WO2017085119A1

WO2017085119A1 - Method and assay for the assessment of a cholestatic risk of a compound

Info

Publication number: WO2017085119A1
Application number: PCT/EP2016/077849
Authority: WO
Inventors: Delilah HENDRIKS; Lisa FREDRIKSSON PUIGVERT; Magnus Ingelman-Sundberg; Simon MESSNER
Original assignee: Insphero Ag
Priority date: 2015-11-16
Filing date: 2016-11-16
Publication date: 2017-05-26

Abstract

The present invention relates to a method and assay for the assessment of a cholestatic risk of one or more test compounds, which method comprises providing at least two hepatocyte cell cultures, exposing one of the said cell cultures to said one or more test compounds, exposing one other of the said cell cultures to said one or more test compounds and to a Bile Acid mixture (BA mixture), determining an impairing effect of the earlier steps on the respective cell cultures, and comparing these at least two impairing effects to assess the cholestatic risk of the one or more test compounds.

Description

Method and assay for the assessment of a cholestatic risk of a compound

Field of the invention

The present invention relates to the assessment of a cholestatic risk of a compound. Introduction

Drug induced cholestasis (DIC) is one of the major subtypes of Drug-Induced Liver Injury (DILI) and may account for up to 50% of all DILI cases.

Cholestasis, in general, is a condition where bile cannot flow from the liver to the duodenum. The two basic distinctions are an obstructive type of cholestasis, where there is a mechanical blockage in the duct system that can occur from a gallstone or malignancy, and metabolic types of cholestasis which are disturbances in bile formation that can occur because of genetic defects, acquired as a side effect of many medications or drugs.

Drug-induced liver injury (DILI) constitutes a significant problem for patient safety and is the most common reason for denial of drug approval and withdrawal of marketed drugs [1]. Drug-induced cholestasis (DIC) is one of the major subtypes of DILI and may account for up to 50% of all DILI cases [2]. Moreover, DIC has been implicated in the hepatotoxicity of nefazodone and troglitazone, which were both withdrawn from the market after reports of fulminant hepatic failure [3, 4]. As discussed, DIC is primarily associated with impaired bile acid (BA) homeostasis, which leads to the intrahepatic retention and accumulation of BAs [5]. Direct inhibition of the canalicular bile salt export pump (BSEP) has emerged as one of the main mechanisms of DIC [6] and has been reported for numerous cholestatic compounds, including bosentan [7], cyclosporin A [8], nefazodone [3] and troglitazone [4]. The majority of existing in vitro models determine the cholestatic potential of compounds based on their ability to directly interfere with BA disposition in membrane vesicles from BSEP-expressing Sf9 cells [9] or sandwich-cultured hepatocytes [10]. However, a major limitation of these systems is their inability to identify compounds that induce cholestasis through mechanisms other than BSEP inhibition, or in the case of the in vitro BSEP-inhibition assay, inability to identify compounds that are dependent on metabolic activation for induction of cholestatic toxicity.

DIC often manifests only weeks or months after the beginning of drug treatment [11]. Yet, the current in vitro models assess the cholestatic potential of compounds only in an acute setting, and most are focused on identifying inhibitors of the bile salt export pump, BSEP, which is the most common cause of the DIC, although not the only one.

Furthermore, the drug concentrations required to elicit an acute toxic response in vitro often greatly exceed the reported plasma Cmax, which could mask the true toxicity mechanisms of the drug, such as the involvement of reactive drug metabolites. It is clear that there is an urgent need for novel in vitro models to study and predict DIC in a more chronic setting, which allows using drug concentrations closer to the plasma Cmax. To this end, it is crucial to be able to maintain long-term human liver functionality in vitro.

It is one object of the present invention to provide a new and alternative method for the assessment of the cholestatic risk of a compound.

Summary of the invention

These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.

