Patent Application
20190257817
The instant disclosure is directed to screening candidate compounds or chemical entities for their cholestatic hepatotoxicity potential. More particularly, the instant disclosure is directed to methods and systems for screening candidate compounds for susceptibility to causing, or their potential to cause, systemic or hepatic toxicity.
In developing new therapeutics, drugs and pharmaceutical compounds there is a need to screen new chemical entities (NCE) to determine their cholestatic hepatotoxicity potential. Drug induced cholestatic hepatotoxicity is the result of impaired bile flow of bile acids resulting in the accumulation of toxic concentrations of bile acids in the hepatocyte. NCEs that are likely to cause drug induced cholestatic hepatotoxicity may be removed from consideration in further drug development trials.
There is a need for improved in vitro methodologies and systems to assess the potential of a NCE, compound or drug candidate to cause cholestatic hepatotoxicity in vivo. Such needs are addressed by the presently disclosed subject matter.
This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
In some embodiments, provided herein are methods of screening a compound for its potential to cause systemic and/or hepatic toxicity, the methods comprising the steps of: providing a compound to be screened, establishing a hepatic cell system (HCS) comprising a capacity for bile acid synthesis, bile acid transport and/or bile acid regulation, exposing the HCS to a range of concentrations of a bile acid to determine a toxicity profile of the bile acid, wherein the bile acid toxicity profile comprises a toxicity potency, exposing the 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, wherein the bile acid toxicity profile comprises a toxicity potency, and comparing the toxicity potencies of the bile acids to determine the susceptibility of the compound to cause systemic and/or hepatic toxicity. In some embodiments, provided herein are in vitro systems for predicting in vivo hepatotoxic potential of a compound, comprising in vitro cultured HCS comprising a capacity for bile acid synthesis, bile acid transport and/or bile acid regulation, one or more bile acids with an established toxicity potency within the HCS, and an assay for determining the hepatotoxicity of a compound when exposed to the HCS in the presence of the one or more bile acids.
In some embodiments, provided herein are methods of screening a compound for susceptibility to causing systemic and/or hepatic toxicity, the methods comprising providing a compound to be screened, establishing a HCS comprising a capacity for bile acid synthesis, bile acid transport and/or bile acid regulation, exposing the 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 in the presence of the compound to be screened, wherein the toxicity profile of the bile acid is known across the range of concentrations, wherein the toxicity profile of the bile acid comprises a toxicity potency of the bile acid, and comparing the toxicity 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 hepatic toxicity.
Accordingly, it is an object of the presently disclosed subject matter to provide methods and systems for screening a compound for susceptibility to causing systemic and/or hepatic toxicity. This and other objects are achieved in whole or in part by the presently disclosed subject matter.
Further, an object of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Drawings and Examples.
The presently disclosed subject matter can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figures, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawings. Although the illustrated embodiment is merely exemplary of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and exemplify the presently disclosed subject matter.
For a more complete understanding of the presently disclosed subject matter, reference is now made to the following drawings in which:
NCE inhibiting bile acid uptake, resulting in reducing the amount of bile acids presented to the hepatocyte leading to systemic cholestasis;
The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
All technical and scientific terms used herein, 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 herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.
Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, 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 indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of a composition, dose, sequence identity (e.g., when comparing two or more nucleotide or amino acid sequences), mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of 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% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
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 of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct 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 “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
As used herein, the term “and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
As used herein, a hepatic cell system (HCS) refers to a hepatic cell system comprising a capacity for bile acid synthesis, bile acid transport and/or bile acid regulation. In some embodiments, the HCS can comprise a two (2)-dimensional culture, such as but not limited to sandwich culture of hepatocytes (SCH), including a sandwich culture of human hepatocytes (SCHH). In some embodiments, the HCS can comprise a three (3)-dimensional culture (such as but not limited to 3D scaffold-based cultures, spheroidal cultures and the like. The HCS can comprise primary hepatocytes or other relevant hepatic cell systems, such as but not limited to HepaRG, Huh7, co-cultured systems, and/or stem-cell derived hepatocytes. In some embodiments, one or more free fatty acids and/or a preselected glucose concentration is/are employed in the HCS, such as in the incubation media of the HCS.
