In Vitro Cholestatic DILI & Mitochondrial Toxicity Studies to ...

In Vitro Cholestatic DILI & Mitochondrial Toxicity

Studies to Assess Hepatotoxicity

Presented by

Miki FujishimaFrom the Drug Development

Solutions Center

Background

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Chemical Research in ToxicologyVolume 25, Issue 8, 20 August 2012

Toxicology and Applied Pharmacology 394 (2020) 114958

A survey of papers conducting comprehensive evaluations reveals that many studies focus on cholestasis and mitochondrial toxicity.

Drug-induced liver injury (DILI) is caused by various developmental mechanisms and it is difficult to accurately predict it in one type of assay. Therefore conducting various experiments and making a comprehensive judgment leads to accurate risk evaluation.

Background

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From the point of view of clinical safety information ...

In many cases, mitochondrial toxicity and bile acid transporter inhibition are observed in drugs that are withdrawn or classified for BBW.

HEPATOLOGY, Vol. 60, No. 3, 2014 (modified)

Cholestatic Drug-Induced Liver InjuryROC Analysis: Predicting Clinical Hepatotoxicity / CholestasisFunctional Assay: Bile Acid-Dependent Hepatotoxicity

Mitochondrial Toxicity

In Vitro Hepatotoxicity Studies

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4

5

Bile acid (BA)

Sandwich cultured hepatocytes

BSEP inhibition by drugs-> BA accumulation-> Liver injury

➢BSEP is one of transporters that relates bile acid (BA) excretion➢BA accumulation in liver occurred when BSEP is inhibited by drugs

= Drug-induced cholestatic liver injuryRef) Susukida et al., Drug Metab Disp, 2015

SMD has established an in vitro test system that assess the drug-induced cholestatic livery injury by patent* licensing from Chiba University

*patent application 2011-152087

Cholestatic DILI Assessment

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＜Features of the sandwich-cultured hepatocytes＞

Sandwich-cultured hepatocytes Transporter expression vesicle

・ Has metabolic capacity・ Has a function to regulate the expression level of transporters and metabolic enzymes

・ No metabolic capacity・ No function to regulate the expression level of transporters and metabolic enzymes

Frozen hepatocyte sandwich culture is thought to be able to evaluate phenomena involving biological mechanisms such as metabolism.

ATP

ADP

Vesicle

ABCTransporter


7

Thu

Day 1Cell seeding

Day 2Matrigeloverlay

Day 3Medium change

Day 4BA and TA Exposure

Day 5LDH measurement

Mon Tue Wed Fri＜Methodology＞


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- Day 5 of sandwich cultured hepatocyte- Bile canaliculi-like structure is visualized by CDFDA (5 (and 6)-carboxy-2,7-

dichlorofluorescein diacetate) exposure and obtain photomicrograph by confocal microscope (ImageXpress, Molecular Devices)

CDFDA

CDF; Fluorescent

hydrolysis


9

＜ Validation (positive, negative control evaluation) ＞


Cyclosporin A (μM) Flutamide (μM)

Rosuvastatin (μM) Fexofenadine (μM)

10

１. ROC (Receiver Operating Characteristics) analysis・ Test substance 1 concentration (100 µM)・ Bile acid presence・ 24 compounds for ROC analysis

(1) Set the cutoff value with the compound for ROC analysis(2) Positive hepatotoxicity if the LDH value of the test substance exceeds the cutoff value

２. Functional Assay (Bile Acid-Dependent Hepatotoxicity)・ 7 concentrations of test substance・ Bile acid with / without・ Positive control 1 concentration 1 type (Cyclosporin A)・ Negative control 1 concentration 1 type (Fexofenadine)

Procedure1 Calculate EC50 with and without bile acid2 Evaluate whether there is a difference in EC50 with or without bile acid⇒ If there is a difference, positive for cholestasis

＜Sample Protocol＞


ALP; >1 % is considered as hepatotoxicity

ROC (Receiver Operating Characteristics) analysisRelation between in vitro tox test (LDH release) and clinical biomaker (ALP)Ref) Susukida et al., Drug Metab Disp, 2015

x Cut off value

Positive

Negative

hepatotoxicityNo toxicity

ROCanalysis

In vitro study

LDH release

Clinical toxicity information


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LDH measurement was performed on 3 Lots of hepatocytes after exposure to the drug/bile acidfor 24 hours.