Embodiments of the invention Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word "comprising" does not exclude other elements or steps. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

According to one embodiment of the invention, a method and assay for the assessment of a cholestatic risk of one or more test compounds is provided, which method comprises

a) providing at least two hepatocyte cell cultures,

b) exposing one of the said cell cultures to said one or more test compounds, c) exposing one other of the said cell cultures to said one or more test compounds and to a Bile Acid mixture (BA mixture), d) determining impairing effects of steps b) and c), respectively, on the respective cell cultures, and

e) comparing these at least two impairing effects to assess the cholestatic risk of the one or more test compounds

The claimed method and assay tests the inhibitors effect of one or more test compounds alone against the impairing effect of one or more test compounds combined with a Bile Acid mix.

The rationale behind is that many compounds have an impairing effect on bile acid (BA) transporters, like MRP2 and BSEP (canalicular bile salt export pump), hence interfering with biliary clearance of bile acids, thus causing. Cholestasis, including drug-induced cholestasis (DIC), is a condition where bile cannot flow from the liver to the duodenum. Therefore the addition of bile acids to the text compounds mimicks the increased bile concentration in the liver as a result of that- The assay assesses the toxicological impact of compounds under those conditions.

Even when incubated with subtoxic levels of Bile Acids, the coincubation thereof with a test agent that affects bile acid clearance can lead to impairment of a cell culture, due to increase of bile acid concentration. Hence, such coincubation can lead to synergistic effects of both the test agent and the Bile Acids, wherein in one embodiment the actual impairment of the cells is caused by increased concentration of Bile Acids rather that by the test agent per se.

As used herein the term "cholestatic risk" hence means the potential of a given compound to cause, or increase the risk of, choleostasis (either alone or in combination with Bile Acids).

As used herein, the term "Bile Acid mixture", refers to a mixture of two or more bile acids. Preferably, the cell cultures are exposed to non-toxic concentrations thereof.

It is important to understand that said model, can distinguish cholestatic from non-cholestatic, yet hepatotoxic, compounds, preferably when under a long-term repeated dose setting. The model is further able to accurately recapitulate previously shown mechanisms of cholestatis. The inventors have shown that the claimed model is able to accurately recapitulate previously shown mechanisms of choleostasis, and to faithfully predict cholestatic potential of different test compounds.

In one embodiment, the hepatocyte cell culture is a primary cell culture. Compared to tumor cells or immortalized cells, primary cells have not accumulated mutations, and is thus more representative, or predictive, for the behavior of in vivo tissue. The transfer of primary cells into 3 -dimensional cell cultures or tissues has two significant advantages: a) it further increases the physiologic similarity of the cells to in vivo tissues, and thus makes them even more representative, or predictive, and b) it increases the longevity of the primary cells compared to 2D cell cultures or cell suspensions.

In one embodiment of the invention, the hepatocyte cell culture is a spheroidal cell culture.

3 -dimensional cell culture or tissue have a significant advantage in the present context because they are a more faithful reproduction of a natural tissue, and behave more physiologically than, e.g., a 2D cell culture or a cell suspension. Further, they have a longer lifetime than 2D cell cultures or cell suspensions, which facilitates handling and allows for the testing of long term effects and off-target effects of candidate compounds. This is especially useful in screening of compounds that may later be used for long term treatments, or to which humans are exposed over long stretches of time.

The inventors have realized that cultivation of hepatocytes as 3D organotypic spheroids in vitro has significant advantages for cholestatic risk assessment over other assay types, which for example use 2D cell cultures.

Chatterjee et al. recently published a model based on sandwich-cultured rat and human hepatocytes which identifies cholestatic compounds based on their potential to interfere with BA homeostasis [13, 14]. However, this approach has some shortcomings which the above embodiment surprisingly overcomes.

Hepatocyte spheroids show extensive cell-cell contacts, cell-extracellular matrix interactions, cell polarity and functional bile canaliculi. In particular, hepatocyte spheroids formed via self- aggregation of the cells is considered useful, since it represents a scaffold-free system that is applicable to high-throughput screening.

