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Drug-Induced Liver Injury (DILI)

• Common reason for failure of pharmaceuticals during drug development, common reason for removing approved medications from the market

• Interfering with normal bile flow can result in mild & transient cholestasis… or severe hepatocellular damage

• Often difficult to predict


The effect of drugs on bile salt clearance in hepatocytes

Bile Salts:• Clearance is heavily dependent on

transporters.• Drug induced inhibition of transport ->

cholestasis.• Increased hepatic accumulation ->

hepatocellular damage.

Drugs & Metabolites:• Clearance is heavily dependent on

transporters• BSEP binding site is inside cells –

intracellular concentration (not plasma concentration) is important.

• Non-bile salt transporters also involved in mediating drug concentrations.


Understanding Cholestatic Liability of Drugs

• We have combined two approaches to better understand potential DILI:

• Mechanistic transporter models: MDCK-II cells transfected with various combinations of transporters involved in hepatic clearance of drugs and bile salts (NTCP, OATPs, BSEP, P-gp, etc.)

• Hepatocyte co-culture models


Contribution of Individual Transporters to Taurocholate Transport

B>A transport rate of 1uM TCA

Transporter(s)

Tra

nsce

llula

r tr

ansp

ort r

ate

(pm

ole/

min

/cm

2)

GFP contro

l

OATP1B1/

NTCP/BSEP

OATP1B1/

NTCP

OATP1B1/

BSEP

NTCP/BSEP

NTCP

OATP1B1

BSEP0

1

2

3

Cellular unbound concentration of TCA

Transporter(s)

Cce

ll,u

(u

M)

GFP Contro

l

OATP1B1/

NTCP/BSEP

OATP1B1/

NTCP

OATP1B1/

BSEP

NTCP/BSEP

NTCP

OATP1B1

BSEP0

5

10

15

Transcellular Transport Intracellular Accumulation


Contribution of Individual Transporters to Taurocholate Transport

B>A transport rate of 1uM TCA

Transporter(s)

Tra

nsce

llula

r tr

ansp

ort r

ate

(pm

ole/

min

/cm

2)

GFP contro

l

OATP1B1/

NTCP/BSEP

OATP1B1/

NTCP

OATP1B1/

BSEP

NTCP/BSEP

NTCP

OATP1B1

BSEP0

1

2

3

Cellular unbound concentration of TCA

Transporter(s)

Cce

ll,u

(u

M)

GFP Contro

l

OATP1B1/

NTCP/BSEP

OATP1B1/

NTCP

OATP1B1/

BSEP

NTCP/BSEP

NTCP

OATP1B1

BSEP0

5

10

15

Transcellular Transport Intracellular Accumulation

Apical efflux permeability of TCA

Transporter(s)

Api

cal e

fflux

per

mea

bilit

y

(uL

/min

/cm

2)

GFP contro

l

OATP1B1/

NTCP/BSEP

OATP1B1/

NTCP

OATP1B1/

BSEP

NTCP/BSEP

NTCP

OATP1B1

BSEP0.0

0.5

1.0

1.5

2.0

ND ND ND ND

Apical Efflux


Mechanistic study of drugs with DILI liability

Effect on TCA secretion Effect on Cellular TCA retention Effect on Apical TCA efflux

% o

f co

ntr

ol

DMSO

50uM

Rifa

mpic

in

30uM

Tro

glita

zone

30uM

Ros

iglita

zone

50uM

Ben

zbro

mar

one

50uM

Bos

enta

n

50uM

Suli

ndac

50uM

Suli

ndac

Sulf

ide

50uM

Suli

ndac

Sulf

one

300u

M P

yraz

inam

ide

20uM

Fen

ofibr

ate

50uM

Fen

ofibr

ic ac

id

50uM

Glim

epirid

e

20uM

Tam

oxife

n

50uM

chlor

prom

azine

50uM

Clav

ulanic

acid

50uM

ery

thro

myc

in

50uM

Glyb

uride

0

20

40

60

80

100

120

OATP1B1/NTCP/BSEP mediated transport of 1uM TCAin presence of drugs with reported DILI liability