Disclosed herein are methods and systems for screening candidate compounds for susceptibility to causing, or their potential to cause, systemic or hepatic toxicity. As used herein, terms used to refer to a candidate compound's “ability”, “susceptibility”, “potential”, “probability”, “likelihood”, or the like, are used interchangeably and are generally indicative of a likelihood that a compound can impact, cause or contribute to system or hepatic toxicity and related conditions as disclosed and discussed herein.
The presently disclosed subject matter provides in some embodiments in vitro methodologies and systems to assess the potential of a new chemical entity (NCE), compound or drug candidate to cause cholestatic hepatotoxicity in vivo. Primary bile acids are synthesized in the hepatocyte by one or more hepatic enzymes, including for example by Cyp7A1, and then rapidly conjugated to both taurine and glycine, and may be further altered through glucuronidation and sulfation. Bile acids and conjugates are excreted in the bile by the Bile Salt Export Protein (BSEP) and MRP2 and into the portal circulation by MRP3/4 or OSTα/β. Bile acids undergo further metabolism in the intestine and are absorbed in the enterocyte by ASBT, and effluxed from the enterocyte into the portal vein by OSTα/β. Bile acids are taken up from the portal circulation primarily by NTCP; however, some studies have also suggested that OATPs may be responsible for some amount of uptake. Bile acids can act as detergents, and high intracellular concentrations (ICC) of bile acids have been shown to be hepatotoxic.
Drug induced cholestatic hepatotoxicity is generally thought to be the result of impaired bile flow of bile acids resulting from inhibition of only BSEP by the NCE. However, impairment of the hepatocyte bile acid homeostasis adaptive response, mediated by the farnesoid X receptor (FXR), likely plays a pivotal role in the drug induced liver injury. Without being bound by any particular theory or mechanism of action and/or the current dogma, it is currently believed that inhibition of BSEP by the NCE is only the initiating event resulting in an increase in the intracellular concentrations of bile acids. When the intracellular concentration of bile acids is high enough, they activate FXR, which triggers a decrease in the expression of CYP7A1, the rate limiting enzyme in bile acid production, resulting in decreased bile acid synthesis and induction of both BSEP and OSTα/β expression. The increase in BSEP expression will have minimal effect since the NCE is inhibiting transport by that route; however, the induction of OSTα/β will greatly increase the bile acid clearance from the hepatocyte into the portal circulation, thereby allowing the hepatocyte to decrease the intracellular concentration of bile acids, and decrease the potential for hepatotoxicity.
Therefore, the proposed mechanism for cholestatic hepatotoxicity is that a potential cholestatic hepatotoxicant must inhibit bile acid efflux routes (e.g. BSEP and/or MRP3/4) and 1) prevent (antagonize) FXR from sensing the rising intracellular concentrations of bile acids; or 2) inhibit bile acid efflux through OSTα/β, a bile acid efflux compensatory pathway or 3) a combination of both.
In order to accurately predict an in vivo effect on the hepatic disposition of bile acids, an integrated system as provided herein can be important, if not required. In some embodiments, the integrated system comprises synthesis, transport (uptake, basolateral efflux, and canalicular efflux), and regulation (transport, metabolism and synthesis). The sandwich-cultured human hepatocyte (SCHH) model is one system that is characterized and capable of combining all of these pathways.