Recent characterization of primary human hepatocyte (PHH) spheroids has furthermore shown stable liver-specific functionality during long-term cultivation, such as hepatic protein expression, cytochrome P450 enzyme activity and albumin secretion.

Without being bound to theory, the inventors speculate that this is one reason for the fact that 3D hepatocyte spheroids have a much longer lifetime than 2D cell cultures, hence allowing prolonged assay duration. This is extremely useful for the purpose of the invention, because oftentimes cholestatic agents develop their harmful impact only after a prolonged exposure.

In fact, most drug-induced cholestasis manifests only weeks after onset of treatment. Therefore, primary human hepatocyte spheroids offers a unique system to test the ability of compounds to induce cholestasis in a chronic setting, with exposure times of up to 3 weeks and more. Additionally, the inventors show that prolonged exposure to test compounds results in a clearer discrimination between cholestasis positive and negative compounds (see Table 3).

For the above reasons, the 3D hepatocyte spheroid system provides significant advantages and is hence superior to previously published 2D system, in particular in case where compounds are to be tested on a potential cholestatic effect.

In several embodiments of the invention, the hepatocyte cell culture is a hepatocyte cell culture comprising

• human cells

• cynomolgus cells

• pig cells

• canine cells, and/or

• rat cells.

In one embodiment, the spheroidal cell culture is grown and cultivated in a hanging drop system, as distributed by InspheroAG of Schlieren, Switzerland, under the brand "GravityPLUS™ Hanging Drop System", and disclosed in international patent application WO2010031194A1. This approach allows the generation of 3-dimensional cell cultures or tissues in more complex 3D cell culture scenarios, such as when using primary cells, cell lines that are resistant to self-aggregation, or when generating co-culture microtissues.

In another embodiment, the spheroidal cell culture is grown and cultivated in a low adherence well.

Such low adherence well system is for example provided by Insphero (CH), who distribute the GravityTRAP™ ULA Plate. As an alternative, Multititer plates treated with a cell repellent as for example provided by Greiner can be used.

In a preferred embodiment, the impairing effect that is assessed is at least one selected from the group consisting of

• growth inhibition of the cell culture,

• residual viability of cells within the cell culture, and/or

• cytotoxic effect on cells within the cell culture.

Growth inhibition can for example be determined by size determination of the cell culture treated according to steps b) and c), optionally compared with an untreated cell culture.

Preferably, the size determination refers to at least one parameter selected from the group consisting of:

• diameter

• perimeter

• volume

• area of an optical cross section

The size can thus either be a parameter that has directly been measured, or a parameter which has been calculated on the basis of such measurements. Preferably, the size determination of the 3 -dimensional cell culture or tissue is carried out by means of an imaging device.

One example of such imaging device is the Cell³iMager distributed by, InSphero AG, Schlieren, CH, and manufactured by SCREEN, Japan. It allows analysis of spheroids by scanning multi-well plates in a bright-field. It computes estimated values based on spheroid size and density, together with spheroid number and area in each well. With easy and efficient operability, its vibration-free design protects cells from damage. An excellent application is also available to determine spheroid proliferation over time and to measure the granular distribution in 3D culture.

Residual Cell viability/cytotoxic effect can for example be determined by quantitation of the ATP present in the cells. One such assay is the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, US), while other cell viability/cytotoxicity assays exist on the market and can likewise be used.

Preferably, the impairing effect is determined as inhibitory concentration, preferably as half maximal inhibitory concentration (IC₅₀).

Such IC50 can preferably relate to any of growth inhibition of the cell culture, residual viability of cells within the cell culture, and/or cytotoxic effect on cells within the cell culture.

