those with the possibility of clinical cholestasis being observed. (3) (systemic bile acid concentrations are elevated, but hepatic cholestasis is unlikely), or can be classified into combinations of these.

[0024] Thus, in some embodiments, the compounds described herein may be used without causing systemic and / or hepatotoxicity. Methods for screening compounds for their potential to induce inflammatory bowel disease are provided. In embodiments, the method comprises the steps of: (a) providing a compound to be screened; (b) a hepatocyte cell line (HCS) capable of bile acid synthesis, bile acid transport, and / or bile acid regulation; (c) determining the toxicity profile of bile acids at a range of bile concentrations. exposing the hepatic cell line (HCS) to bile acid, wherein the bile acid toxicity profile is (d) potency of the hepatic cell line (HCS) in the presence of the compound being screened. exposing the animal to a range of concentrations of bile acids to determine the toxicity profile of the bile acids; (e) comparing the toxic potencies of the bile acids, wherein the bile acid toxicity profile comprises a toxic potency. and determining the potential of the compound to cause systemic and / or hepatotoxicity. do.

[0025] In some embodiments, the compound is used to determine its potential to cause systemic and / or hepatotoxicity. The determining step may further comprise determining, in the presence of the compound, as compared to the absence of the compound, determining that there is no change in the toxic potency of the bile acid in the presence of the compound; determining an increase in the toxic potency of the bile acid compared to the absence of the compound; or In the presence of the compound, the toxic potency of the bile acid is reduced compared to the absence of the compound. Compounds that do not cause a change in the toxic potency of bile acids are known to be effective in reducing the systemic toxicity of bile acids. It is characterized as not causing toxicity or hepatotoxicity.

[0026] However, compounds that cause an increase in the toxic potency of bile acids may induce cholestatic hepatotoxicity. This cholestatic hepatotoxicity is characterized by impaired bile acid excretion. This can be caused by the inhibition of bile acid excretion (in which case the compound is characterized as a bile acid excretion inhibitor). This cholestatic hepatotoxicity is due to the farnesoid X receptor (FX R) antagonism (in this case, the compound acts against the farnesoid X receptor) (FXR) antagonists), and / or a combination of both. In some embodiments, the inhibition of bile acid excretion comprises inhibition of bile salt excretion protein (BSEP). include. Nevertheless, compounds that cause a decrease in the toxic potency of this bile acid may result in systemic toxicity. This systemic toxicity is characterized as having the potential to cause cholestasis. Cholestasis, which can occur, is caused by inhibition of bile acid uptake.

[0027] In some embodiments, determining the potential of the compound to cause systemic or hepatotoxicity described this compound as a bile acid export inhibitor, a farnesoid X receptor (FXR) antagonist, Some embodiments include characterizing the compound as a bile acid uptake inhibitor, or the absence of the above. In an embodiment, the compound can be a candidate drug, and the candidate drug has systemic toxicity and and / or liver toxicity, or as being unlikely to cause systemic and / or liver toxicity. It is characterized as having a high likelihood of rubbing. In some embodiments, bile acid transport is determined by bile acid uptake, basolateral excretion, and / or vasoconstriction. This range of bile acid concentrations is consistent with intracellular fasting and / or postprandial excretion in vivo. This range of bile acid concentrations includes intracellular concentrations that mimic the farnesoid X receptor (FRX) Contains intracellular concentrations of bile acids sufficient to activate the FXR feedback mechanism.

[0028] In some embodiments, determining the toxicity profile of a bile acid comprises using a cytotoxicity assay. Such cytotoxicity assays may include measuring hepatotoxic responses. Selected from the group consisting of: ATP, APOTOX Glo, and combinations thereof This enzyme leakage assay can be performed on, but is not limited to, ALT, AS It may be selected from the group consisting of T and LDH. In some embodiments, the potential for causing systemic and / or hepatotoxicity The method for screening compounds further involves exposing a hepatic cell line (HCS) to multiple bile acids. determining a plurality of toxicity profiles for the plurality of bile acids; The acid toxicity profile may include multiple toxicity potencies, which may include: To predict the hepatotoxic potential of a compound, hepatocyte cell lines were cultured in the absence and presence of the compound. (HCS).

[0029] In some embodiments, the potential for causing systemic and / or hepatotoxicity Methods for screening compounds include secondary hepatocytes using primary hepatocytes or other hepatocyte cell lines. Hepatocyte cell lines (HCS) are available in either original culture or three-dimensional culture. The culture can include a sandwich culture of hepatocytes (SCH), the SCH being derived from human liver. The three-dimensional cultures include three-dimensional (3D) scaffold-based cultures or spheroid cultures. Other hepatocyte cell lines include HepaRG, Huh7, co-cultures, and stem cells. Cells that are useful for the treatment of hepatic encephalopathy include, but are not limited to, stem cells, hepatocyte-derived cells, and combinations thereof. In some embodiments, the compound inhibits hepatocyte cell lines (H CS). This bile acid or acids can be GCA, GCDCA, , GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLC A, or any combination thereof. Furthermore, in this hepatic cell line (HCS), Further concentrations of free fatty acids and / or predetermined glucose can be employed. .

[0030] In some embodiments, an index for predicting the in vivo hepatotoxic potential of a compound is provided. An in vitro system comprising: (i) a cell capable of bile acid synthesis, bile acid transport, and / or bile acid regulation; (ii) an in vitro cultured hepatocyte cell line (HCS) that is capable of expressing the hepatocyte cell line (HCS); one or more bile acids having toxic effects, and (iii) in the presence of one or more bile acids an assay for measuring the hepatotoxicity of the compound when exposed to said hepatocyte cell line (HCS) at A system is provided that includes: In some embodiments, the in vitro cultured hepatic cell line (HCS) is a cell line that expresses bile. It contains an integrated hepatocyte system with bile acid synthesis, transport, and bile acid homeostasis feedback mechanisms. This in vitro cultured hepatocyte cell line (HCS) can be cultured using primary hepatocytes or their This may include two-dimensional or three-dimensional cultures using other hepatocyte cell lines. In vitro cultured hepatocyte cell lines (HCS) include sandwich cultures of hepatocytes (SCH). The SCH may contain human hepatocytes. This in vitro cultured hepatocyte cell line (HC) S) can include three-dimensional (3D) scaffold-based cultures or spheroid cultures. The in vitro cultured hepatocyte cell lines (HCS) include HepaRG, Huh7, co-culture systems, stem cell-derived hepatocytes, and combinations thereof.

