IN VIVO AND IN VITRO ASSESSMENT OF ASUNAPREVIR (ASV; BMS-650032) AS AN

INHIBITOR AND SUBSTRATE OF ORGANIC ANION TRANSPORT POLYPEPTIDE (OATP) TRANSPORTERS

IN HEALTHY VOLUNTEERS

T Eley, Y-H Han, S-P Huang, B He, W Li, W Bedford, M Stonier, D Gardiner, K Sims, P Balimane, D Rodrigues, RJ Bertz

7th International Workshop on Clinical Pharmacology of Hepatitis Therapy Cambridge MA, USA

27 - 28 June 2012 Oral presentation O_04

Disclosures

■ All authors are full-time employees of Bristol-Myers Squibb

■ TE, Y-HH, S-PH, BH, WL, MS, DG, KS, PB, DR, RJB are stockholders of Bristol-Myers Squibb

Background: Asunaprevir (ASV, BMS-650032)

■ Potent, selective inhibitor of the HCV NS3 protease ■ In vitro activity against HCV GT 1 and 4 ■ Currently in Phase 3 trials as part of DUAL/QUAD therapy in patients with

HCV GT 1 and 4 with daclatasvir (DCV) +/- peg-alfa/RBV – Preclinical data demonstrated ASV inhibits OATP1B1 and OATP1B3 with IC50

values of 0.3µM and 3.0µM, respectively

■ Steady state plasma Cmax for 200 mg BID Phase 2 tablet with food ~300-500 ng/mL (~400-660nM) with CV in the 60-80% range – Plasma protein binding of >99% – High liver:plasma ratio in animals (40–359x); NS3 PI class related – High SVR rates with low plasma concentrations; liver distribution important

clinically ■ ≥ 83% SVR24 with ASV/peg-alfa/RBV in treatment naive HCV patients ■ ≥ 90% SVR4 with ASV+DCV+peg-alfa/RBV in interferon null responders

BID, twice daily; CV, coefficient of variation; HCV, hepatitis C virus; GT, genotype OATP: Organic anion-transporting polypeptide; Peg-alfa, pegylated interferon alfa; RBV, ribavirin

■ OATPs known to transport – Bile acids, bilirubin – Steroid and bilirubin conjugates – Thyroid hormones – HMGCo-A reductase inhibitors – Methotrexate – Glyburide – Rifampin – Angiotensin receptor blockers

■ Multiple clinically relevant DDIs

■ Preferential liver distribution suggests involvement of OATP

Organic Anion Transporting Polypeptides

Image excerpted from Giacomini, et al, for ITC, Nature Reviews Drug Discovery Mar 2010

0 20 40 60 80 100 120 Uptake (% of control)

Control

50 µM BSP

100 µM Taurocholate

1 mM PAH

1 mM TEA

ASV (1 µM)

■ No significant inhibition by transporter inhibitors a t 1 µM ■ ASV showed high permeability in CaCo-2 and PAMPA assays ■ No difference in uptake in over-expressed systems (not shown)

No Apparent Involvement of Active Uptake into Hepatocytes at ≥ 1 μM

TEA, tetraethylamine ; PAH, para-aminohippuric acid , BSP: sulfobromophthalein

0

10

20

30

40

0 20 40 60 80 100 Concentration (µM)

Upt

ake

(nm

ol/m

g/m

in)

ASV (lowest conc. 1 µM)

Aim: to assess the effect of ASV on the PK of rosuvastatin 20 healthy male and female subjects, ages 18-49, BMI 18-32 kg/m2 Single sequence crossover study design

Study AI447015: Evaluation of ASV as an OATP Inhibitor

Day

7 SD RST 10 mg 72 hr PK Da

ys 1

0-19

9 days of ASV 200 mg BID with meals Da

ys 2

0-22

SD RST 10 mg 72h PK with ASV continuing at 200 mg BID throughout

BID, twice daily; SD, single dose; RST, rosuvastatin

2

4

6

8

10

12

0 0 8 16 24

Study AI447015: ASV Increases Rosuvastatin Exposures

ASV effect on rosuvastatin pharmacokinetics

Parameter Treatment C (RST)

Treatment E (BMS + RST)

Geo. LSM Ratio (90 % CI)

Cmax (ng/mL) Geo. LSM

2.63 (n=20)

5.11 (n=20)

1.946 (1.469-2.576)

AUCt (ng*hr/mL) Geo. LSM

25.33 (n=20)

32.32 (n=20)

1.276 (1.017-1.601)

AUCinf (ng*hr/mL) Geo. LSM

29.56 (n=16)

41.57 (n=18)

1.406 (1.257-1.573)

LSM, least-squares mean, RST, rosuvastatin

Mea

n (+

/- S

D) R

ST

conc

entra

tion

(ng/

mL)