The use of the complete concentration range, and deduction of the IC50 value therefrom, for determining the Cholestatic Index (CI) gives less room for false positive (and negative) errors than the use of isolated concentrations only as previously published in the shape of the Drug- Induced Cholestasis Index (DICI). [13]

In a preferred embodiment of the invention, wherein the Bile Acid mixture comprises at least two of

• cholic acid

• chenodeoxycholic acid

• deoxycholic acid

• glycochenodeoxycholic acid, and/or

• lithocholic acid. In one preferred embodiment of the invention, the cholestatic risk of the one or more test compounds is determined by means of a cholestatic index (CI).

The concept of the cholestatic index (CI) has been newly developed by the inventors. The CI is defined as the ratio of the ICso-value resulting from co-exposure to a compound and the BA mixture and the ICso-value of exposure to the same compound alone (ICso(+BA)/ICso(-BA)). The classification of the cholestatic liabilities of compounds can be found in Table 2.

Table 2. Definition of a compounds cholestatic risk as

determined by the Cholestatic Index (CI).

In preferred embodiments, the CI is defined as the ratio of a) the ICso-value resulting from co-exposure to one or more test compounds and the BA mixture, and

b) the ICso-value of exposure to the same one or more test compounds alone.

In a preferred embodiment of the invention, it is provided that a Cholestatic Index (CI) of a) > 0.90 signifies no or low cholestatic risk

b) 0.70 - 0.90 indicates a moderate cholestatic risk, and

c) < 0.70 indicates a high cholestatic risk, and of the one or more test compounds.

According to one embodiment of the invention, the use of a spheroidal hepatocyte cell culture in a method according to the above description is provided.

In one embodiment, the test compound is a natural, synthetic or recombinant substance or any combination thereof. The latter would be a substance produced by recombinant expression technology or synthetic peptide synthesis, such as, e.g., a recombinant antibody, fragment or derivative thereof, fusion protein, peptide, or antibody-drug conjugate. According to a preferred embodiment, the test compound is a chemotherapeutic substance.

In one embodiment of the invention, the use or method described above serves for the determination of the therapeutic window of the test compound. As used herein, the term therapeutic window, also called therapeutic index, refers to a comparison of the amount or dosis of a test compound that causes toxicity to the amount or dosis that causes the therapeutic effect. A high therapeutic window or index is preferable for a drug to have a favorable safety profile. During clinical development, the therapeutic window can be expressed as ratio of the dosis that causes adverse effects at an incidence/severity not compatible with the targeted indication (e.g., toxic dose in 50% of subjects) divided by the dosis that leads to the desired pharmacological effect (e.g., efficacious dose in 50% of subjects).

In one embodiment of the invention, a library of test compounds is screened with the use or method described above.

Said library of test compounds is understood herein as a set of several, many or a large number of test compounds (or combinations of test compounds) which are screened consecutively, or simultaneously, for the respective effect. Such libraries can be, for example, libraries of synthetic chemical compounds, libraries of complex natural compounds, libraries of extracts from plants or microorganisms, phage display libraries, yeast display libraries, or ribosomal display libraries.

In one embodiment of the invention, the use or method described above is carried out consecutively, or simultaneously, with two or more test compounds, or two or more test combinations each comprising two or more test compounds.

Experiments and Figures

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Any reference signs should not be construed as limiting the scope.

Materials and Methods

Cell culture medium, medium supplements, and compounds were obtained from Sigma- Aldrich (Sweden) or Life Technologies (Sweden) unless otherwise stated. Bosentan was obtained from Sequoia Research Products, Ltd. Cholic acid was obtained from Millipore (Sweden) and antibodies against MRP2 and BSEP were retrieved from Abeam (UK) and Atlas antibodies (Sweden) respectively. N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,l,3- benzoxadiazole)]-amino-3a,7a,12a-trihydroxy-27-nor-5P-cholestan-26-oyl)-2- aminoethanesulfonate (tauro-nor-THCA-24-DBD) was obtained from Genomembrane Company, Ltd. (Yokohama, Japan).