[0031] In some embodiments, the bile acid or acids are GCA, GCDCA, GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA , or any combination thereof. The established toxic potency of one or more bile acids can be assessed by hepatotoxicity assays using cytotoxicity assays. The toxicity profile of the one or more bile acids may be based on a physiological response. Cytotoxicity assays such as enzyme leakage assay, ATP, APOTOX Glo, and The enzyme leakage assay includes, but is not limited to, For example, ALT, AST and LDH.

[0032] In some embodiments, such an in vitro system is used to alter the toxic potency of bile acids. Compounds that do not cause systemic or hepatotoxicity are considered to be compounds that are unlikely to cause systemic or hepatotoxicity. In some embodiments, such interfaces can be configured to be characterized as In vitro systems were used to identify compounds that cause an increase in the toxic potency of bile acids, and to identify cholestatic hepatotoxins. It can be constructed to characterize compounds that may cause toxicity. In some embodiments, such in vitro systems can be used to reduce the toxic effects of bile acids. Compounds that cause cholestasis leading to systemic toxicity were identified as compounds that could cause cholestasis. It can be configured to characterize.

[0033] In some embodiments, the potential for causing systemic and / or hepatotoxicity Methods for screening compounds are provided. In some embodiments, the methods The method comprises the steps of (a) providing a compound to be screened; (b) determining whether bile acid synthesis, bile acid (c) establishing a hepatocyte cell line (HCS) capable of transporting and / or regulating bile acids; Hepatocyte cell lines (HCS) were exposed to a range of bile acid concentrations in the presence of the compound to be screened. determining the toxicity profile of the bile acid in the presence of the compound being screened. Therefore, the toxicity profile of the bile acid is known over the concentration range, and the bile acid (d) the bile acid toxicity profile comprises the toxic potency of the bile acid in the presence of the compound and Compare the toxic potency of bile acids in the absence, causing systemic and / or hepatotoxicity In such a method, the compound is The compounds can be exposed to hepatic cell lines (HCS) over a range of concentrations.

[0034] In some embodiments, the bile acid may comprise a combination of bile acids, The toxicity profile of this bile acid combination is known, and this toxicity profile is This combination of bile acids includes the potency of the bile acids provided in a predetermined ratio. This combination of bile acids can be used to control the concentration of bile acids in vivo. The composition may comprise a plurality of bile acids in concentrations and ratios configured to mimic the ratios of bile acids in the composition. The steps to determine the susceptibility of a compound to cause this systemic and / or hepatotoxicity are several. In some embodiments, the present invention further provides a method for determining the amount of hydroxybenzoates in the presence of the compound compared to the absence of the compound. and determining that there is no change in bile acid toxicity potency in the presence of the compound, compared to the control. determining an increase in bile acid toxicity compared to the absence of the compound, or determining a reduction in bile acid toxicity in the presence of the compound compared to the absence of the compound. may include:

[0035] Compounds that do not cause changes in the toxic potency of bile acids are classified as compounds that do not cause systemic 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 ( the compound is characterized as a bile acid export inhibitor), and / or Stagnant hepatotoxicity is caused by antagonism of the farnesoid X receptor (FXR) In this case, the compound acts as a farnesoid X receptor (FXR) antagonist. This inhibition of bile acid excretion is characterized by: This may include inhibition of bile salt export protein (BSEP). Nevertheless, compounds that cause a decrease in the toxic potency of this bile acid are not known to cause systemic toxicity. This systemic toxicity can be characterized as having the potential to cause cholestasis. Cholestasis resulting from cholestasis can be caused by inhibition of bile acid uptake.

[0036] In some embodiments, the potential of the compound to cause systemic or hepatotoxicity is determined. The first step is to identify this compound as a bile acid export inhibitor, a farnesoid X receptor (FXR) antagonist, and The compound is characterized as a bile acid uptake inhibitor, a bile acid uptake inhibitor, or the absence of the above. The drug may be a candidate drug, which may cause systemic toxicity and / or hepatotoxicity. are particularly categorized as having a low potential to cause systemic and / or hepatotoxicity or as having a high potential to cause systemic and / or hepatotoxicity. be assigned. Bile acid transport can include bile acid uptake, basolateral excretion, and / or canalicular excretion. The bile acid concentration range is determined to mimic fasting and / or postprandial concentrations in vivo. This range of bile acid concentrations is due to the farnesoid X receptor (FXR) ) It can contain intracellular concentrations of bile acids sufficient to activate the feedback mechanism. In some embodiments, the bile acid or acids are GCA, GCDCA, GD CA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA, or may be any combination thereof, but is not limited to these.

[0037] In some embodiments, the determination of the toxicity profile of the bile acid is performed using a cytotoxicity assay. The cytotoxicity assay can include measuring hepatotoxicity responses using a cytotoxicity assay. For example, enzyme leakage assays, ATP, APOTOX Glo, and combinations thereof. The enzyme leakage assay may be, for example, ALT, AST, and LDH. stomach. In such methods, the hepatocyte cell line (HCS) may be a primary hepatocyte or other hepatocyte cell line. This two-dimensional culture can include two-dimensional culture or three-dimensional culture utilizing The invention also includes a sandwich culture of hepatocytes (SCH), which can include human hepatocytes. On the other hand, this three-dimensional culture is also called a three-dimensional (3D) scaffold-based culture or a spheroid culture. Other liver cell lines include HepaRG, Huh7, co-culture systems, stem cells, etc. Finally, such methods may include hepatocyte-derived hepatocytes, and combinations thereof. In this hepatic cell line (HCS), one or more free fatty acids and / or a predetermined group The concentration of the glucose may be employed. [Example]

[0038] The following examples are included to further illustrate various embodiments of the invention. Those skilled in the art will appreciate that, in light of the present disclosure, many modifications can be made to the specific embodiments disclosed. and still achieve the same or similar results without departing from the spirit and scope of the present invention. can be obtained.