% o

f co

ntr

olDM

SO

50uM

Rifa

mpic

in

30uM

Tro

glita

zone

30uM

Ros

iglita

zone

50uM

Ben

zbro

mar

one

50uM

Bos

enta

n

50uM

Suli

ndac

50uM

Suli

ndac

Sulf

ide

50uM

Suli

ndac

Sulf

one

300u

M P

yraz

inam

ide

20uM

Fen

ofibr

ate

50uM

Fen

ofibr

ic ac

id

50uM

Glim

epirid

e

20uM

Tam

oxife

n

50uM

chlor

prom

azine

50uM

Clav

ulanic

acid

50uM

ery

thro

myc

in

50uM

Glyb

uride

0

200

400

600

800

1000

Cellular retention of TCA in presence of drugswith reported DILI liability

% o

f co

ntr

ol

DMSO

50uM

Rifa

mpic

in

30uM

Tro

glita

zone

30uM

Ros

iglita

zone

50uM

Ben

zbro

mar

one

50uM

Bos

enta

n

50uM

Suli

ndac

50uM

Suli

ndac

Sulf

ide

50uM

Suli

ndac

Sulf

one

300u

M P

yraz

inam

ide

20uM

Fen

ofibr

ate

50uM

Fen

ofibr

ic ac

id

50uM

Glim

epirid

e

20uM

Tam

oxife

n

50uM

chlor

prom

azine

50uM

Clav

ulanic

acid

50uM

ery

thro

myc

in

50uM

Glyb

uride

0

20

40

60

80

100

120

Apical efflux of TCA in presence of drugswith reported DILI liability


Rifampicin

• BDDCS Class I, Cmax ~10µM, fu ~20%

• Extensive metabolized in liver to Desacetyl-Rifampicin

• Clearance: hepatic ~70%, renal: ~30 (~7% parent)

• High biliary concentrations of DES-RIF and RIF

• Rifampicin is known to cause liver injury (~1% of patients),

incl. fatal liver failure

• A potent inhibitor of OATPs, MRPs and BSEP

• Does not inhibit NTCP (up to 300µM) or ASBT*

• PXR activator

• Induce metabolic enzymes

• Up-regulate OATPs, MRPs and P-gp

Rifampicin

25-desacetyl rifampicin


Rifampicin is not transported by NTCP, BSEP, OATP1B1

• Mechanistic study using clearly defined MDCK models expressing various transporters revealed that Rifampicin is not transported by NTCP or BSEP

GFP contro

l

MDR1

1B1/

NTCP

1B1/

NTCP/BSEP

1B1/

NTCP/BSEP/M

DR10.0

0.5

1.0

1.5

2.0

2.5

B>A transcellular transport of 1uM Rifampicin

Tra

nsce

llula

r tr

ansp

ort r

ate

(pm

ole/

min

/cm

2 )

GFP contro

l

MDR1

1B1/

NTCP

1B1/

NTCP/BSEP

1B1/

NTCP/BSEP/M

DR10

5

10

15

20

Total cellular concentration of Rifampicin at 90min

Cce

ll,to

tal (

uM

)

GFP contro

l

MDR1

1B1/

NTCP

1B1/

NTCP/BSEP

1B1/

NTCP/BSEP/M

DR10.0

0.2

0.4

0.6

0.8

Apical efflux permeability of 1uM Rifampicin

Api

cal e

fflux

per

mea

bilit

y

(pm

ole/

min

/cm

2)

Transcellular Transport Intracellular Accumulation Apical Efflux


Rifampicin is a substrate of P-gp

• Mechanistic study using clearly defined MDCK models expressing various transporters revealed that Rifampicin is a substrate of P-gp, but is not transported by NTCP nor BSEP, and its intracellular concentration heavily depends on P-gp

