Investigation of the effect of hepatic metabolism on off-target cardiotoxicity in a multi-organ human-on-a-chip system

Abstract

Regulation of cosmetic testing and poor predictivity of preclinical drug studies has spurred efforts to develop new methods for systemic toxicity. Current in vitro assays do not fully represent physiology, often lacking xenobiotic metabolism. Functional human multi-organ systems containing iPSC derived cardiomyocytes and primary hepatocytes were maintained under flow using a low-volume pumpless system in a serum-free medium. The functional readouts for contractile force and electrical conductivity enabled the non-invasive study of cardiac function. The presence of the hepatocytes in the system induced cardiotoxic effects from cyclophosphamide and reduced them for terfenadine due to drug metabolism, as expected from each compound's pharmacology. A computational fluid dynamics simulation enabled the prediction of terfenadine-fexofenadine pharmacokinetics, which was validated by HPLC-MS. This in vitro platform recapitulates primary aspects of the in vivo crosstalk between heart and liver and enables pharmacological studies, involving both organs in a single in vitro platform. The system enables non-invasive readouts of cardiotoxicity of drugs and their metabolites. Hepatotoxicity can also be evaluated by biomarker analysis and change in metabolic function. Integration of metabolic function in toxicology models can improve adverse effects prediction in preclinical studies and this system could also be used for chronic studies as well.

Keywords: Cardiotoxicity; Functional readouts; Human-on-a-chip; Metabolism; Non-invasive.

Figure 1.. Cardiac and liver co-culture in a pumpless microfluidic system.

A schematic of the microfluidic platform and the interface used to measure the functional activity. Two laser cut acrylic layers housed two laser cut PDMS gaskets that define the microfluidic flow pathway and the compartments for each organ chip (A). Cells were cultured on the respective surfaces: hepatocytes on glass coverslip (compartment 1) and cardiomyocytes on cantilever (compartment 2), and on MEA chips (compartment 3) (A-B). Medium exchange was performed through both reservoirs (R1 and R2), and drug addition through R1. Signals on the MEA chips were recorded utilizing an amplifier via a printed circuit board and an elastomeric connector (C). Cardiac contractile function was measured using a laser-deflection based apparatus (D). Drug compound quantification was performed via HPLC-MS (E).

Figure 2.. Characterization of human cardiomyocytes and hepatocytes static co-culture in serum-free medium.

Human iPSc derived cardiomyocytes were co-cultured with human primary hepatocytes in serum-free medium (HSL2, 2 mL) for 7 - 14 days in wells. At day 7, mono-culture (MC; top) or co-culture (CC; bottom) morphology images of cardiomyocytes (A-left) and hepatocytes (A-right), and their viability (p=0.6 and p=0.6, respectively) (B). Spontaneous beat frequency of cardiomyocytes tracked over 14 days of culture appears affected by the time and the presence of the hepatocytes (p=0.002) (C). Albumin (red) and DAPI (blue) staining of hepatocytes at day 7 (D). Hepatocyte albumin (p=0.8) and urea (p=0.4) daily productions, and cytochrome p450 1A1 (p=0.3) and 3A4 (p=0.6) activities at day 7 (E). (50 μm scale). (** p<0.01)

Figure 3.. Characterization of the heart-liver system –serum-free and flow- with non-invasive measurements along seven days.

Human cardiomyocytes and hepatocytes were studied over 7 days in HSL3 medium. Representative morphology images are shown for human cardiomyocytes (A) in mono-culture (top) or co-culture (bottom) (80 μm scale) and hepatocytes in co-culture (B) after 7 days in the housing (50 μm scale). Cardiac function was measured throughout 7 days, in the presence (red square) or absence (blue diamond) of hepatocytes. Cardiac function is plotted as conduction velocity, spontaneous beat frequency, mISI (or QT interval) and contractile force (C). Two-way ANOVA was performed to study the effects of culture time and the presence of the hepatocytes on the different cardiac functional parameters; conduction velocity (p=0.8, 0.03), beat frequency (p=0.8, 0.2), mISI (p=0.3, 0.2) and force (p= 0.7, 0.9). Hepatic function was studied after 7 days in the system with cardiomyocytes and compared to the static mono-culture condition. No significant differences were evident through a t-test for the 1A1 (p=0.09) and 3A4 (p=0.7) enzymes (D).