The presently disclosed methods and systems combine the utility of the SCHH model for determination of BSEP inhibition and bile acid regulation through FXR. In addition, it was observed that the intracellular concentration of bile acids is the driving force for the activation of the FXR feedback mechanism. Since the intracellular concentration of bile acid is a factor for the development of an in vitro methodology to predict cholestatic hepatotoxicity, it was determined that the intracellular concentration (ICC) of bile acids was required to activate the FXR feedback mechanism. In addition, different bile acids are known to have different potentials for hepatotoxicity, therefore multiple bile acids and combinations thereof (bile acid pool) were evaluated for their potential to elicit a hepatotoxic response using standard assays for toxicity (for example, but not limited to, ATP, LDH, Caspase 3/7 and APOTOX Glo), and various mixtures of bile acids, including deoxycholic acid (DCA) identified as one of the most potent hepatotoxic bile acids. Use of these assays allowed for the differentiation between cell viability, necrosis and apoptosis in the system.
Generally, the presently disclosed methods can in some embodiments comprise culturing SCHH with a concentration range of a bile acid (e.g. DCA), or bile acid pool to identify the toxicity profile of the bile acid (such as by measurement of ATP and/or LDH; dotted line in
The NCE could then be classified as having no effect on bile acid homeostasis (1), or a high potential to cause cholestatic hepatotoxicity which may or may not be associated with increased systemic concentrations of bile acids (2), or having a potential for observation of clinical cholestasis (3) (increased systemic concentrations of bile acids with little potential for hepatic cholestasis), or a combination thereof.
Thus, in some embodiments, provided herein are methods of screening a compound for its potential to cause systemic and/or hepatic toxicity. In some embodiments, the methods comprise providing a compound to be screened, establishing a hepatic cell system (HCS) comprising a capacity for bile acid synthesis, bile acid transport and/or bile acid regulation, exposing the HCS to a range of concentrations of a bile acid to determine a toxicity profile of the bile acid, wherein the bile acid toxicity profile comprises a toxicity potency, exposing the 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, wherein the bile acid toxicity profile comprises a toxicity potency, and comparing the toxicity potencies of the bile acids to determine the potential of the compound to cause systemic and/or hepatic toxicity.
In some aspects, determining the potential of the compound to cause systemic or hepatic toxicity further comprises identifying no change in the bile acid toxicity potency in the presence of the compound as compared to the absence of the compound, identifying an increase in the bile acid toxicity in the presence of the compound as compared to the absence of the compound, or identifying a decrease in the bile acid toxicity in the presence of the compound as compared to the absence of the compound. A compound causing no change in the bile acid toxicity potency is characterized as not causing systemic or hepatic toxicity.
However, a compound causing an increase in the bile acid toxicity potency is characterized as having a potential to cause cholestatic hepatic toxicity. The cholestatic hepatic toxicity can be caused by inhibition of bile acid efflux, wherein the compound is characterized as a bile acid efflux inhibitor, and/or the cholestatic hepatic toxicity is caused by antagonism of farnesoid X receptor (FXR), wherein the compound is characterized as a FXR antagonist, and/or a combination of both. In some aspects, inhibition of bile acid efflux comprises inhibition of a bile salt export protein (BSEP).
Still yet, a compound causing a decrease in the bile acid toxicity potency is characterized as having a potential to cause cholestasis resulting in systemic toxicity. The cholestasis can result in systemic toxicity is caused by inhibition of bile acid uptake.
In some aspects, determining the potential of the compound to cause systemic or hepatic toxicity comprises characterizing the compound as a bile acid efflux inhibitor, an FXR antagonist, an inhibitor of bile acid uptake, or none of the above. In some embodiments, the compound can be a drug candidate, wherein the drug candidate is characterized as having a low probability of causing systemic and/or hepatic toxicity, or a high probability of causing systemic and/or hepatic toxicity.
In some embodiments, bile acid transport comprises bile acid uptake, basolateral excretion and/or canalicular excretion. The range of concentrations of the bile acid can comprise intracellular concentrations mimicking in vivo intracellular fasting and/or postprandial concentrations. The range of concentrations of the bile acid can comprise a bile acid intracellular concentration sufficient to activate an FXR feedback mechanism.