[0039] Example 1 Bile acid homeostasis As demonstrated herein, drug-induced cholestatic hepatotoxicity was significantly associated with the administration of new chemical entities (NCs). Inhibition of bile salt export protein (BSEP) by E) and hepatocyte bile acid homeostasis adaptation This is the result of impaired bile acid flow through impaired bile acid response. Drug-induced cholestatic hepatotoxicity One possible mechanism of action that may cause this is the biliary 1) inhibiting the steroid efflux protein (BSEP) (early injury), and 2) farnesoid X receptor 2. Preventing the receptor (FXR) from sensing an increase in intracellular bile acid concentration (antagonism) ) inhibiting all bile acid excretion pathways (basolateral and canalicular), or 3) a combination of both. It can consist of a combination of:

[0040] 1-6 illustrate tight junctions 14 and one or more bile ducts 16 therebetween. 1 is a schematic diagram of a hepatocyte cell line 10 (in vivo or in vitro) comprising a plurality of hepatocytes 12 having The components, enzymes and transporters within this cell line include: bile acids ( BA); adenosine triphosphate (ATP); farnesoid X receptor (FXR); bile salt export protein 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 transporter (OST); and bile acid synthase (Cyp7A1). These are discussed and defined here. . Figure 1A shows the normal bile acid homeostasis pathway in vivo, while Figure 1B shows the latent Figure 1 shows compromised bile acid homeostasis ultimately leading to drug-induced cholestatic hepatotoxicity. In B, farnesoid X receptor (FXR), bile salt export protein (BSEP) and and / or one or more of the basolateral efflux transporters (OSTs) are blocked or inhibited, resulting in the release of bile acids This causes interference with the homeostasis and transport of

[0041] Thus, in some embodiments, the screening assays, methods and systems of the present invention The system includes the following conceptual components: -Bile salt export protein (BSEP) inhibition -Bile acid homeostasis (farnesoid X receptor (FXR)) feedback mechanism M -Utilizing an integrated hepatocyte system that maintains bile acid transport and bile acid homeostasis feedback mechanisms The sandwich culture of human hepatocytes (SCHH) system is described above. Meet the first two items. -Sandwich culture of human hepatocytes (SCHH) system to behave more like in vivo Recognition of the need for additional bile acids (e.g., fasting and postprandial); the system Bile acids in response to fasting and postprandial model conditions in the presence and absence of substances (NCE) (BA) dose is used. - physiologically relevant bile acids or bile acid pools (i.e., DCA) that induce toxic reactions ) and / or -Relate cholestasis to toxicity in initial in vivo assays.

[0042] Example 2 Bile acid homeostasis feedback mechanism Figure 2 shows normal bile acid (BA) uptake (NTCP), synthesis (CYP7A1), and excretion in vivo. (bile salt export protein (BSEP)). Figure 3 shows that the new chemical entity (NCE) acts as a bile salt export protein (BSEP) inhibitor. , bile acid homeostasis feedback mechanisms (e.g., farnesoid X receptor (FXR) activation of the basolateral efflux transporter OSTα, which results in compensatory mechanisms / β) and a decrease in intracellular bile acid concentration. Initiating mechanisms prevent bile acid hepatotoxicity (also known as cholestatic hepatotoxicity). Figure 4 shows that the new chemical entity (NCE) is a bile salt export protein (BSEP) inhibitor and a farnesone- It acts as a pseudox receptor (FXR) antagonist and acts as a feedback regulator of bile acid homeostasis. Prevents activation of the blocking mechanism, resulting in an increase in intracellular bile acid concentration and the prevention of bile acid hepatotoxicity. It has been shown to have a detrimental effect on liver function (also known as cholestatic hepatotoxicity). Figure 5 shows that new chemical entities (NCEs) inhibit multiple bile acid excretion pathways, resulting in hepatotoxicity. The bile acid feedback mechanism remains intact, but compensatory mechanisms (e.g., OSTα / β basolateral export) and other bile acid export mechanisms are inhibited, resulting in increased intracellular bile acid concentrations. This results in increased severity and hepatotoxicity (i.e., cholestatic hepatotoxicity). Figure 6 shows that new chemical entities (NCEs) inhibit the uptake of bile acids, resulting in their presentation to hepatocytes. This reduces the amount of bile acids released, resulting in systemic cholestasis.

[0043] Example 3 Development of a cholestatic hepatotoxicity screening assay Here, we identify new chemical entities (NCEs) that may interfere with the ability of hepatocytes to process bile acids. In some embodiments, assays, methods, and systems for detecting the presence of β-glucan in the blood are provided. Such assays, methods and systems are useful for toxicity assays (e.g., ATP / LDH / caspase 3 / 7 / 8) and / or clinical measurements typically used to monitor liver function (A By monitoring LDH related to ST / ALT, it is possible to link cholestasis to hepatotoxicity. In some embodiments, such assays, methods, and The system categorizes new chemical entities (NCEs) into those that are (i) cholestatic and (ii) ineffective. or (iii) causing cholestatic hepatotoxicity. .

[0044] The possible outcome (bile acids (BA) in the presence of new chemical entities (NCEs)) has been demonstrated in several experiments. In an embodiment, it can be represented 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 ) to determine Bile acid (BA) dose response for - System ID normal response calibration - Demonstrate how hepatocytes respond to a wide range of bile acid (BA) concentrations No change in the potency of bile acid (BA) toxicity in the presence of new chemical entities (NCE) = predicted No effect -Dashed line: Increased bile acid (BA) toxic potency in the presence of new chemical entities (NCE) = cholestatic May cause liver toxicity Bile acid (BA) excretion inhibitors, farnesoid X receptor (FXR) antagonists, or Both -solid line: In the presence of new chemical entities (NCEs), the potency of bile acid (BA) toxicity is reduced, resulting in cholestasis. This is equivalent to the possibility of causing systemic toxicity. R Bile acid (BA) uptake inhibitors

[0045] The bile acids GCA, GCDCA, and ATP (Figure 9) and LDH (Figure 10) contents were evaluated at increasing concentrations of GDCA. As shown in Figures 9 and 10, the results of these evaluations showed that the human hepatocyte sandwich Schizosaccharomyces cerevisiae (SCHH) cultures are fairly resistant to bile acid (BA) toxicity after exposure for up to 24 hours. More specifically, GCA is not cytotoxic, but rather inhibits GCDC in the ATP assay. A was comparable to that of GDCA, and GDCA was greater than GCDCA in the LDH assay.