GFP contro

l

MDR1

1B1/

NTCP

1B1/

NTCP/BSEP

1B1/

NTCP/BSEP/M

DR10.0

0.5

1.0

1.5

2.0

2.5

B>A transcellular transport of 1uM Rifampicin

Tra

nsce

llula

r tr

ansp

ort r

ate

(pm

ole/

min

/cm

2 )

GFP contro

l

MDR1

1B1/

NTCP

1B1/

NTCP/BSEP

1B1/

NTCP/BSEP/M

DR10

5

10

15

20

Total cellular concentration of Rifampicin at 90min

Cce

ll,to

tal (

uM

)

GFP contro

l

MDR1

1B1/

NTCP

1B1/

NTCP/BSEP

1B1/

NTCP/BSEP/M

DR10.0

0.2

0.4

0.6

0.8

Apical efflux permeability of 1uM Rifampicin

Api

cal e

fflux

per

mea

bilit

y

(pm

ole/

min

/cm

2)

Transcellular Transport Intracellular Accumulation Apical Efflux


Effect of P-gp on Rifampicin and Metabolite

• P-gp dramatically reduces the intracellular concentrations of Rif and Des-Rif in cells expressing P-gp.

• P-gp may be a key determinant of the hepatic concentration and liver toxicity of Rif and Des-Rif.

Rifam

picin

Desac

etyl

-Rif

0

5

10

15

20

Total cellular concentration in MDCK cellsexpressing OATP1B1/NTCP/BSEP

Cce

ll,to

tal (

uM

)

w/o MDR1 w/ MDR1

5.2x

12.7x


P-gp protects against rifampicin hepatotoxicity

0

5

10

15

20

0.1 1 10 100 1000

Cellular unbound concentration of TCAin MDCK cells expressing various transporter

Basal Rifampicin (M)

Cce

ll,u

(uM

)

OATP1B1/NTCP/BSEPOATP1B1/NTCP/BSEP/MDR1

0

5

10

15

20

0.1 1 10 100 1000

Cellular unbound concentration of TCAin MDCK cells expressing various transporter

Basal Desacetyl-Rifampicin (M)

Cce

ll,u

(uM

)

OATP1B1/NTCP/BSEPOATP1B1/NTCP/BSEP/MDR1

P-gp decreases intracellular concentrations of potentially hepatotoxic agents… This suggests that P-gp inhibitors may potentiate hepatotoxic BSEP inhibition.


Potential Synergistic Effects of RIF Co-Meds on Blocking Hepatic Bile Salt Clearance?

• The incidence of liver injury in patients taking Pyrazinamide/Rifampicin is ~4 times higher than taking Rifampicin alone (Yew & Leung 2006)

• Protease inhibitors (Lopinavir/Ritonavir, Saquinavir/Ritonavir) caused unexpected liver injury in HIV infected TB patients (Nijland et. al. 2008, Schmitt et. al. 2009)

Yew WW, Leung CC. Antituberculosis drugs and hepatotoxicity. Respirology 2006; 11(6):699-707 Nijland HM et al. High incidence of adverse events in healthy volunteers receiving rifampicin and adjusted doses of lopinavir/ritonavir tablets. AIDS. 2008;22(8):931–935Schmitt C et al. Unexpected hepatotoxicity of rifampin and saquinavir/ritonavir in healthy male volunteers. Archives of Drug Information. 2009;2(1):8–16.


Protease Inhibitors Inhibit P-gp and Aggravate Rifampicin Toxicity

• Ritonavir + Lopinavir cause an increase in intracellular concentrations of Rif and Des-Rif in MDCK models by inhibition of P-gp.

• These results suggest that if co-administrated with Rif, they may aggravate Rif hepatotoxicity.