・ By adding BA, more toxicity can be detected.・ Similar tendency was seen between lots

＜Toxicity test for 24 chemicals＞


0 50 100 150

DMSO

Voriconazole

Tranilast

Ticlopidine

Tacrolimus

Simvastatin

Rosuvastatin

Pravastatin

Pioglitazone

Methotrexate

Losartan

Levofloxacin

Leflunomide

Lamivudine

Flutamide

Fluoxetine

Fexofenadine

Famotidine

Everolimus

Duloxetine

Cyclosporin A

Clopidogrel

Carbamazepine

Atorvastatin

Amiodarone

LDH release (%)

BA x150

BA x0

HC10-10

0 50 100

DMSO

Voriconazole

Tranilast

Ticlopidine

Tacrolimus

Simvastatin

Rosuvastatin

Pravastatin

Pioglitazone

Methotrexate

Losartan

Levofloxacin

Leflunomide

Lamivudine

Flutamide

Fluoxetine

Fexofenadine

Famotidine

Everolimus

Duloxetine

Cyclosporin A

Clopidogrel

Carbamazepine

Atorvastatin

Amiodarone

LDH release (%)

BA x150

BA x0

HC4-38

0 50 100

DMSO

Voriconazole

Tranilast

Ticlopidine

Tacrolimus

Simvastatin

Rosuvastatin

Pravastatin

Pioglitazone

Methotrexate

Losartan

Levofloxacin

Leflunomide

Lamivudine

Flutamide

Fluoxetine

Fexofenadine

Famotidine

Everolimus

Duloxetine

Cyclosporin A

Clopidogrel

Carbamazepine

Atorvastatin

Amiodarone

LDH release (%)

BA x150

BA x0

HC7-25

■ BA +■ BA -

■ BA +■ BA -

■ BA +■ BA -

ALP HC10-10 HC4-38 HC7-25cutoff (%) 8.2 14.2 7.7

真陽性率(Sensitivity) 54.5 54.5 54.5真陰性率(Specificity) 84.6 76.9 76.9

AUC 0.615 0.678 0.601

Voriconazole

Tranilast

Ticlopidine

Tacrolimus

Simvastatin

Rosuvastatin

Pravastatin

Pioglitazone

Methotrexate

Losartan

Levofloxacin

Leflunomide

Lamivudine

Flutamide

Fluoxetine

Fexofenadine

Famotidine

Everolimus

Duloxetine

Cyclosporin A

Clopidogrel

Carbamazepine

Atorvastatin

Amiodarone

0

10

20

30

40

50

60

70

80

90

100

0.01 0.1 1 10

LD

H t

oxic

ity (

%)

ALP (%)

HC10-10

Voriconazole

Tranilast

Ticlopidine

Tacrolimus

Simvastatin

Rosuvastatin

Pravastatin

Pioglitazone

Methotrexate

Losartan

Levofloxacin

Leflunomide

Lamivudine

FlutamideFluoxetine

Fexofenadine

Famotidine

Everolimus

Duloxetine

Cyclosporin A

Clopidogrel

Carbamazepine

Atorvastatin

Amiodarone

0

10

20

30

40

50

60

70

80

90

100

0.01 0.1 1 10

LD

H t

oxic

ity (

%)

ALP (%)

HC4-38

Voriconazole

Tranilast

Ticlopidine

Tacrolimus

Simvastatin

Rosuvastatin

Pravastatin

Pioglitazone

Methotrexate

Losartan

Levofloxacin

Leflunomide

Lamivudine

Flutamide

Fluoxetine

Fexofenadine

Famotidine

Everolimus

Duloxetine

Cyclosporin A

Clopidogrel

Carbamazepine

AtorvastatinAmiodarone

0

10

20

30

40

50

60

70

80

90

100

0.01 0.1 1 10

LD

H t

oxic

ity (

%)

ALP (%)

HC7-25

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HC10-10 HC7-25

HC4-38

Cut off (%) 8.2%

Sensitivity 54.5%

Specific 84.6%

AUC 0.615

Cut off (%) 14.2%

Sensitivity 54.5%

Specific 76.9%

AUC 0.678

Cut off (%) 7.7%

Sensitivity 54.5%

Specific 76.9%

AUC 0.601

- We totally have 5 lot data

- Day-to-day reproducibility data was also obtained (data not shown)

High reproducibility was observed


Lot A

Lot C

Lot B


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Normal Study Design・ 7 concentrations of test substance・ Bile acid with / without・ Positive control 1 concentration 1 type (Cyclosporin A)・ Negative control 1 concentration 1 type (Fexofenadine)

Procedure1 Calculate EC50 with and without bile acid2 Evaluate whether there is a difference in EC50 with or without bile acid⇒ If there is a difference, positive for cholestasis

Functional Assay (Bile Acid-Dependent Hepatotoxicity)