[0046] In sandwich cultures of human hepatocytes (SCHH), cholestatic agents (e.g., troglitazone) In the absence and presence of acetaminophen (azolam and sitaxsentan), glucose concentration increased with increasing bile acid pool toxicity. Glucose concentrations were assessed to examine the effect (i.e., shift) on sexual profiles. Using the same method as in the previous section, ApoTox-Glo TM Hepatotoxicity was evaluated using a triplex assay. The results are shown in Figures 11 and 12. Human hepatocyte samples were analyzed for troglitazone effects on cell viability and bile acid efficacy relationships. Low glucose conditions were required to enhance the sensitivity of the German culture (SCHH) system ( (Figures 11-12). Low glucose conditions likely improve hepatic mitochondrial function. This increases ATP production and promotes mitochondrial function, thereby improving cell viability. The dynamic range of the assay was improved. The addition of sitaxsentan reduced the hepatotoxicity of the bile acid pool. I didn't change my gender.

[0047] Example 4 Potential of cholestatic hepatotoxicity screening assays Disclosed assays, methods and systems distinguish between ambrisentan and sitaxsentan A feasibility study was also conducted to evaluate the ability of the system to The characteristics of sitaxsentan and ambrisentan are listed below. Sitaxentan (2-200 μM) -Reduced toxic potential of bile acids (BAs) - In vitro assays identify potent / effective normal bile acid uptake (NTCP) inhibitors showed. -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 probably not the primary site of toxicity. Toxicity at 3 months none Ambrisentan (2-200 μM) -No change in efficacy of bile acid (BA) toxicity -Cmax = 3 μM - Well tolerated and non-toxic -In vitro assay for normal bile acid uptake (NTCP) / bile salt export protein (BSEP) showed no effect.

[0048] Individual bile acids and bile acid mixtures (bile acid pools) were treated with troglitazone (100 μM) and sirolimus. The potential for modifying the hepatotoxicity of taxenthen (60 μM) was investigated in the presence of 5 mM glucose for 24 hours. The effects of test compounds were assessed in sandwich-cultured human hepatocytes after short-term exposure. The effects of bile acid treatment alone were evaluated in the presence of bile acids and in the presence of a bile acid pool. The effects of DMSO treatment alone were used as a control for hepatotoxicity. ApoTox-Glo to assess viability, cytotoxicity, and apoptosis TM Evaluation was performed using a triplex assay.

[0049] [Table 1]

[0050] A bile acid mixture was evaluated. This bile acid mixture contained the physiologically significant amounts of each bile acid as shown in Table 1. It is composed of four different bile acids maintained in appropriate ratios for the individual bile acids. was administered in the concentrations shown in this mixture. Only in the presence of bile acid pools was a left-shift of troglitazone observed, which may be related to cell viability. This indicates that fewer bile acids are required to reduce the level of bile acid in the blood (Figure 14). Exposure to HCl did not result in a left-shift in cell viability (Figures 15-18). These data suggest that the bile acid pool may, in some embodiments, be It is a necessary component to be able to distinguish the effect of compounds on hemostasis. Troglitazone appears to inhibit the ability of hepatocytes to respond to increased intracellular bile acid concentrations. interferes with the ATP synthesis, 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, we analyzed the cellulose in the presence of two free fatty acid ratios (oleic acid:palmitoyl oleic acid). acid = 2:1 and 0:3), troglitazone (100μ The effects of free fatty acid treatment (60 μM) and sitaxenten (60 μM) were evaluated. Hepatotoxicity was assessed using ApoTox-Glo, which assesses viability, cytotoxicity, and apoptosis. TM T The results were evaluated by riplex assay. In the absence of free fatty acids, the hepatotoxicity of bile acids was increased in the presence of troglitazone (Fig. 19).TC 50 (the concentration of bile acids that is 50% toxic to hepatocytes) was 44% of the control. The concentration of ATP in the IL-100 / ...

[0052] [Table 2]

[0053] Addition of free fatty acids at both oleic acid:palmitic acid ratios significantly increased the The hepatotoxicity of the bile acid pool was further altered in the presence of troglitazoline. See Figure 20. and bile in the presence of oleic acid and palmitic acid (oleic acid:palmitic acid ratio 2:1). TC on Juicy Acid 50 was reduced to 9.5% of the control (from 1.32 μM to 0.13 μM to) (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). Troglitazone and oleic acid and palmitic acid (oleic acid:palmitic acid ratio 0:3) TC of bile acids in the presence of 50 was reduced to 17% of the control (from 1.28 μM to 0.2 to 2 μM) (Table 4).

[0056] [Table 4]

[0057] At any ratio of oleic acid to palmitic acid, the addition of free fatty acids alone resulted in a pool The DMSO control had little effect on the hepatotoxicity profile of the bile acids administered. TC of bile acids 50 The ratio of free fatty acids (oleic acid: palmitic acid 2:1) When 0:1 or 0:3) was added, the reduction was only 89% and 86% of the control, respectively. See Table 5 below.

[0058] [Table 5]

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

[0060] Example 6 Applications of cholestatic hepatotoxicity screening assays As mentioned above, drug exposure may disrupt bile acid excretion pathways (e.g., bile salt excretion protein (BSEP) and / or MRP3 / 4), and 1) the farnesoid X receptor (FXR) is activated in cells. 2) by blocking (antagonizing) the sensing of elevated intracellular concentrations of bile acids; or 3) by blocking (antagonizing) the basolateral efflux Inhibits bile acid efflux via the α / β bile acid transporter (OST), or 3) both of these. Hepatocytes become more susceptible to bile acid toxicity (e.g., cholestatic hepatotoxicity). To further illustrate this, sandwich cultures of human hepatocytes (SCHH) were cultured in a low glucose ( about 2 mM to about 10 mM, or about 5 mM) standard medium or low glucose (about 2 mM to about 1 0 mM, or about 5 mM), or in some embodiments, other suitable a suitable energy source (e.g., galactose), free fatty acids (from 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 and bile acid pool (about 5 μM to about 1 mM, or about 50 μM to about 500 μM, or about 100 μM 250 μM to about 250 μM) to inhibit bile salt export protein (BSEP) drugs (e.g., cyclosporine A (CsA); Ansede et al., 2010), or bile salt excretion inhibitors. It has the characteristics of both a steroid protein (BSEP) inhibitor and a farnesoid X receptor (FXR) antagonist. Compounds with vasopressin-releasing hormone receptors (e.g., troglitazone; Marion et al., 2007 and Kaimal et al., 2009) 9) for 24 hours.