In some aspects, determination of a toxicity profile of the bile acid can comprise measuring a hepatotoxic response using a cytotoxicity assay. Such a cytotoxicity assay can be, but is not limited to, an enzyme leakage assay, ATP, APOTOX Glo and a combination thereof. The enzyme leakage assay can comprise, for example, ALT, AST and LDH.
In some embodiments, methods of screening a compound for its potential to cause systemic and/or hepatic toxicity can further comprise exposing the HCS to a plurality of bile acids to determine toxicity profiles for the plurality of bile acids, wherein the bile acid toxicity profiles comprise a plurality of toxicity potencies. The plurality of toxicity potencies can be measured in HCS in the absence and presence of the compound to predict a hepatotoxic potential of the compound.
In some embodiments, methods of screening a compound for its potential to cause systemic and/or hepatic toxicity can utilize an HCS including a 2-dimensional culture or 3-dimensional culture, utilizing primary hepatocytes or other hepatic cell systems. The 2-dimensional culture can comprise a sandwich culture of hepatocytes (SCH), wherein the SCH comprises human hepatocytes. The 3-dimensional culture can comprise a 3D scaffold-based culture or spheroidal culture. Other suitable hepatic cell systems include, but are not limited to, HepaRG, Huh7, co-cultured systems, stem-cell derived hepatocytes and combinations thereof.
In some embodiments the compound can be exposed to the HCS across a range of concentrations. The bile acid or bile acids is/are GCA, GCDCA, GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof. Moreover, in some aspects one or more free fatty acids and/or a predetermined glucose concentration can be employed in the HCS.
In some embodiments, provided herein are in vitro systems for predicting in vivo hepatotoxic potential of a compound, comprising in vitro cultured HCS comprising a capacity for bile acid synthesis, bile acid transport and/or bile acid regulation, one or more bile acids with an established toxicity potency within the HCS, and an assay for determining the hepatotoxicity of a compound when exposed to the HCS in the presence of the one or more bile acids.
In some embodiments, the in vitro cultured HCS can comprise an integrated hepatic cell system with bile acid synthesis, transport, and bile acid homeostasis feedback mechanisms. The in vitro cultured HCS can comprise a 2-dimensional culture or 3-dimensional culture, utilizing primary hepatocytes or other hepatic cell systems. The in vitro cultured HCS can comprise a sandwich culture of hepatocytes (SCH), wherein the SCH comprises human hepatocytes. The in vitro cultured HCS can comprise a 3D scaffold-based culture or spheroidal culture. The in vitro cultured HCS can comprise HepaRG, Huh7, co-cultured systems, stem-cell derived hepatocytes and combinations thereof.
In some aspects, in these systems the bile acid or bile acids is/are 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 comprise a toxicity profile of the one or more bile acids based on a hepatotoxic response using a cytotoxicity assay. Such cytotoxicity assays can comprise, but are not limited to, an enzyme leakage assay, ATP, APOTOX Glo and a combination thereof. The enzyme leakage assay can be, for example, ALT, AST and LDH.
In some aspects, such an in vitro system can be configured to characterize a compound causing no change in the bile acid toxicity potencies as a compound not likely to cause systemic or hepatic toxicity. In some aspects, such an in vitro system can be configured to characterize a compound causing an increase in the bile acid toxicity potencies as a compound having a potential to cause cholestatic hepatic toxicity. In some aspects, such an in vitro system can be configured to characterize a compound causing a decrease in the bile acid toxicity potencies as a compound having a potential to cause cholestasis resulting in systemic toxicity.
In some embodiments, provided herein are methods of screening a compound for its potential to cause systemic and/or hepatic toxicity. In some embodiments, the methods comprise providing a compound to be screened, establishing a HCS comprising a capacity for bile acid synthesis, bile acid transport and/or bile acid regulation, exposing the 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 in the presence of the compound to be screened, wherein the toxicity profile of the bile acid is known across the range of concentrations, wherein the toxicity profile of the bile acid comprises a toxicity potency of the bile acid, and comparing the toxicity potency of the bile acid in the presence and absence of the compound to determine the susceptibility of the compound to cause systemic and/or hepatic toxicity. In such methods, the compound can be exposed to the HCS across a range of concentrations.