Rifam

picin

Desac

etyl

-Rif

0

5

10

15

20

Total cellular concentration in MDCK cellsexpressing OATP1B1/NTCP/BSEP

Cce

ll,to

tal (

uM

)

w/o MDR1 w/ MDR1

w/ MDR1+LPV/r

3.3x3.9x


Methods: Hepatocyte Co-Cultures

Culture System Hurel Hepatic Co-Cultures

Lot/Species Human (Hurel H1004)

Test Compounds Cyclosporin A (0-50 µM), Ritonavir (0-200 µM)Troglitazone (0-629 µM), Bosentan (0-199 µM)Erythromycin Estolate (0-250 µM)

Bile Acids Mix (40x) 52.8 µM glycochenodeoxycholic acid15.6 µM chenodeoxycholic acid15.2 µM glycodeoxycholic acid16 µM deoxycholic acid14 µM glycocholic acid

Incubation Time 1 hr (compound incubation), 24hr, 48hr

Toxicity Assays CellTiter-Blue, CellTiter-GloAlbumin Assay (Liver-Specific Function)

Replica n = 2


Albumin Assay for Liver-Specific Function

• Albumin secretion was measured in Hurel hepatic co-culture models.

• Bile salts alone have no effect

• Hepatotoxic drugs decrease albumin secretion in this assay.

• Albumin is a good marker for hepatotoxicity


Cyclosporine A Hepatotoxicity is Potentiated by Bile Salts

• Albumin secretion was measured in the presence of Cyclosporine A with and without bile salts.

• After 24 hours, increased hepatotoxicity is seen in the presence of drug + bile salts compared to drug alone.


Ritonavir Hepatotoxicity is also Potentiated by Bile Salts

• Albumin secretion was measured in the presence of ritonavir with and without bile salts.

• After 24 hours, increased hepatotoxicity is seen in the presence of drug + bile salts compared to drug alone.


Hepatotoxicity of Bosentan

• For compounds like bosentan, the toxicity can take up to 48 hours to present• Toxicity is enhanced in the presence of bile salts.


Rifampicin in Hepatic Co-Cultures

• Hepatotoxicity of rifampicin was measured with bile salts +/- ritonavir.• Rifampicin hepatotoxicity is greatly enhanced in the presence of ritonavir.


Summary

• The clearance of bile salts is heavily dependent on transporters. Numerous transporters are involved, directly or indirectly.

• Drug induced inhibition of bile salt uptake and/or efflux can cause blockage of bile salt hepatic clearance (resulting in cholestasis), and potentially result in increased hepatic bile salt accumulation (resulting in hepatocellular damage).

• Mechanistic transporter models can show potential cholestatic risk of drugs, while hepatic co-cultures can confirm potential hepatotoxicity.


Conclusions

• Several drugs (cyclosporine, rifampicin, bosentan) showed increased hepatotoxicity in the presence of bile salts, suggesting that blocking bile salt clearance via inhibiting bile salt transporters likely attributes to their liver toxicity.

• Using mechanistic transporter models, rifampicin (and desacetyl-rifampicin) potently inhibit BSEP but not NTCP, while their intracellular concentrations are mediated by P-gp. Therefore, P-gp inhibition by a co-medication can increase hepatotoxicity.

• The hepatocyte co-culture model demonstrates that ritonavir potentiates rifampicin hepatotoxicity in a bile salt dependent manner. These results may provide a mechanistic explanation on the high incidence of unexpected liver injuries in HIV-infected TB patients.

• Well-defined transporter models are useful tools for mechanistic studies of bile salt transport and its inhibition by drugs, while the resultant hepatotoxicity can be confirmed with the hepatocyte co-culture model. This combination allows for the ability to run detailed mechanistic transporter studies that can identify potential transporter interactions and confirm hepatotoxicity.


Thank you!

Eric NovikCheul ChoEric PludwinskiMatt Shipton

Yong HuangJason BaikDawn Stricker

www.optiviabio.com | [email protected] | +1.650.324.3177 an integrated approach to more accurately...

Documents

hepatic clearance of

bile salt clearance

nonbile salt transporters

tca secretioneffect

apical tca efflux

mechanistic study of

effect of drugs

cellular tca retentioneffect