[0061] Briefly, hepatocytes were established in a sandwich culture configuration four times. On day 4 of culture, human hepatocytes were cultured in a sandwich culture configuration four times. Sandwich cultures of cytoplasmic vesicles (SCHH) were treated with cyclosporine A (CsA) (10 μM; 13 × CsA) x) or troglitazone (100 μM; 16 × Cmax) for 24 hours, followed by standard The cells were cultured in medium or sensitizing medium. The concentrations of cyclosporine A (CsA) and troglitazone were D Based on the Cmax values reported in awson et al. 2012. Intraportal administration of test compound after oral administration Taking into account the potential convergence effect, drug exposure ranged from approximately 8-fold to approximately 16-fold Cmax. After 4 hours, LDH leakage and ATP content were assessed using commercially available assays. Under standard culture conditions, cyclosporine A (CsA) (10 μM; 13 × Cmax) or Three human hepatocytes treated with either lorglitazone (100 μM; 16×Cmax) In both sandwich culture (SCHH) donors, a significant increase in LDH secretion and A No significant decrease in TP content was observed (Fig. 22A and Fig. 22B). Under culture conditions, troglitazone exposure, but not cyclosporine A (CsA), significantly increased the risk of developing rhesus monkeys in three different cultures. In sandwich cultures of rat hepatocytes (SCHH) donors, LDH secretion increased to more than 490% of the control. Addition of ATP to ATP-containing medium (Tukey's; p<0.05) reduced ATP content to less than 0.9% of the control (Tukey's; p<0.05). ) (Figures 22A and 22B).

[0062] Sensitized media inhibits free fatty acids and hepatocyte bile acid homeostasis mechanisms while minimizing toxicity. Challenge with appropriate concentrations (250 μM) of the above primary and secondary bile acids (GCDCA, GCA After 24 hours of exposure to the sensitizing medium, three LDH secretion was significantly increased in two sandwich culture (SCHH) preparations of human hepatocytes. In the same donor, ATP was not added in the sensitization medium (Tukey's p value < 0.05) (Fig. 23A). The content of IgG decreased by 24.8% (Fig. 23B). These results suggest that the sensitized medium was showed high tolerance across the entire range.

[0063] Under standard culture conditions, in the presence of the cholestatic hepatotoxic agent troglitazone, The cells do not have sufficient concentrations of bile acids or the appropriate mixture to induce toxicity. Treatment with rhoglitazone resulted in a significant increase in the number of rhoglitazone-treated donors in all three donors examined under standard culture conditions. This was demonstrated by the lack of effect on LDH and ATP in the presence of ATP (Fig. 24A and 24B). To fully assess the function of the bile acid homeostasis mechanism, hepatocytes After a meal, more than 95% of the bile acids secreted are absorbed into the portal vein from the ileum. They are reabsorbed in vivo (Chiang, 2009). Therefore, in vivo bile acid portal vein concentrations are dynamic. , which increased more than threefold between fasting and postprandial states, represented by standard and sensitized media, respectively. It has been reported that this occurs (Angelin B et al, 1982).

[0064] The main toxicities associated with cyclosporine A (CsA) therapy in humans are nephrotoxicity and neurotoxicity. It is not hepatotoxic, with a relatively low incidence compared to other uses (LiverTox Database, Magnasco et al. In contrast, clinical studies of troglitazone have shown that the incidence of liver damage The incidence may be as high as 1:1000 patients, indicating that troglitazone may be a potential cause of liver damage. These two potent bile salt excretors have been shown to induce The clear difference in the incidence of clinical liver injury between bile salt export protein (BSEP) inhibitors In addition to inhibiting bile acid esterase inhibitors (BSEP), interference with bile acid homeostasis mechanisms (e.g., farnesoids) This suggests that other processes, such as FXR antagonism, may be involved. of experimental liver injury and results of in vivo assays of cyclosporine A (CsA) and troglitazone. The results are in complete agreement, indicating that the cholestatic hepatotoxicity assay is a valuable tool for assessing non-drug hepatic toxicity. Cyclosporine A (CsA), a drug that causes liver injury (DILI), and cholestatic drug-induced liver injury (DILI) This suggests that it is possible to distinguish between troglitazone and cerebrospinal fluid.

[0065] 50 μM (8 × Cmax), 75 μM (12 × Cmax) and 100 μM (16 × Cmax) ax) in three different human hepatocyte sandwich culture (SCHH) preparations. Titration of thiazolidinedione (a thiazolidinedione compound) to determine the toxic dose only in sensitized culture medium. A drug-dependence was established (Figures 24A and 24B). ax) In all three troglitazone-treated donors, the mean age was 590% or more of the control. LDH leakage and loss of ATP content of more than 95% of the control were observed (Fig. 24B). These results suggest that the hepatotoxic pathway is not idiosyncratic and that dose-dependent toxicity is preserved in donors. demonstrated that troglitazone was effective under standard culture conditions at all concentrations evaluated. In exposed sandwich cultures of human hepatocytes (SCHH), no LDH leakage or ATP loss was observed. These results were consistent with those of the cholestatic liver injury (DILI) agent. To determine this, sandwich cultures of human hepatocytes (SCHH) were cultured under specific conditions (e.g., These results demonstrate that hepatocytes are able to process large amounts of bile acids. In conditions requiring this (e.g., sensitization conditions), troglitazone may be used to inhibit bile acid phosphatase activity. These findings suggest that the steroid hormone may disrupt hemostatic mechanisms, resulting in bile acid or cholestatic hepatotoxicity. Ta.