Primary human hepatocyte spheroid cultures

Cryopreserved primary human hepatocytes (PHHs) (lot SSR, Bioreclamation IVT, USA) were thawed according to manufacturer's protocol. Cells were seeded in PHH medium (Williams' medium E containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L- glutamine, 10 μg/ml insulin, 5.5 μg/ml transferrin, 6.7 ng/ml sodium selenite, 100 nM dexamethasone) supplemented with 10% fetal bovine serum at a density of 1,500 cells per well in ultra-low attachment (ULA; Corning, Sigma-Aldrich, Sweden) plates to allow spheroid formation. At day 5 and 7, 50% of the medium was exchanged with serum-free PHH medium.

Bile acid mixture

A bile acid (BA) mixture consisting of two primary BAs (cholic acid and chenodeoxycholic acid) and three secondary BAs (deoxycholic acid, glycochenodeoxycholic acid, and lithocholic acid) was used. The BA mixture was for each BA 30-5 Ox (PHH spheroids) concentrated compared to the respective concentration found in human plasma (Table 1) based on the toxicity profile (Fig. 7). These concentrations are although elevated, still in the range of what can be seen in cholestatic patients [15]. Concentration in human plasma

Acronym Full name

(μΜ)

CA cholic acid 0.41

CDCA chenodeoxycholic acid 0.64

DCA deoxycholic acid 0.48

LCA lithocholic acid 0.008

UDCA ursodeoxycholic acid 0.14

GCDCA glycochenodeoxycolic acid 0.8

SUM 2.478

Table 1. Declaration of the bile acid mix used and the concentrations found in normal human plasma [16]

Toxicity studies

Stock solutions of all compounds, except acetaminophen, were prepared in DMSO and the final DMSO concentration did not exceed 0.5%. Toxicity studies were started on day 8 after seeding. Spheroids were treated every other day with the compounds in the presence or absence of the BA mixture. After 8 and 14 days of treatment, cellular ATP content of single spheroids (n=5) was determined as a marker of cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Sweden) according to manufacturer's protocol. IC50 values were determined using GraphPad Prism.

Bile acid accumulation

Chlorpromazine-induced BA accumulation was assessed with a fluorescent labelled- taurocholate (tauro-nor-THCA-25-DBD). On day 6 of treatment with spheroids were additionally exposed to 20 μΜ tauro-nor-THCA-25-DBD. On day 8 of treatment, the spheroids were washed twice with HBSS and incubated with 1 mg/ml Hoechst 33342 for 1 h at 37°C. Spheroids were washed twice with HBSS and fiuorescence was assessed by confocal microscopy (Zeiss LSM 710, Germany) using a 10 x objective. Immunohistochemistry

Spheroids were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose and embedded in Tissue-Tek O. C. T. compound. Immunohistochemical staining was performed on spheroid cryosections (8 μιη) for MRP2 and BSEP. F-actin was visualized utilizing Alexa Fluor 488 Phalloidin. Slides were mounted with ProLong Gold Antifade Mountant with DAPI. Fluorescence was assessed with a confocal microscope using a 20 x objective.

Gene expression

Total RNA was extracted from PHH spheroids using Qiazol, according to manufacturer's protocol. RNA was reverse-transcribed into cDNA with Superscript III reverse transcriptase using Oligo (dT)₂o primers and RT-qPCR analysis was performed with a 7500 Fast Real-Time PCR system using a TaqMan Universal mix or SYBR Green mix.

RESULTS

Bile acid transporter expression in PHH spheroids

The competence of the primary human hepatocyte (PHH) spheroid system as a model to predict drug-induced cholestasis (DIC) was initially assessed by evaluating the protein expression of two key bile acid (BA) transporters, MRP2 and BSEP. Immunohistochemical analysis of PHH and HepaRG spheroids on day 16 revealed that MRP2 was abundantly expressed, whereas BSEP expression was only marginally detected at the rim of the spheroids (Fig. 1). However, importantly the protein expression of both MRP2 and BSEP could be induced using a non-toxic BA mixture for 8 days (Fig. 7).