BA +

BA -

BA +

BA -

No difference: Negative for cholestasis Big difference: Positive for cholestasis

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Differences in toxicity between the presence of bile acids (BA +) and the absence of bile acids (BA-)⇒ Suspected cholestatic-type hepatotoxicity

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0

20

40

60

80

100

0 10 20 30

Concentration (μmol/L)

BA(+)

BA(-)

LD

H%

Cyclosporin ACyclosporin A (μmol/L)-20

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100 110

Concentration (μmol/L)

BA(+)

BA(-)

LD

H%

FlutamideFlutamide (μmol/L)

＜Prediction of the presence or absence of cholestasis＞


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- Someone who observed hepatotoxicity in in vivo animal test-> To get the data regarding human liver

- Someone who observed strong BSEP inhibition in vesicle study-> This study shows an “actual hepatotoxicity”. The data obtained in this study may save NCEs that have BSEP inhibition potency.

This study could be helpful for…





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Crabtree effect: In vivo hepatocytes produce ATP by aerobic respiration, but when cultured in a glucose-rich medium (general medium), aerobic respiration is suppressed and ATP is produced mainly in glycolysis. It is possible to shift to a state in which aerobic respiration is dominant by culturing in a galactose medium.

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Glucose cultureATP is mainly produced by glycolysis.ATP reduction is unlikely to occur even if mitochondrial toxicity occurs

Galactose cultureATP is produced mainly in mitochondria.When mitochondrial toxicity occurs, ATP production decreases directly

Nat Biotechnol. 2010 March ; 28(3): 249–255

＜Overview＞

Mitochondrial Toxicity Assessment

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Mitochondrial Toxicity Assessment ◆ Mechanism of mitochondrial toxicity

Journal of Hepatology 2011 vol. 54 j 773–794

Long-term exposure assessment (6-14 days)- Ribosome inhibition : chloramphenicol, linezolid- Inhibition of mtDNA synthesis : (under development)

Short-term exposure (1day)- ATP synthesis inhibitor : oligomycin- Respiratory chain inhibition, uncoupling : rotenone, antimycin, FCCP- β-oxidation inhibition : (under development)

β-oxidationATP synthesis

Respiratory chainUncoupling

DNA synthesis

Ribosome

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Start maintenance culture of HepG2 in flask

1-2 weeks Cell seeding on 96well plate

1day drug exposure

1 dayATP measurement(Short-term exposure assessment)

Glucose medium

Galactose medium

Glucose medium

6-14 daysATP measurement(Long-term exposure assessment)

＜Method timeline＞


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0

20

40

60

80

100

120

0.0001 0.001 0.01 0.1

ATP

% C

ontr

ol

μM

◆ Respiratory chain inhibition, uncoupling, ATP synthesis inhibition

◆ Other cytotoxicity (negative control)

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10

ATP

% C

ontr

ol

nM

Antimycin

0

20

40

60

80

100

120

0.1 10 1000

ATP

% C

ontr

ol

μM

FCCP

0

20

40

60

80

100

120

0.001 0.1 10

ATP

% C

ontr

ol

nM

Oligomycin

0

20

40

60

80

100

120

0.1 1 10 100 1000

ATP

% C

ontr

ol

μM

Imipramine

0

20

40

60

80

100

120

140

0.01 0.1 1 10 100

ATP

% C

ontr

ol

μM

Digitonin

Rotenone

・ Differences in toxicity expression in glucose / galactose cultures are observed for mitochondrial toxicity-positive compounds・ If the main toxicity mechanism is different than mitochondrial toxicity, no difference can be seen.

◆ glucose media■ galactose media

＜Short-term exposure assessment＞

◆ glucose media■ galactose media


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020406080

100120140

10 100 1000

% o

f con

trol

μM

Linezolid

◆ Ribosome synthesis inhibition (long period exposure)

0

20

40

60

80

100

120

140

10 100 1000

% o

f con

trol

μM

day 10

0

20

40

60

80

100

120

140

10 100 1000

% o

f con

trol

μM

day 10

0

20

40

60

80

100

120

140

10 100 1000

% o

f con

trol

μM

Chloramphenicolday 1day 1

Toxicity is hard to see with short-term exposure

Detection of mitochondrial toxicity by

long-term exposure

Toxicity that can only be detected by long-term exposure

＜Long-term exposure assessment＞


Conclusion

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DILI is caused by many and complex mechanism

Drug-induced liver cholestasis and Mitochondrial toxicity are important for DILI prediction

The Drug Development Solutions Center offers contract services of

these two in vitro studies

Thank you for your attentionFor questions regarding the presentation, study details, or how to get a quote for contracted Hepatotoxicity Studies please use the Contact Us form and a service specialist will get in touch.

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