[0066] This conclusion is supported by sandwich cultures of human hepatocytes (SCHH) in the presence of troglitazone. The inhibition of biliary clearance of d8-TCA (Figure 25) and farnesoids observed in This was further supported by the antagonism of the X receptor (FXR) (Figure 26). Biliary clearance of 8-TCA was measured using a sandwich of human hepatocytes treated with troglitazone. The effect of troglitazone on the expression of stromal cells in SCHH cultures was significantly reduced in a dose-dependent manner (Fig. 25A). Biliary excretion of d8-TCA in exposed sandwich cultures of human hepatocytes (SCHH) The biliary excretion index (e.g., biliary excretion index) was also significantly decreased in a dose-dependent manner (Figure 25B). In the presence of troglitazone, canalicular dome size increased, consistent with previous reports (Marion et al., 2007). This suggests that bile acid excretion across the intestine was reduced. Troglitazone (75-100 μM) exposure also significantly increased the risk of developing CDCA and cyclosporine A (Cs A) OSTβ mRNA in sandwich cultures of human hepatocytes (SCHH) after co-treatment The synergistic induction response of CDCA, CsA, and DY268 (a novel In humans treated with a combination of 2 potent farnesoid X receptor (FXR) antagonists Similar effects were observed in sandwich cultures of hepatocytes (SCHH) (Yu et al., 20 14) Apart from this, the decrease in OSTβ mRNA content was not associated with cytotoxicity under the conditions evaluated. However, under the conditions investigated, there was no significant decrease in ATP content or LDH leakage. No increase in OSTβ mRNA was observed, and the decrease in OSTβ mRNA content was not related to the activation of farnesoid X receptor (FXR). It was suggested that this was due to the antagonistic effect of activating the ATP (Figure 27).

[0067] Other thiazolidinedione compounds, including pioglitazone and rosiglitazone, have been used A preliminary evaluation of the predictive accuracy of cholestatic hepatotoxicity screening was performed. It is a thiazolidinedione compound and a well-established drug for drug-induced liver injury (DILI). The incidence of liver damage was 1:1000 patients and it was removed from the market due to liver damage (LiverTox D In contrast, both pioglitazone and rosiglitazone are widely used. Despite its use, fewer than 12 cases of liver injury have been reported (LiverTox Database). For either pioglitazone or rosiglitazone, the results were generally ALT elevations of more than three times the upper limit of the serotonin-dependent pathway were not different from placebo recipients. This suggests that the potential for liver damage is low for any of the azolidinedione compounds. glitazone (Cmax: 6.4 μM; Dawson et al. 2012), pioglitazone (Cmax: 2.9 μM; Daw son et al. 2012) and rosiglitazone (Cmax: 1.0 μM; Dawson et al. 2012). All three thiazolidins were tested over a wide range of concentrations (1, 5, 10, 25, 50, 100 μM) including Troglitazone was evaluated in a cholestatic hepatotoxicity screening assay. In sandwich cultures of human hepatocytes (SCHH) treated with (100 μM), the sensitized medium Only under nutrient conditions did LDH leakage increase significantly (863% of the control) and ATP content decrease significantly. Loss (>99% of control) was observed (Figures 28A and 28B), which is consistent with previous results. Under all culture conditions tested, any concentration of pioglitazone or rosiglitazone A significant increase in LDH leakage was also observed in sandwich cultures of human hepatocytes (SCHH) treated with acetaminophen. No increase or decrease in ATP content was observed (Figures 28A and 28B). The Toro results demonstrate the predictive accuracy of the disclosed cholestatic hepatotoxicity screening assay. This was generally consistent with the incidence of clinical liver damage demonstrated with thiazolidinedione compounds.

[0068] References Patents, patent applications and publications, scientific journal articles, and database entries (e.g., GENBANK(R) database entries and all annotations available therein (but All references, including but not limited to, the methodologies, techniques, and / or compositions thereof, to the extent that such content supplements, explains, provides background to, or teaches the The entirety of which is incorporated herein by reference. ○ 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 sandwi ch-cultured rat hepatocytes. Drug Metab and Dispos 38:276-280. ○ Marion TL, Leslie EM and Brouwer KLR (2007) Use of sandwich-cultured hepatocy tes to evaluate impaired bile acid transport as a mechanism of drug-induced hepa totoxicity. 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 li ver injury in humans. Drug Metab and Dispos 40: 130-138. ○ Angelin B, Bjorkhem I, Einarsson K, and Ewerth S (1982) Hepatic uptake of bil e 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, pharmacogeneti cs and perspectives. Curr Clin Pharmacol. 3(3):166-73. ○ Yu DD, Lin W, Forman BM, and Chen T (2014) Identification of trisubstituted-p yrazol carboxamide analogs as novel and potent antagonists of farnesoid X recept or. 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 li ver 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 can be changed without departing from the scope of the invention. Furthermore, the above description is for illustrative purposes only and is not intended to be limiting. Not yet. [Explanation of symbols]

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

Claims

1. Methods for screening compounds for their potential to cause systemic and / or hepatotoxicity A method comprising the steps of: (a) providing a compound to be screened; (b) Hepatocyte cell lines (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation. Establishing the (c) Exposing the hepatocyte cell line to a range of bile acids to determine their toxicity profile. (HCS), wherein the bile acid toxicity profile comprises a toxic effect; (d) incubating the hepatic cell line (HCS) in the presence of the compound to be screened at a range of concentrations. exposing the subject to a bile acid and determining a toxicity profile of the bile acid; the file contains a toxic effect; and (e) comparing the toxic potency of the bile acid between step (c) and step (d) to determine whether the compound has systemic toxicity; Determining the potential to cause toxicity and / or hepatotoxicity

2. The step of determining the potential for the compound to cause systemic and / or hepatotoxicity may further comprise the steps of: In the presence of the compound, the toxic effect of the bile acid is reduced compared to the absence of the compound. determining whether there is any change in the presence of the compound compared to the absence of the compound; and comparing the bile acid to determine an increase in the toxic potency of the bile acid in the presence of the compound. and determining a reduction in the toxic effect of the bile acid compared to the absence of the compound. The method of claim 1 , comprising:

3. Compounds that do not cause changes in the toxic potency of bile acids are also known to cause systemic or hepatotoxicity.