The presence of bile acids selectively sensitizes PHH spheroids towards the toxicity of cholestatic hepatotoxicants

Recent studies in sandwich-cultured rat and human hepatocytes have shown that the toxicity of cholestatic hepatotoxicants is increased in the presence of BAs, whereas the toxicity of non-cholestatic hepatotoxicants remains unaffected by such a co-exposure [13, 14]. Knowing that the hepatocyte spheroids present a more liver-like system (Mora, submitted), which accurately express the major bile acid transporters (Fig. 1) we here wanted to investigate whether this strategy could be extrapolated to the PHH spheroid systems in which also long- term repeated dose toxicity could be evaluated.

In order to determine the potential of these systems to predict DIC we used three cholestatic hepatotoxicants (chlorpromazine, troglitazone and bosentan) and two non-cholestatic hepatotoxicants (acetaminophen and tetracycline). The concentration of each BA in the BA mixture was 30x concentrated as compared to the physiological plasma concentration of each BA (Table 1) to ensure that the BA exposure alone caused no significant toxicity.

To be able to evaluate the cholestatic liability of the compounds tested we formulated the concept of a cholestatic index (CI). The CI is defined as the ratio of the ICso-value resulting from a compound and BA mix co-exposure and the ICso-value of the same compound alone. Compounds with a CI exceeding 0.90 were considered having a low or absent risk of cholestatic liability, while values between 0.90 and 0.70 were deemed moderate, and compounds with CI below 0.70 were considered as having a high risk for cholestasis.

In PHH spheroids, the toxicity of bosentan substantially increased in a dose-dependent manner in the presence of BAs resulting in a CI of 0.19 ± 0.12 whereby bosentan was deemed having a high cholestatic liability (Fig. 2). The toxicity of troglitazone (CI = 0.80 ± 0.17) and chlorpromazine (CI = 0.90 ± 0.12) was slightly increased when PHH spheroids were co- exposed to BAs, resulting in the classification of these hepatotoxicants as having low-to- moderate cholestatic risk. Importantly, the presence of BAs did not sensitize the PHH spheroids towards the toxicity of the acetaminophen (CI = 1.89 ± 0.37). Rather, a consistent protective effect of the BAs was observed at normally toxic concentrations of this compound (Fig. 2). Similarly, the presence of BAs did not sensitize the PHH spheroids towards the toxicity of tetracycline (CI = 1.00 ± 0.08).

Chronic dosing results in improved identification of cholestatic hepatotoxicants in PHH and spheroids

Recently it was shown that the toxicity of various hepatotoxicants increased in a time- dependent manner (Mora, submitted). Hence, subsequently we evaluated whether extending the treatment time to 14 days would improve the ability of the PHH spheroid system to distinguish cholestatic from non-cholestatic hepatotoxicants.

Regardless of the presence of the BA mixture, the toxicity of all hepatotoxicants was more pronounced after 14 days than 8 days (compare Fig. 2 and 3). Furthermore, the BAs sensitized the PHH spheroids to a greater extent towards the toxicity of the cholestatic compounds after 14 days compared to 8 days (Fig. 3). Most notably, an increase in the toxicity of chlorpromazine in the presence of BAs was observed after 14 days, leading to the classification of chlorpromazine as a compound with moderate cholestatic risk in the PHH spheroids (CI = 0.78 ± 0.05. The toxicity of acetaminophen and tetracycline remained unaffected in the presence of BAs after 14 days (Fig. 3).

Importantly for the model the results from the BA co-exposure experiments are reproducible and the PHH spheroid systems is able to distinguish cholestatic hepatotoxicants from non- cholestatic hepatotoxicants based on the CI values in a consistent manner (Table 3).