Figure 4.. Dose-response of cyclophosphamide and its metabolite acrolein in human cardiomyocytes and hepatocytes.

The viability and function of human cardiomyocytes (A-black diamonds: beat frequency and A-green squares: viability) and hepatocytes (B-blue diamonds: 1A1 activity, B-red squares: 3A4 activity and B-green triangles: viability) in mono-cultures were studied using increasing concentrations of cyclophosphamide (CP; 0-9000 μM) (left) and the metabolite acrolein (ACR; 0-500 nM) (right) for 48 hours in HSL2 medium. The concentrations achieved in the dose-response were studied as a statistical factor influencing cellular function or viability (square brackets). CP reduced beat frequency significantly only at 9000 μM, compared to the control. (* p<0.05, ** p<0.01; *** p<0.001).

Figure 5.. Dose-response of terfenadine and its metabolite fexofenadine in human cardiomyocytes and hepatocytes.

The viability and function of human cardiomyocytes (A-black diamonds: beat frequency and A-green squared: viability) and hepatocytes (B-blue diamonds: 1A1 activity, B-red squares: 3A4 activity and B-green triangles: viability) in mono-cultures upon increasing concentrations of terfenadine (TER; 0-10 μM) (left) and its metabolite fexofenadine (FEX; 0-10 μM) (right) for 48 hours in HSL2 medium. The concentrations achieved in the dose-response were used as a statistical factor influencing cellular function or viability (square brackets). (** p<0.01; *** p<0.001).

Figure 6.. Heart-liver system and cardiac outcome upon cyclophosphamide treatment.

Human iPSc derived cardiomyocytes electrical (A-C) and mechanical (D) activities were evaluated non-invasively before and after 24 and 72 hours of an acute administration of cyclophosphamide (CP, 9 mM), with and without hepatocytes. All values are normalized to the control immediately before drug addition (A-D). As an endpoint analysis, cardiac viability was compared to the control (E). (^a p<0.08, * p<0.05, ** p<0.01; *** p<0.001).

Figure 7.. Heart-liver system and cardiac outcome upon terfenadine treatment.

Human iPSc derived cardiomyocytes electrical (A-C) and mechanical (D) activities were evaluated non-invasively after 24 and 72 hours of an acute administration of terfenadine (TER, 10 μM) in the heart-liver system, with and without hepatocytes. All values are normalized to the control immediately before drug addition (A-D). As an endpoint analysis, cardiac viability was compared to the control (E). (^a p<0.08, * p<0.05, ** p<0.01, *** p<0.001).

Figure 8.. Heart-liver system and hepatic outcome upon drug treatment.

Human primary hepatocytes were evaluated after the acute administration of cyclophosphamide (CP) (A), and terfenadine (TER) (B) in a dose-response fashion. p450 1A1 and 3A4 enzymatic activities (top), and cellular viability (bottom) were analyzed at the endpoint (at day 7, 72 h after drug addition). The concentrations achieved in the dose-response were used as a statistical factor influencing hepatic function and viability (square brackets). (*** p<0.001).

Figure 9.. Terfenadine and fexofenadine quantifications and correlation to the pharmacokinetic model.

The predicted concentration of terfenadine in the system over time changes with the system dynamic inputs of mixing, medium change, absorption and adsorption (A). The incorporation of the metabolic input into (A) generates the prediction of terfenadine and fexofenadine concentrations in the system. The experimental quantifications form the heart-liver and the model values are plotted together (B).