3. The method of claim 2, characterized as:

4. Compounds that cause an increase in the toxic potency of bile acids may cause cholestatic hepatotoxicity. The method of claim 2, characterized as having

5. The cholestatic hepatotoxicity is caused by inhibition of bile acid excretion (in this case, the compound is characterized as a bile acid export inhibitor), and / or the cholestatic hepatotoxic The sexual dysfunction is caused by antagonism of the farnesoid X receptor (FXR) (in this case, The compounds are characterized as farnesoid X receptor (FXR) antagonists. and / or a combination of both.

6. 6. The method of claim 5, wherein the inhibition of bile acid excretion comprises inhibition of bile salt excretion protein (BSEP). How to do it.

7. Compounds that cause a decrease in the toxic potency of bile acids can induce cholestasis, which leads to systemic toxicity.

3. The method of claim 2, characterized as having the potential to cause

8. The cholestasis resulting in the systemic toxicity is caused by inhibition of bile acid uptake. The method of claim 7.

9. The step of determining the susceptibility of a compound to causing systemic or hepatotoxicity comprises treating the compound with a bile acid Efflux inhibitors, farnesoid X receptor (FXR) antagonists, bile acid uptake inhibitors or the absence of said step. Law.

10. The compound is a candidate drug, and the candidate drug causes systemic toxicity and / or hepatotoxicity. as unlikely or likely to cause systemic and / or hepatotoxicity The method according to any one of claims 1 to 9, characterized in that

11. 11. The method of claim 1, wherein the bile acid transport comprises bile acid uptake, basolateral excretion and / or canalicular excretion.

10. The method according to any one of claims 1 to 9.

12. The bile acid concentration range mimics fasting and / or postprandial concentrations in vivo within cells. The method of any one of claims 1 to 11, comprising an intracellular concentration.

13. This range of bile acid concentrations activates the farnesoid X receptor (FXR) feedback mechanism.

13. The method of any one of claims 1 to 12, comprising an intracellular concentration of bile acids sufficient to

14. Determining the toxicity profile of the bile acids may involve measuring the hepatotoxic response using a cytotoxicity assay. The method of any one of claims 1 to 13, comprising:

15. The cytotoxicity assay includes enzyme leakage assay, ATP, APOTOX Glo and the like.

15. The method of claim 14, wherein the compound is selected from the group consisting of:

16. 10. The enzyme leakage assay of claim 1, wherein the enzyme leakage assay is selected from the group consisting of ALT, AST, and LDH.

5. The method according to claim 5.

17. Furthermore, hepatic cell lines (HCS) were exposed to multiple bile acids to generate multiple responses to the multiple bile acids. determining a toxicity profile of the bile acid, the toxicity profile being indicative of a plurality of toxic effects; The method of any one of claims 1 to 16, comprising a force.

18. The multiple toxicity potencies are used to predict the hepatotoxic potential of the compound.

18. The method of claim 17, wherein the measurement is performed in a hepatic cell line (HCS) in the presence and absence of ribosomal protein.

19. The hepatocyte cell line (HCS) is a two-dimensional culture using primary hepatocytes or other hepatocyte cell lines. or a method according to any one of claims 1 to 18, comprising a three-dimensional culture.

20. The two-dimensional culture comprises a sandwich culture (SCH) of hepatocytes, the SCH comprising human hepatocytes.

20. The method of claim 19, comprising administering to said subject a therapeutically effective amount of steroid hormone.

21. The three-dimensional culture comprises a three-dimensional (3D) scaffold-based culture or a spheroid culture.

20. The method of claim 19.

22. The other hepatocyte cell lines include HepaRG, Huh7, co-culture systems, stem cell-derived hepatocytes, and 20. The method of claim 19, including combinations thereof.

23. The compound is exposed to a hepatocyte cell line (HCS) over a range of concentrations.

23. The method of any one of claims 22.

24. The bile acid or bile acids are GCA, GCDCA, GDCA, DCA, CA, CDC A, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof The method according to any one of claims 1 to 23, wherein

25. 1. An in vitro system for predicting the in vivo hepatotoxic potential of a compound, comprising: (i) a gallbladder; In vitro cultured hepatocytes capable of bile acid synthesis, bile acid transport and / or bile acid regulation (ii) one or more of the following that have established toxic efficacy in the hepatocyte cell line (HCS): and (iii) in the presence of one or more bile acids. and an assay for measuring the hepatotoxicity of the compound upon exposure.

26. The in vitro cultured hepatic cell line (HCS) is involved in bile acid synthesis, transport, and bile acid homeostasis.

26. The in vivo method of claim 25, comprising an integrated hepatocyte cell system with an osteogenesis feedback mechanism. Toro System.

27. The in vitro cultured hepatocyte cell line (HCS) may be a primary hepatocyte or other hepatocyte cell line.

27. The in vitro method according to claim 25 or 26, comprising two-dimensional or three-dimensional culture. system.

28. The in vitro cultured hepatocyte cell line (HCS) is a sandwich culture of hepatocytes (SC H), wherein the SCH comprises human hepatocytes. In vitro system.

29. The in vitro cultured hepatic cell line (HCS) is a three-dimensional (3D) scaffold-based culture. or spheroid cultures, Tem.

30. The in vitro cultured hepatocyte cell line (HCS) is HepaRG, Huh7, co-culture system 30. Any one of claims 25 to 29, comprising stem cell-derived hepatocytes, and combinations thereof. The in vitro system described in

31. The bile acid or bile acids are GCA, GCDCA, GDCA, DCA, CA, CDC A, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof The in vitro system according to any one of claims 25 to 30,

32. The established toxic potency of the one or more bile acids may be assessed by measuring hepatotoxicity using a cytotoxicity assay. Claims 25 to 31, comprising a reaction-based toxicity profile of said one or more bile acids.

1. The in vitro system according to any one of claims 1 to 9.

33. The cytotoxicity assay includes enzyme leakage assay, ATP, APOTOX Glo, and the like.

33. The in vitro system of claim 32, wherein the in vitro system is selected from the group consisting of:

34. 3. The enzyme leakage assay is selected from the group consisting of ALT, AST and LDH.

3. The in vitro system according to claim 3.

35. Compounds that do not cause changes in the toxic potency of bile acids are considered to be compounds that do not cause systemic or hepatotoxicity. Any one of claims 25 to 34 configured to characterize a compound as having a high probability of Item 1. The in vitro system described in Item 1.