Table 3. The PHH spheroids can distinguish cholestatic from non-cholestatic compounds in a reproducible manner by determining the cholestatic index (CI) after 8 and 14 days of repeated exposure.

Typical mechanisms associated with drug-induced cholestasis can be detected in PHH spheroids Next, the PHH spheroids were evaluated for their use to mechanistically study DIC. Since one of the major hallmarks of DIC is hepatocellular BA accumulation [17], we assessed whether chlorpromazine induced BA accumulation in the spheroids using a fluorescently labelled taurocholic acid derivative (tauro-nor-THCA-24-DBD). In the PHH spheroids, a dose- dependent increase in BA accumulation was observed after 8 days of chlorpromazine exposure (Fig. 4).

Subsequently, we evaluated the effect of chlorpromazine on BSEP mRNA expression. In line with the observed accumulation of fluorescent bile acid, and consistent with previous findings [18], chlorpromazine down-regulated BSEP expression 5-fold in the PHH spheroids, as determined by quantitative PCR (Fig. 5 A). In addition, as previously described [18], disruption of especially the pericanalicular F-actin could be observed in the PHH spheroid system upon chlorpromazine exposure possibly leading to a destabilized bile canalicular structure and thereby further limiting the ability to secrete the excessive amounts of bile acids leading to their accumulation.

Fig. 1. Spheroids of primary human hepatocytes express the key bile acid transporters BSEP and MRP2 and both can be induced by the addition of a non-toxic dose of a bile acid mix for 8 days.

Fig. 2. PHH spheroids were exposed to compounds that are determined positive and negative for cholestasis with (+BA) and without (-BA) a non-toxic bile acid mix for 8 days. Viability was measured by quantifying the ATP levels. Data presented are means +/- SEM of 5 spheroids.

Fig. 3. The long-term (14 days) toxic effects of compounds that are determined positive and negative for cholestasis was investigated in the PHH spheroids with (+BA) and without (-BA) a non-toxic bile acid mix. Viability was quantified by measuring the ATP levels. Data presented are means +/- SEM of 5 spheroids.

Fig. 4. PHH spheroids show a concentration dependent accumulation of the fluorescent taurocholic acid derivative tauro-nor-THCA-24-DBD after 8 days exposure to chlorpromazine. The images are representative examples from three independent experiments. Fig. 5. The PHH spheroids can accurately recapitulate known underlying mechanisms of cholestasis induction. Repeated exposure to chlorpromazine for 8 days leads to a reduction of BSEP mRNA expression (A) as well as decreased F-actin expression (B). Data presented are means of 3 independent experiments +/- SEM and representative images from 3 different experiments respectively.

Fig. 6. Repeated dosing (8 days) of a bile acid mix concentrated according to the respecitve concentrations in human plasma (lx = 2.5 μΜ total concentration of bile acids, see Table 1) leads to a concentrantion dependent toxicity in PHH spheroids as determined by ATP levels (A). The maximal non-toxic conentration was defined as 3 Ox that of normal and it remained non-toxic for up to 14 days (B).

Fig. 7. Titration of bile acid toxicity in PHH and HepaRG spheroids. The toxicity of the BA mixture, concentrated according to the respective concentration of each BA in human plasma, was titrated in PHH spheroids (A) and HepaRG spheroids (B) after 8 days of repeated exposure. The maximal non-toxic concentration of the BA mixture was determined to be 30- 50x for PHH and 50x for HepaRG spheroids, respectively. The BA mixture remained nontoxic after 14 days of repeated exposure.

Fig. 8. ATP and albumin as toxicity read-outs have similar sensitivity in determining the cholestatic risk of compounds. PHH spheroids were repeatedly exposed to chlorpromazine or tetracycline for 8 days in the presence or absence of the BA mixture. Cholestatic risk classification of both compounds was compared using cellular ATP content (A) or albumin secretion (B) as toxicity read-outs.

