Lamarre et al. Manuscript # 2003-08-08873
Supplementary information 1
Reversible Inhibition of NS3-NS4A Protease Genotype 1b with BILN 2061
A
9500
Fluorocescence Units
9000
8500
B
8000
7500
7000
6500
6000
0
300
600
900
1200
1500
1800
time (sec)
Representative progress curves from (A) the association and (B) the dissociation of the BILN 2061. HCV
genotype 1b NS3-NS4A serine protease showed a reversible inhibition and noncovalent binding of BILN
2061. The reversibility study was conducted at 23C at a final NS3-NS4A protein concentration of 0.5
nM, a final substrate (anthranilyl-DDIVPAbu[C(O)-O]-AMY(3-NO2)TW-OH) concentration of 10 µM and
a final BILN 2061 concentration of 6 nM in 50 mM Tris-HCl, pH 8.0, 0.25 M sodium citrate, 0.01% ndodecyl--D-maltoside, 1 mM TCEP, 5% DMSO. The association reaction was initiated by addition of the
NS3-NS4A protein to a solution containing the substrate and BILN 2061. The dissociation reaction was
initiated by diluting the pre-formed enzyme-inhibitor complex, obtained by incubating for up to 60 min the
enzyme and the inhibitor at concentrations ten-fold higher than the desired final concentrations, in the
substrate solution. The increase in fluorescence upon substrate cleavage was monitored at an excitation
wavelength of 325 nm and an emission wavelength of 415 nm on a SLM-Aminco Spectrofluorimeter
model 8100 (Spectronic Instruments, US). The steady-state velocity in Fluorescent Units/sec was
calculated from the linear regression of the fluorescence signal observed between 600 and 1800 seconds
following initiation of the enzymatic reaction and was corrected for spontaneous hydrolysis of the substrate
determined under the same conditions. Attainment of similar rates of product formation at the same final
concentrations of inhibitor and substrate demonstrated the reversibility of the inhibition and the absence of
enzyme inactivation.
Lamarre et al. Manuscript # 2003-08-08873
Supplementary information 2
Pre-Clinical Evaluation of BILN 2061
BILN 2061 exhibited a favorable profile across the panel of assays performed. Overall, BILN 2061 (10300 mg/kg, PO) was well tolerated in the in vivo general pharmacology profile (mouse and rat) at doses up
to 300 mg/kg, PO. This dosing regimen provided peak plasma concentrations of BILN 2061 that
substantially exceeded (25-6,700 fold) its in vitro cellular EC50 (3-4 nM). BILN 2061 did not exhibit any
physiologically relevant CNS, cardiovascular, pulmonary, renal or gastrointestinal effects at any of the
doses tested. Furthermore, BILN 2061 did not exhibit any significant effect on action potential duration
when tested at concentrations up to 10 µM in an isolated guinea pig papillary muscle assay. BILN 2061
exhibited only minimal effects in a small number of in vitro receptor binding and enzyme preparations but
only at assay concentrations (10 - 30 µM) that were well above the cellular EC50 for BILN 2061 observed
in the HCV replicon model. BILN 2061 showed a weak inhibitory activity (IC50 > 7 µM) when evaluated
in a panel of human P450 isozymes suggesting a low potential for cytochrome P450 dependent drug-drug
interactions in vivo. BILN 2061 was non-mutagenic in a comprehensive battery of genotoxicity assays
(Ames, mouse lymphoma and rat micronucleus). Clinical studies were also supported by the general
pharmacology evaluation of safety and by the pre-clinical safety profile obtained from single and repeated
doses toxicology studies in different animal species (Patrick Lilly, unpublished observation).