36. Compounds that cause an increase in the toxic potency of bile acids are classified as compounds that can cause cholestatic hepatotoxicity. The compound according to any one of claims 25 to 35, characterized as a compound having antibacterial properties. The in vitro system described above.

37. Compounds that cause a reduction in the toxic potency of bile acids are used to induce cholestasis leading to systemic toxicity. Any of claims 25 to 36 characterized as a compound that may cause 1. The in vitro system described in claim 1.

38. Methods for screening compounds for their potential to cause systemic and / or hepatotoxicity A method comprising the steps of: (a) providing a compound to be screened; (b) Hepatocyte cell lines (HCS) capable of bile acid synthesis, bile acid transport and / or bile acid regulation. Establishing the (c) Incubating a hepatocyte cell line (HC) with a range of bile acids in the presence of the compound to be screened. S) to determine the toxicity profile of bile acids in the presence of the compound being screened. wherein the toxicity profile of the bile acid is known over the concentration range; wherein the bile acid toxicity profile of the bile acid comprises a toxic potency of the bile acid; and (d) comparing the toxic potency of bile acids in the presence and absence of the compound to determine systemic toxicity and determining the susceptibility of said compound to causing hepatotoxicity and / or liver toxicity

39. 39. The method of claim 38, wherein the compound is exposed to a hepatocyte cell line (HCS) over a range of concentrations. How to post.

40. The bile acid is a combination of a plurality of bile acids, and the toxicity profile of the combination of bile acids is 40. The method of claim 38 or 39, wherein the toxicological profile includes a toxic potency. How to post.

41. 10. The combination of bile acids according to claim 9, wherein the combination of bile acids comprises bile acids provided in a predetermined ratio.

40. The method according to claim 40.

42. The combination of bile acids is designed to mimic the concentrations and ratios of bile acids in vivo.

42. The method of claim 40 or 41, comprising a plurality of bile acids in concentrations and ratios configured as follows: 。

43. The step of determining the susceptibility of the subject to compounds causing systemic toxicity and / or hepatotoxicity may further comprise the steps of: In the presence of the compound, there is no change in bile acid toxicity potency compared to the absence of the compound. determining whether or not a compound is present in the presence of the compound compared to its absence; and determining an increase in bile acid toxicity, or determining whether or not the compound is present in the presence of the compound. The method of any one of claims 38 to 42, further comprising a step of confirming a reduction in bile acid toxicity compared to the absence of the compound. The method according to any one of claims 1 to 5.

44. Compounds that do not cause changes in the toxic potency of the bile acids are considered to be compounds that do not cause systemic or hepatotoxicity.

44. The method of claim 43, wherein the method is characterized as not

45. The compound causing an increase in the toxic potency of the bile acid is selected from the group consisting of compounds causing cholestatic hepatotoxicity.

44. The method of claim 43 characterized as having potential.

46. The cholestatic hepatotoxicity is caused by inhibition of bile acid excretion (in this case, the compound is characterized as a bile acid export inhibitor), and / or the cholestatic hepatotoxic The sexual dysfunction is caused by antagonism of the farnesoid X receptor (FXR) (in this case, The compounds are characterized as farnesoid X receptor (FXR) antagonists.

46. The method of claim 45, wherein the hydroxyl group is hydroxypropyl methylcellulose, ...

47. 47. The method of claim 46, wherein the inhibition of bile acid excretion comprises inhibition of bile salt excretion protein (BSEP). How to post.

48. Compounds that cause a decrease in the toxic potency of bile acids can induce cholestasis, which leads to systemic toxicity.

44. The method of claim 43, characterized as having the potential to cause

49. The cholestasis resulting in the systemic toxicity is caused by inhibition of bile acid uptake.

49. The method of claim 48.

50. The step of determining the potential of the compound to cause systemic or hepatotoxicity comprises administering the compound to the bile duct. Bile acid excretion inhibitor, farnesoid X receptor (FXR) antagonist, bile acid uptake inhibitor 44. The method of claim 43, comprising characterizing the agent as the absence of said agent.

51. The compound is a candidate drug, and the candidate drug causes systemic toxicity and / or hepatotoxicity. as unlikely or likely to cause systemic and / or hepatotoxicity The method according to any one of claims 38 to 50, characterized in that

52. 52. The method of claim 38, wherein bile acid transport comprises bile acid uptake, basolateral excretion, and / or canalicular excretion.

10. The method according to any one of the preceding claims.

53. The bile acid concentration range mimics fasting and / or postprandial concentrations in vivo within cells.

53. The method of any one of claims 38 to 52, comprising an intracellular concentration.

54. This range of bile acid concentrations activates the farnesoid X receptor (FXR) feedback mechanism.

54. The method of any one of claims 38 to 53, comprising an intracellular concentration of bile acids sufficient to

55. The bile acid or bile acids are GCA, GCDCA, GDCA, DCA, CA, CDC A, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof The method according to any one of claims 38 to 54, wherein

56. Determining the toxicity profile of the bile acids may involve measuring the hepatotoxic response using a cytotoxicity assay.

56. The method of any one of claims 38 to 55, comprising:

57. The cytotoxicity assay includes enzyme leakage assay, ATP, APOTOX Glo and the like.

57. The method of claim 56, wherein the compound is selected from the group consisting of:

58. 6. The enzyme leakage assay is selected from the group consisting of ALT, AST and LDH.

7. The method according to claim 7.

59. The hepatocyte cell line (HCS) is a two-dimensional culture using primary hepatocytes or other hepatocyte cell lines. or a method according to any one of claims 38 to 58, comprising a three-dimensional culture.

60. The two-dimensional culture comprises a sandwich culture (SCH) of hepatocytes, the SCH comprising human hepatocytes.

60. The method of claim 59, comprising administering to said subject a therapeutically effective amount of steroid hormone.

61. The three-dimensional culture comprises a three-dimensional (3D) scaffold-based culture or a spheroid culture.

60. The method of claim 59.

62. The other hepatocyte cell lines include HepaRG, Huh7, co-culture systems, stem cell-derived hepatocytes, and 60. The method of claim 59, including combinations thereof.

63. In the hepatic cell line (HCS), one or more free fatty acids and / or a predetermined glycoprotein are added.

63. The method of any one of claims 1 to 24 and 38 to 62, wherein a concentration of 100% ethanol is employed.

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