In some aspects, the bile acid can comprise a combination of bile acids, wherein the toxicity profile of the combination of bile acids is known, wherein the toxicity profile comprises a toxicity potency. The combination of bile acids can comprise a plurality of bile acids provided in predetermined ratios. The combination of bile acids can comprise a plurality of bile acids at concentrations and ratios configured to mimic in vivo concentrations and ratios of the plurality of bile acids.
Determining the potential of the compound to cause systemic or hepatic toxicity can in some aspects further comprise identifying no change in the bile acid toxicity potency in the presence of the compound as compared to the absence of the compound, identifying an increase in the bile acid toxicity in the presence of the compound as compared to the absence of the compound, or identifying a decrease in the bile acid toxicity in the presence of the compound as compared to the absence of the compound.
A compound causing no change in the bile acid toxicity potency can be characterized as not causing systemic or hepatic toxicity. Alternatively, a compound causing an increase in the bile acid toxicity potency can be characterized as having a potential to cause cholestatic hepatotoxicity. The cholestatic hepatotoxicity can be caused by inhibition of bile acid efflux, wherein the compound is characterized as a bile acid efflux inhibitor, and/or the cholestatic hepatic toxicity is caused by antagonism of FXR, wherein the compound is characterized as a FXR antagonist, and/or a combination of both. The bile acid efflux inhibition can comprise inhibition of a bile salt export protein (BSEP).
Still yet, a compound causing a decrease in the bile acid toxicity potency can be characterized as having a potential to cause cholestasis resulting in systemic toxicity. The cholestasis resulting in systemic toxicity can be caused by inhibition of bile acid uptake.
In some aspects, determining the potential of the compound to cause systemic or hepatic toxicity comprises characterizing the compound as a bile acid efflux inhibitor, an FXR antagonist, an inhibitor of bile acid uptake, or none of the above. The compound can be a drug candidate, wherein the drug candidate is characterized as having a low probability of causing systemic and/or hepatic toxicity, or a high probability of causing systemic and/or hepatic toxicity.
Bile acid transport can comprise bile acid uptake, basolateral excretion and/or canalicular excretion. The range of concentrations of the bile acid can comprise intracellular concentrations mimicking in vivo intracellular fasting and/or postprandial concentrations. The range of concentrations of the bile acid can comprise a bile acid intracellular concentration sufficient to activate an FXR feedback mechanism. In some aspects, the bile acid or bile acids can be, but are not limited to, GCA, GCDCA, GDCA, DCA, CA, CDCA, TCA, TCDCA, LCA, GLCA, TLCA, or any combination thereof.
In some embodiments, determination of a toxicity profile of the bile acid can comprise measuring a hepatotoxic response using a cytotoxicity assay. The cytotoxicity assay can be, for example, an enzyme leakage assay, ATP, APOTOX Glo and a combination thereof. The enzyme leakage assay can include, for example, of ALT, AST and LDH.
In such methods, the HCS can comprise a 2-dimensional culture or 3-dimensional culture, utilizing primary hepatocytes or other hepatic cell systems. The 2-dimensional culture can comprise a sandwich culture of hepatocytes (SCH), wherein the SCH comprises human hepatocytes. Alternatively, the 3-dimensional culture can comprise a 3D scaffold-based culture or spheroidal culture. Other hepatic cell systems can comprise HepaRG, Huh7, co-cultured 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 HCS.
The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed subject matter.
As demonstrated herein, drug induced cholestatic hepatotoxicity is the result of impaired bile flow of bile acids via inhibition of BSEP by the NCE and impairment of the hepatocyte bile acid homeostasis adaptive response. One possible mechanism of action causing drug induced cholestatic hepatotoxicity can comprise a cholestatic hepatotoxicant inhibiting BSEP function (initial insult) and 1) preventing (antagonize) Farnesoid X Receptor (FXR) from sensing the rising intracellular concentrations of bile acids; or 2) inhibiting all bile acid efflux pathways (basolateral and canalicular); or 3) a combination of both.
