ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2009, p. 2544–2552
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 6
Preclinical Characterization of PF-00868554, a Potent Nonnucleoside
Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase?†
Stephanie T. Shi,* Koleen J. Herlihy, Joanne P. Graham, Jim Nonomiya, Sadayappan V. Rahavendran,
Heather Skor, Rebecca Irvine, Susan Binford, John Tatlock, Hui Li, Javier Gonzalez,
Angelica Linton, Amy K. Patick, and Cristina Lewis
Pfizer Global Research and Development, La Jolla Laboratories, 10724 Science Center Drive, San Diego, California 92121
Received 2 December 2008/Returned for modification 23 January 2009/Accepted 17 March 2009
PF-00868554 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, which exerts its
inhibitory effect by binding to the thumb base domain of the protein. It is a potent and selective inhibitor, with
a mean 50% inhibitory concentration of 0.019 ?M against genotype 1 polymerases and a mean 50% effective
concentration (EC50) of 0.075 ?M against the genotype 1b-Con1 replicon. To determine the in vitro antiviral
activity of PF-00868554 against various HCV strains, a panel of chimeric replicons was generated, in which
polymerase sequences derived from genotype 1a and 1b clinical isolates were cloned into the 1b-Con1 sub-
genomic reporter replicon. Our results indicate that PF-00868554 has potent in vitro antiviral activity against
a majority (95.8%) of genotype 1a and 1b replicons, with an overall mean EC50of 0.059 ?M. PF-00868554
showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 ?M).
Furthermore, the antiviral activity of PF-00868554 was retained in the presence of human serum proteins. An
in vitro resistance study of PF-00868554 identified M423T as the predominant resistance mutation, resulting
in a 761-fold reduction in susceptibility to PF-00868554 but no change in susceptibility to alpha interferon and
a polymerase inhibitor that binds to a different region. PF-00868554 also showed good pharmacokinetic
properties in preclinical animal species. Our results demonstrate that PF-00868554 has potent and broad-
spectrum antiviral activity against genotype 1 HCV strains, supporting its use as an oral antiviral agent in
The CDC estimates that 4.5 million individuals in the United
States and more than 170 million worldwide are infected with
the hepatitis C virus (HCV), with about 25,000 new infections
occurring each year. Approximately 80% of hepatitis C pa-
tients become persistently infected, among which 10% to 20%
develop progressive liver disease during their lifetime, includ-
ing cirrhosis, hepatocellular carcinoma, and liver failure, thus
making hepatitis C the leading indication for liver transplan-
tation (38, 44). HCV was primarily transmitted via a contam-
inated blood supply before the discovery of HCV in 1989 and
the development of sensitive detection methods in 1992. Since
then, intravenous drug use has become the major route of
HCV transmission and the incidence of HCV infection has
decreased rapidly. However, the large number of patients in-
fected before 1992 are now in the later stages of the disease, in
which treatment becomes necessary to prevent liver damage,
transplantation, or death.
The current standard of care for HCV is pegylated alpha
interferon (IFN-?) in combination with ribavirin. The success
rate of this therapy with genotype 1 infection, which is the most
prevalent in the United States and worldwide, is only about
40% to 50% (9, 11, 32). In addition, IFN-? therapy is associ-
ated with significant side effects, including fatigue, headache,
myalgia, fever, nausea, and insomnia in ?30% of patients (9,
11, 32). Ribavirin also causes hemolytic anemia in 10% to 20%
of patients (28, 40). Due to the tolerability and safety concerns
of current therapies, as well as the slow progression and un-
certain course of this disease, physicians generally withhold
treatment from patients until symptoms manifest or liver func-
tion begins to be impaired. Currently, fewer than 10% of
chronically infected patients in the United States are receiving
HCV treatment. Thus, there remains a significant unmet need
for more effective, more tolerable, and safer HCV therapies.
The HCV genome is a single-stranded, positive-sense RNA
of approximately 9.6 kb (7). The genomic RNA encodes a
polyprotein that is processed by host and viral proteases into at
least 10 structural and nonstructural (NS) proteins (10, 14, 15).
Most of the HCV NS proteins are required for viral RNA
replication (1). The NS5B protein, encoding the viral RNA-
dependent RNA polymerase (RdRp), is an essential compo-
nent of the HCV RNA replication complex (20). Due to its
apparent sequence and structural differences from human
DNA and RNA polymerases, the HCV RNA polymerase is
considered an attractive target for antiviral drug discovery. In
addition to nucleoside analogs (4) and pyrophosphate mimics
(41) that target the active site, a number of structurally diverse
nonnucleoside polymerase inhibitors have been reported (19).
