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ADME PK

3D Hepatotoxicity Assay using HepaRG Spheroids

Detect hepatotoxicity of novel therapeutics with enhanced in vivo relevance using Cyprotex’s 3D HepaRG spheroids combined with high content screening (HCS).

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Hepatotoxicity Assessment using 3D Microtissues

  • Drug induced hepatotoxicity is a leading cause of attrition during drug development. In vitro three-dimensional (3D) cell cultures allow better recapitulation of the complex in vivo microenvironment than traditional 2D monolayer models.
  • 3D models also permit long term compound exposures allowing a closer replication of clinical dosing strategies.
  • Glutathione depletion, reactive oxygen species (ROS) formation, mitochondrial disruption and cellular ATP depletion are key mechanisms involved in drug induced hepatotoxicity.
  • Confocal HCS allows the simultaneous detection of each cell health parameter within a 3D spheroid in combination with a measure of cellular ATP content

Protocol

3D Microtissue based hepatotoxicity assay protocol

Spheroid Cryopreserved HepaRG™ cells
Analysis Platform Confocal Cellomics ArrayScan® XTI (Thermo Scientific).
Test Article Concentration 8 point dose response curve with top concentration based on 100x Cmax or solubility limit
3 replicates per concentration*
Test Article Requirements 150 µL of a DMSO* solution to achieve 100x Cmax (200 x top concentration to maintain 0.5% DMSO) or equivalent amount in solid compound.
Time Points 14 days (336 hrs)*
Quality Controls Negative control: 0.5% DMSO (vehicle)
Positive controls: L-buthionine sulfoximine (GSH depletion and ATP) and rotenone (ROS formation and spheroid size)
Data Delivery Minimum effective concentration (MEC) and AC50 value for each measured parameter (spheroid count, spheroid size, DNA structure (DNA), mitochondrial mass (Mito Mass), mitochondrial membrane potential (MMP), glutathione content (GSH), oxidative stress (ROS) and cellular ATP content (ATP))*

*other options available on request

Data

Data from Cyprotex's 3D hepatotoxicity assay

 
Figure 1
Representative 3D confocal high content screening (HCS) images of known hepatotoxins, troglitazone and acetaminophen, labeled with Syto11 (green) to detect DNA structure, monochlorobimane (mBCL) (blue) to detect GSH content, dihydroethidium (DHE) (yellow) to detect ROS formation and MitoTracker deep red (red) to detect mitochondrial function.
DrugHuman exposure Cmax (µM)In vivo DILI category (P/N)3D HepaRG DILI prediction: multi-endpoint assay (MEC; µM)3D HepaRG DILI prediction: ATP alone (MEC; µM)Most sensitive feature
Amiodarone 5.3 p 2.41 5.12 DNA
Trovafloxacin 19.7 P 7.27 7.27 ATP
Diclofenac 10.1 P 30.5 38.8 DNA
Flutamide 5.4 P 7.43 7.75 SIZE
Lapatinib 19.2 P 0.77 1.21 GSH
Nitrofurantoin 21 P 4.89 9.27 SIZE
Sunitinib 0.25 P 0.28 1.1 GSH
Troglitazone 6.29 P 1.69 25 DNA
Fialuridine 1 P 1.41 1.41 ATP
Perhexiline 2.16 P 1.69 1.76 DNA
Tolcapone 21.96 P 18.2 20.5 MMP
Acetaminophen 165.4 P 240 342 SIZE
Bosentan 4.7 P 10.4 35.2 DNA
Ticlopidine 8.1 P 34 36.2 Mito Mass
Azathioprine 2.22 P 0.28 0.28 ATP
Chlorpromazine 0.94 P 1.07 3.48 SIZE
Tamoxifen 1.18 P 3.52 10.8 SIZE
Buspirone 0.01 N NR NR -
Entacapone 3.28 N 45.4 45.5 GSH
Metformin 7.74 N NR NR -
Table 1
Hepatotoxicity prediction of 20 reference compounds categorized according to literature data.

HepaRG spheroids were exposed to test compound for 14 days. During the 14 day period re-dosing occurs on 4 occasions with the final re-dose occurring 16 hours prior to the assay. The cell models were analyzed using the confocal mode of Cellomics ArrayScan® XTI (Thermo Scientific) following which cellular ATP content was measured using CellTiterGlo® (Promega). MEC = Minimum effective concentration. NR = No response. DILI = Drug induced liver injury. P = Positive, N = Negative. Red ≤ 5x Cmax, Green ≥ 5x Cmax.
A.
Raw traces for control DMSO and test compound troglitazone
B.
 
Figure 2
Graphical representation of (a) DNA structure (HCS) and cellular ATP response to troglitazone and (b) cellular ATP response to acute (16 hr) and chronic (14 day) fialuridine exposure in HepaRG spheroids.

All reference compound toxicities were correctly predicted by the HepaRG spheroid chronic exposure model using the 3D liver toxicity assay with a 5x Cmax cut off (table 1). Bosentan and tamoxifen are categorized as false negative responders with ATP alone (table 1) highlighting the enhanced sensitivity of a combined assay. Troglitazone response is one example of the improved assay sensitivity with HCS (ATP MEC 25 µM; DNA MEC 1.69 µM) (figure 2a), while fialuridine highlights the need for long term exposures in vitro (14 day MEC 1.41 µM; 16 hr MEC 451 µM) (figure 2b).

The combination of an in vitro 3D model that better recapitulates the in vivo cellular physiology of hepatic tissue with a multiparametric HCS and cytotoxicity assay presents a viable screening strategy for the accurate in vivo relevant detection of novel therapeutics that cause drug induced liver injury early in drug development.

References

1 Persson M et al., (2014). High-content analysis/screening for predictive toxicology: Application to hepatotoxicity and genotoxicity. Basic & Clin Pharma & Tox 115(1); 18-23.

2
Hornberg JJ et al., (2014). Exploratory toxicology as an integrated part of drug discovery. Part II: Screening strategies. Drug Discovery Today. 19(8); 1137-1144.

3 Sakatis MZ et al., (2012). Preclinical strategy to reduce clinical hepatotoxicity using in vitro bioactivation data for >200 compounds. Chem Res Toxicol 25(10); 2067 –82.

4 Thompson RA et al., (2012). In vitro approach to assess the potential for risk of idiosyncratic adverse reactions caused by candidate drugs. Chem Res Toxicol 25(8); 1616-32.

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