Thus, in some embodiments, the screening assays, methods and systems provided herein include components of the following concepts:
Assays, methods and systems are provided herein to identify NCE that have the potential to interfere with the hepatocyte's bile acid handling capacity. 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 which is relatable to clinical measurements (AST/ALT) typically used to monitor liver function. In some embodiments, such assays, methods and systems are configured to categorize an NCE as being cholestatic, having no effect, or causing cholestatic hepatotoxicity.
Potential Outcomes (BA in the presence of NCE) can in some embodiments be expressed as illustrated in
Bile acids GCA, GCDCA and GDCA were evaluated at increasing concentrations in SCHH over 24 hours. ATP (
Glucose concentrations were evaluated to examine the impact of glucose concentrations on bile acid pool toxicity profiles, i.e., shifts, in the absence and presence of cholestatic agents, e.g. troglitazone and sitaxsentan, in SCHH. Utilizing similar methodology from the previous sections, hepatotoxicity was evaluated with ApoTox-Glo™ Triplex Assay. The results are shown in
Low glucose conditions were needed to increase the SCHH system susceptibility to the troglitazone effect on cell viability and bile acid potency relationship (
A feasibility study was also conducted to determine the ability of the disclosed assays, methods and systems to distinguish Ambrisentan from Sitaxsentan, the results of which are below and in
The effects of individual bile acids and a mixture of bile acids (bile acid pool) were evaluated in sandwich-cultured human hepatocytes following a 24 hour exposure time in the presence of 5 mM glucose, for their potential to alter the hepatotoxicity of troglitazone (100 μM) and sitaxsenten (60 μM). The effects of the test compounds were evaluated in the presence of the individual bile acids and in the presence of a bile acid pool. The effects were compared to bile acid treatment alone. DMSO treatment alone was used as a comparator for hepatotoxicity. Hepatotoxicity was evaluated with ApoTox-Glo™ Triplex Assay, which assesses viability, cytotoxicity and apoptosis.
A bile acid mixture was evaluated, and was composed of 4 different bile acids maintained at a physiological relevant ratio of each bile acid. Table 1. Individual bile acids were dosed using the concentration they represent in the mixture.
A left shift for troglitazone was observed only in the presence of the bile acid pool, indicating that less bile acids were required to decrease cell viability (
These data suggest that a bile acid pool is, in some embodiments, a necessary component to allow the system to differentiate compounds effects on bile acid homeostasis. Troglitazone interferes with the ability of the hepatocyte to respond to increased intracellular concentrations of bile acids, and thereby increasing the hepatotoxicity of the bile acids.
Utilizing similar methodology from the previous section, the effect of troglitazone (100 μM) and sitaxsenten (60 μM) on the hepatotoxicity of a bile acid pool was evaluated in the presence of two free fatty acid ratios (2:1, and 0:3 Oleate:Palmitate). A treatment group without free fatty acids was used as a control. Hepatotoxicity was evaluated with ApoTox-Glo™ Triplex Assay which assessed viability, cytotoxicity and apoptosis.
In the absence of free fatty acids the hepatotoxicity of the bile acids was increased in the presence of troglitazone (
The addition of free fatty acids at both ratios of oleate:palmitate resulted in additional changes in the hepatotoxicity of the bile acid pool in the presence of troglitazone. See
A similar effect was observed in the presence of a 0:3 oleate:palmitate ratio (
Addition of free fatty acids alone at either ratio of oleate:palmitate minimally affected the hepatotoxicity profile of pooled bile acids. The TC50 of bile acids in the DMSO control was minimally reduced by 89% and 86% of Control, respectively, with the addition of either the 2:1 or 0:3 oleate:palmitate ratio of free fatty acids. See Table 5 below.
This unanticipated effect of free fatty acids on the hepatotoxicity of bile acids 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 disposition and to predict hepatotoxicity.