Lamarre et al. Manuscript # 2003-08-08873
Supplementary information 3
Pharmacokinetics of BILN 2061
The oral bioavailability was higher in dog (36%) than rat (13%) and rhesus monkey (7%). The compound
was absorbed rapidly in all species with a T max ranging from 1 to 4 hours. Following a single oral dose of 5
mg/kg, the Cmax and the AUC0-∞ ranged from 250 to 4,000 ng/mL and from 800 to 24,300 ng·hr/mL,
respectively, for all three species. The elimination half-life averaged 0.8 h in male rat, 2.2 h and 4.7 h in
male and female dog, respectively, and 7.0 h in male rhesus monkey. Total plasma clearance (CL) was low
(1-14 mL/min/kg) relative to hepatic blood flow in all three species. After a single oral dose of 5 mg/kg in
rat, the liver concentrations of BILN 2061 were 39, 32 and 5-fold greater than jugular vein plasma
concentrations at 1, 4 and 8 hours post-administration (n = 3 per time point), respectively. Analogous NS3
protease inhibitors that exhibited a high plasma CL were predominantly excreted in the bile as the parental
compound following an intravenous administration in rats22. The in vitro plasma protein binding study
indicated that BILN 2061 exhibited high protein binding in human plasma, averaging 99.1% at
concentrations up to 5 µg/mL. In vitro metabolism of BILN 2061 was evaluated using rat, dog, rhesus
monkey, and human hepatic microsomes. BILN 2061 was slowly metabolized by hepatic microsomes in
all species tested and was more stable in human and rat liver microsomes than beagle dog or rhesus
monkey preparations.
Pharmacokinetic profile and in vitro metabolism of BILN 2061
Pharmacokinetics following a single oral dose of 5 mg/kga
Species
Cmax
Sex
Tmax
AUC0-
Fb

In vitro
Metabolism
(ng/mL)
(hr)
(ng · hr/mL)
(%)
T1/2 (min)
Rat
M
250 ± 200
1.1 ± 0.4
800 ± 500
13 ± 8
Stable
Dog
M
2,100 ± 600
2.3 ± 2.5
13,000 ± 4,600
38 ± 24
105
Dog
F
4,000
1.5
24,300
32
nd
Rhesus
M
300 ± 300
4.0 ± 0.0
900 ± 700
7±6
89
Humanc
M
800 (51)
2.4 (30)
3,300 (55)
-
> 385
Pharmacokinetics following a single intravenous dose of 2 mg/kga
Species
Sex
AUC0-
d
CL
Vss
T1/2
(ng · hr/mL)
(mL/min/kg)
(L/kg)
(hr)

Rat
M
2,500 ± 900
14.3 ± 5.2
1.04 ± 0.67
0.8 ± 0.3
Dog
M
15,000 ± 4,300
2.3 ± 0.7
0.33 ± 0.04
2.2 ± 0.6
Dog
F
29,700 ± 4,600
1.1 ± 0.1
0.24 ± 0.03
4.7 ± 0.2
Rhesus
M
6,200 ± 1,100
5.5 ± 1.0
0.45 ± 0.03
7.0 ± 4.3
a
Vehicle for oral and intravenous administration used for the rat, dog and rhesus studies was 80%
PEG 400:20% water. bF = bioavailability. cSingle oral dose of 200 mg in healthy male volunteers
(n=6) administered as a drinking solution in 80% PEG 400:20% ethanol. Human values are
expressed as geometric mean and % geometric CV.
dHarmonic
mean. Animals were fasted in
all studies. Values for the animal studies (n>2) are mean ± SD except for the oral female dog
study (n=1). The in vitro metabolism studies were performed using hepatic microsomes from
Sprague Dawley rat, beagle dog, rhesus monkey, and human. In vitro half-life was estimated at a
BILN 2061 concentration of 10 µM. T1/2 was determined by the ratio of ln 2 over the first-order
elimination rate constant of the parent compound. Abreviations: AUC0-∞: area under the plasma
concentration-time curve; Cmax: maximum concentration in plasma after a single dose
administration; Tmax: time to reach Cmax following a single dose administration; CL: plasma
clearance; Vss : volume of distribution.