They were shown to interact with at least four distinct allosteric
sites by a combination of crystallographic analysis and in vitro
resistance studies (18, 19).
We have reported the discovery, using high-throughput
screening and structure-based drug design, of a novel and
* Corresponding author. Mailing address: Department of Cancer
Biology, Pfizer Global Research and Development, La Jolla Labora-
tories, 10724 Science Center Drive, San Diego, CA 92121. Phone:
(858) 526-4906. Fax: (858) 526-4349. E-mail: stephanie.shi@pfizer
† Supplemental material for this article may be found at http://aac
?Published ahead of print on 23 March 2009.
potent class of nonnucleoside HCV polymerase inhibitors
characterized by a dihydropyrone core (23, 24). These mole-
cules bind within the thumb base portion of the polymerase
(pocket II) (2) and are noncompetitive inhibitors with respect
to nucleotide. In this report, we characterize the preclinical
activity and pharmacokinetic profile of PF-00868554 (25), a
molecule that retains optimal biochemical potency and selec-
tivity and yet has improved pharmacokinetic and physicochem-
ical properties compared to its predecessors (23, 24). In a
phase I clinical trial with genotype 1 HCV-infected, treatment-
naïve subjects, PF-00868554 potently inhibited viral replica-
tion, with mean maximum reductions in HCV RNA ranging
from 0.97 to 2.13 logs (12). It is currently in phase II clinical
trials. The antiviral activity of PF-00868554 against different
HCV genotype 1 strains was determined by generating chi-
meric replicons containing polymerase sequences derived from
a panel of genotype 1 clinical isolates in the genotype 1b (Con1
strain) backbone. The pharmacokinetic properties of the mol-
ecule were profiled, revealing promising oral bioavailability in
rodent and nonrodent species. In addition, an in vitro resis-
tance study was carried out to identify the resistance changes
associated with exposure to PF-00868554.
MATERIALS AND METHODS
Compounds. PF-00868554 (25) was synthesized at Pfizer Global Research and
Development (La Jolla, CA) (Fig. 1). The benzimidazole compound C (21) and
benzothiadiazine SB-750330 (5) were prepared according to published protocols
(Fig. 1). IFN-? was purchased from Sigma Aldrich (St. Louis, MO). Individual
compounds, except IFN-?, were dissolved in dimethyl sulfoxide (DMSO) to a
concentration of 10 mM or 100 mM and diluted to appropriate concentrations in
buffer or growth medium. IFN-? was dissolved in phosphate-buffered saline to a
stock concentration of 105IU/ml and diluted to appropriate concentrations in
Reagents, enzymes, cells, and plasmids. Recombinant constructs of HCV
NS5B polymerase 1a H77, 1b BK, 1b Con1, 1b Con1 M423T, 2b, and 4a enzymes
each bore a 21-amino-acid C-terminal deletion of the membrane insertion frag-
ment (?21) and a C-terminal six-His tag and were expressed in Escherichia coli
and purified as previously described (29). Recombinant His-tagged NS5B poly-
merase genotypes (?21) 1b J1 and (?21) 3a were expressed in insect cells using
a baculovirus expression system and were similarly purified. Human DNA poly-
merase alpha and delta were cloned, expressed, and purified according to pub-
lished procedures (8, 36, 42). Human DNA polymerase gamma was purchased
from Replizyme Ltd. (Heslington, York, United Kingdom). Recombinant human
immunodeficiency virus (HIV) reverse transcriptase and HIV Q7K protease
were purified from E. coli, as described previously (3, 16). Huh7 and Huh7.5
cells, obtained from Apath LLC (St. Louis, MO), were propagated in Dulbecco’s
modified Eagle medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine
serum (FBS; HyClone, Logan, UT), 0.1 mM nonessential amino acid (Invitro-
gen), 100 units/ml of penicillin (Invitrogen), and 100 ?g/ml of streptomycin
sulfate (Invitrogen). HEK-293, HeLa, and HepG2 cells were propagated in
Dulbecco’s modified Eagle medium containing 10% FBS, 100 IU/ml of penicillin,
and 100 mg/ml of streptomycin sulfate.