Fig. 9. DR5 expression profile of PHH spheroids exposed to toxic concentrations of chlorpromazine or bile acids. PHH spheroids were repeatedly exposed to a toxic concentration of chlorpromazine or BAs. After 8 days, viability was assessed by measuring cellular ATP content (A) and expression levels of DR5 (B) were evaluated by RT-qPCR and normalized to the expression of the housekeeping gene GAPDH. Kaplowitz, N., Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov, 2005. 4(6): p. 489-99.

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Claims

What is claimed is

1. Method and assay for the assessment of a cholestatic risk of one or more test

compounds, which method comprises a) providing at least two hepatocyte cell cultures,

b) exposing one of the said cell cultures to said one or more test compounds, c) exposing one other of the said cell cultures to said one or more test compounds and to a Bile Acid mixture (BA mixture),

d) determining impairing effects of steps b) and c), respectively, on the respective cell cultures, and

e) comparing these at least two impairing effects to assess the cholestatic risk of the one or more test compounds.

2. The method according to claim 1, wherein the hepatocyte cell culture is a primary cell culture.

3. The method according to any one of the aforementioned claims, wherein the

hepatocyte cell culture is a spheroidal cell culture.

4. The method according to any one of the aforementioned claims, wherein the

hepatocyte cell culture is a hepatocyte cell culture comprising

• human cells

• cynomolgus cells

• pig cells

• canine cells, and/or

• rat cells.

5. The method according to any one of claims 3 - 4, wherein the spheroidal cell culture is grown and cultivated in a hanging drop system.

6. The method according to any one of claims 3 - 4, wherein the spheroidal cell culture is grown and cultivated in a low adherence well.

7. The method according to any one of the aforementioned claims, wherein the impairing effect is at least one selected from the group consisting of

• growth inhibition of the cell culture,

• residual viability of cells within the cell culture, and/or

• cytotoxic effect on cells within the cell culture.

The method according to any one of the aforementioned claims, wherein the impairing effect is determined as inhibitory concentration, preferably as half maximal inhibitory concentration (IC₅₀).

The method according to any one of the aforementioned claims, wherein the Bile Acid mixture comprises at least two of

• cholic acid

• chenodeoxycholic acid

• deoxycholic acid

• glycochenodeoxycholic acid, and/or

• lithocholic acid.

10. The method according to any one of the aforementioned claims, wherein the

cholestatic risk of the one or more test compounds is determined by means of a cholestatic index (CI).

11. The method according to any one of the aforementioned claims, wherein the CI is defined as the ratio of

a) the ICso-value resulting from co-exposure to one or more test compounds and the BA mixture, and

b) the ICso-value of exposure to the same one or more test compounds alone.

12. The method according to any one of the aforementioned claims, wherein a Cholestatic Index (CI) of d) > 0.90 signifies no or low cholestatic risk

e) 0.70 - 0.90 indicates a moderate cholestatic risk, and

f) < 0.70 indicates a high cholestatic risk, and of the one or more test compounds.

13. Use of a spheroidal hepatocyte cell culture in a method according to any one of claims

1 - 12

14. The use of claim 13 or method according to any one of claims 1 - 12, wherein the test compound is a natural, synthetic or recombinant substance or any combination thereof.

15. The use according to any one of claims 13 or 14 or method according to any one of claims 1 - 12 or 14, wherein the test compound is a chemotherapeutic substance.

16. The use according to any one of claims 13 - 15 or method according to any one of claims 1 - 12 or 14 - 15 for the determination of the therapeutic window of the test compound.

17. The use according to any one of claims 13 - 16 or method according to any one of claims 1 - 12 or 14 - 16, in which a library of test compounds is screened.

18. A method for screening a library of test compounds, which method encompasses the use according to any one of claims 13 - 17 or method according to any one of claims 1 - 12 or 14 - 17, carried out consecutively, or simultaneously, with two or more test compounds, or two or more test combinations each comprising two or more test compounds.