As discussed above, hepatocytes become susceptible to bile acid toxicity (e.g. cholestatic hepatotoxicity) when drug exposure inhibits bile acid efflux routes (e.g. BSEP and/or MRP3/4) and 1) prevents (antagonize) FXR from sensing the rising intracellular concentrations of bile acids; or 2) inhibits bile acid efflux through OSTα/β; or 3) a combination of both. To further illustrate this discovery, SCHH were exposed to either a BSEP inhibitor (e.g. cyclosporine A (CsA; Ansede et al., 2010) or a compound with both BSEP inhibitor and FXR antagonist characteristics (e.g. troglitazone; Marion et al., 2007 and Kaimal et al. 2009) for 24 hours using a low glucose (about 2 mM to about 10 mM, or about 5 mM) standard culture medium or sensitization 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 a 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). Briefly, hepatocytes were established in a sandwich-cultured configuration for four days. On day four of culture, SCHH were exposed to CsA (10 μM; 13× Cmax) or troglitazone (100 μM; 16× Cmax) for 24 hours cultured in standard culture medium or sensitization medium. Concentrations of CsA and troglitazone were based on Cmax values reported in Dawson et al. 2012. Drug exposures ranged from about 8× to about 16× Cmax to account for concentrating effect that can occur in the portal vein following oral administration of test compounds. Following 24 hours, LDH leakage and ATP content was evaluated utilizing commercially available assays. No marked increases of LDH secretion and no significant decreases in ATP content were observed in any of the three SCHH donors treated with either CsA (10 μM; 13× Cmax) or troglitazone (100 μM; 16× Cmax) under standard culture conditions (
The sensitization medium included free fatty acids and a physiological mixture of primary and secondary bile acids, described above (GCDCA, GCA, DCA, and GDCA) at an adequate concentration (250 μM) that minimized toxicity but challenged the hepatocytes bile acid homeostasis mechanism. Following 24 hours of exposure to sensitization media, LDH secretion was not significantly (Tukey's; p-value<0.05) increased in two out of the three SCHH preparations evaluated (
Hepatocytes under standard culture conditions do not have a sufficient concentration of bile acids or proper mixture to induce toxicity in the presence of a cholestatic hepatotoxicant, troglitazone. This was demonstrated by the lack of an effect on LDH and ATP under standard culture conditions across all three donors examined when treated with troglitazone (
Titration of troglitazone, a thiazolidinedione class compound, of 50 μM (8× Cmax), 75 μM (12× Cmax), and 100 μM (16× Cmax) across three different SCHH preparations established dose-dependency of the toxicity manifesting in only the sensitization media (
This conclusion was further supported by inhibition of biliary clearance of d8-TCA (
Other thiazolidinediones including pioglitazone and rosiglitazone were utilized as a preliminary evaluation of the cholestatic hepatotoxicity screen prediction accuracy. Troglitazone, a thiazolidinediones and well-established DILI agent, has clinical liver injury incidence of 1:1000 patients and was withdrawn from market due to liver injury (LiverTox Database). In contrast, fewer than 12 liver injury cases have ever been reported for either of pioglitazone and rosiglitazone despite extensive use (LiverTox Database).
In large clinical trials, ALT elevations≥3× upper limit normal were not different from placebo recipients for either pioglitazone or rosiglitazone suggesting low potential for liver injury for either of these thiazolidinediones. All three thiazolidinediones were evaluated in the cholestasis hepatotoxicity screening assay across a wide range of concentrations (1, 5, 10, 25, 50, 100 μM) encompassing the Cmax for each 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). A marked increase of LDH leakage (863% of control) and marked loss of ATP content (>99% of control) was observed in SCHH treated with troglitazone (100 μM) under sensitization culture conditions only (
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It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
This application claims benefit of U.S. Provisional Patent Application Ser. No. 62/395,503, filed Sep. 16, 2016, herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/052022 | 9/18/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62395503 | Sep 2016 | US |