Time post dose (hours)

Weak OATP Inhibition by ASV

■ ASV increased rosuvastatin exposure

■ Based on AUCinf this effect can be characterized as weak inhibition of OATP1B1/1B3 by ASV

■ Examples of strong OATP inhibitors – Cylcosporine increases pravastatin AUC 9.9x; rosuvastatin AUC 7.1x – LPV/RTV increases bosentan AUC 5.48x

■ Clinically relevant interactions are unlikely to exist between ASV and most OATP substrates – Still precautionary medications in Phase 3

Study AI447015: Weak OATP Inhibition by ASV

Study AI447018: Evaluation of ASV as an OATP Substrate

Aim: to assess the effect of single dose rifampin on the PK of ASV – ASV given fasted

20 healthy male subjects, ages 18-49, BMI 18-30 kg/m2 Single sequence crossover study design

Day

1 SD RIF 600 mg with 200 mg SD ASV (fasted) • 48h PK

Day

8 200 mg SD ASV (fasted) • 48h PK

RIF, rifampin; SD, single dose

Study AI447018: Rifampin Increases ASV Exposure

Rifampin effect on ASV pharmacokinetics Treatment and Comparison

Treatment A (ASV + RIF)

Treatment B (ASV) GMR (90% CI)

Cmax (ng/mL) adjusted geo. mean

1214 (n=20)

57.49 (n=20)

21.11 (14.27, 31.24)

AUCinf (ng*h/mL) adjusted geo. mean

6045 (n=20)

408.3 (n=20)

14.81 (11.22, 19.53)

Con

cent

ratio

n (n

g/m

L)

Time (h) CI, confidence interval; Geo, geometric; GMR, geometric mean ratio; RIF, rifampin; TRT, treatment

Cmax by Treatment AI447018

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Cm

ax (

ng

/mL

)

AUCinf by Treatment AI447018

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

AU

Cin

f (n

g*h

/mL

)

11

■ Individual changes in Cmax range from ~4.8x to ~222x ■ Individual changes in AUCinf range from ~4.9x to ~67x

With rifampin Control

Study AI447018: Wide Range of Effect of Single Dose Rifampin

With rifampin Control

AUCinf by treatment Cmax by treatment

Study AI447018: ASV Exposure Increased by Single Dose Rifampin

■ Results indicate that ASV is a sensitive substrate of OATP-mediated uptake with a wide range of effect

■ Although passive distribution plays a greater role at higher concentrations, there is clearly an active component at lower concentrations

■ Strong OATP inhibitors may reduce antiviral activity of ASV

■ ASV 200 mg single dose fasted tablet had Cmax well below 1μM

0

30

60

90

120

150

180

0 0.2 0.4 0.6 0.8 1

Upt

ake

rate

(pm

ol/m

in/m

g)

ASV concentration (µM)

Km = 0.68µM

13

Hepatocytes

Saturable, Transporter-mediated Uptake: Hepatic Uptake via OATP1B1 and OATP2B1

HEK-293 cells

ASV (10 nM) ASV (10 nM) + RIF (50 µM)

0

1

2

3

4

5

Mock OATP2B1 OATP1B1 OATP1B3

Upt

ake

rate

(pm

ol/m

in/m

g)

Clinically Relevant Genetic Polymorphisms in OATP1B1

.

■ OATP1B1 allelic frequency varies with ethnic group – 388G present in 40% of Caucasians, 75% of African Americans and 60% of Asians – 521T present in 15% of Caucasians, 2% of African Americans and 15% of Asians

■ Enhanced and reduced function haplotypes likewise vary

■ Correlated with adverse events and pharmacokinetics – E. Link et al (2008): OATP1B1 variants linked to simvastatin-induced myopathy – L. Ramsey, et al (2012): OATP1B1 variants explain 10.7% of population variability in

methotrexate clearance

Transporter (old name)

Gene name Polymorphism rs number Common

haplotype Amino acid change Comment

OATP1B1 (LST1, OATP-C, OATP2)

SLCO1B1 388A→G rs2306283 SLCO1B1*1b 130Asn→Asp Increased activity

521T→C rs4149056 SLCO1B1*5, *15, *17

174Val→Ala

Reduced activity

Summary and Conclusions

In vivo, ASV increases the plasma exposure of the OATP1B1 and OATP1B3 probe rosuvastatin by weakly inhibiting liver uptake

Single dose rifampin markedly increases ASV plasma exposure

ASV has been shown to be a substrate of OATP1B1/OATP2B1 in vitro

OATP-mediated transport affects the disposition of ASV and likely contributes to the observed pharmacokinetic variability of the compound

More data to be collected from ongoing studies to determine OATP1B1 and 2B1 subtypes to evaluate their potential effect on ASV plasma PK

Summary and Conclusions