The dicistronic selectable replicon BB7, containing the HCV genes NS3 to
NS5B derived from the Con1 strain of genotype 1b, was licensed from Apath
LLC. The constructionofthe HCV
BB7M4hRLuc, and the generation of the stable cell lines, dicistronic single
reporter (DSR) and dicistronic dual reporter (DDR), were described previously
(13, 39). DSR is a stable cell line that harbors the BB7M4hRLuc reporter
replicon, whereas DDR also contains the CMV-firefly luciferase (FLuc), allow-
ing for simultaneous monitoring of cytotoxicity. The monocistronic selectable
replicon RB10, containing the same HCV sequences under the control of the
HCV internal ribosome entry site, was licensed from ReBLikon GmbH (Mainz,
Germany). The dicistronic selectable subgenomic replicon H77-SG, containing
the HCV genes NS3 to NS5B (derived from the H77 strain of genotype 1a) under
the control of the encephalomyocarditis virus internal ribosome entry site, was
licensed from Apath LLC.
Biochemical assays. Recombinant HCV NS5B genotypes 1b BK, 1b J1, 1b
Con1, 1b Con1 M423T, and 2b were assayed by measuring the incorporation of
[?-33P]GMP into dG12:poly(rC)350 primer-template substrate [dG12 DNA
primer and poly(rC)350RNA template preannealed in a 12.5:1 primer-to-tem-
plate molar ratio]. NS5B genotypes 1a H77, 3a, and 4a were assayed using either
the GMP incorporation or UMP incorporation format; the latter measured the
incorporation of [?-33P]UMP into dT17:poly(rA)350primer-template [dT17DNA
primer and poly(rA)350RNA template preannealed in a 12:1 molar ratio]. Assays
were performed in 96- or 384-well microplates, with final reaction volumes of 100
?l or 75 ?l, respectively. The reaction mixtures of GMP incorporation assays
contained 20 mM Tris-Cl (pH 7.6), 10 mM MgCl2, 20 mM NaCl, 0.05% Tween
20, 1 mM dithiothreitol (DTT), 30 nM polymerase enzyme, 2% (vol/vol) di-
methyl sulfoxide, 62.5 nM dG12primer, 5 nM poly(rC)350template, 1 ?M GTP,
and 1 nCi/?l [?-33P]GTP. Reaction mixtures for UMP incorporation assays were
similar, except that 60 nM dT17primer, 5 nM poly(rA)350template, 2.5 uM UTP,
and 10 nCi/?l [?-33P]UTP were substituted for primer, template, and nucleotide.
Reactions were routinely initiated by enzyme addition and incubated for 30 min
at room temperature for GMP assays or 60 min at 30°C for UMP assays.
Separate experiments demonstrated that product formation was linear with re-
spect to time, and substrate conversion was ? ?10%, conforming to initial rate
kinetics under these assay conditions. Reactions were stopped by addition of 59
mM EDTA, and the nucleotide product was detected by phosphorimaging after
filtration onto Biodyne B nylon 6,6 membranes (Thermo Fisher Scientific, Roch-
ester, NY). Noncompetitive, tight-binding 50% inhibitory concentration (IC50)
constants were measured by the fitting of the dose-response data to the following
subgenomic reporter replicon,
vi/vo? 1 ? ????E? ? ?I? ? IC50? ? ???E? ? ?I? ? IC50?2? 4?E??I??1/2?/2?E??
where viis the inhibited velocity, vois the uninhibited velocity, [E] is the enzyme
concentration, and [I] is the inhibitor concentration. Unless indicated otherwise,
all reported inhibition data represent averages of at least two measurements in
Human DNA polymerase alpha (20 nM) was assayed in the presence of 20
mM Tris (pH 7.5), 1 uM dTTP (plus 100 nCi/well of [?-33P]dTTP), 10 mM
MgCl2,0.1 mg/ml bovine serum albumin, 1 mM DTT, 8 nM dA-dT primer-
template, and 2% dimethyl sulfoxide. Human DNA polymerase delta (5 nM) was
assayed in the presence of 19 mM Tris (pH 7.6), 1.9 mM MnCl2, 0.85 ?M dATP,
0.43 uM dTTP (plus 200 nCi/well of [?-33P]dTTP), 1.3 mM DTT, 4.2% (vol/vol)
glycerol, 2% (vol/vol) DMSO, and 8.5 ng/?l poly(dA-dT)/poly(dA-dT). Reac-
tions were initiated with enzyme, and the incorporation of [?-32P]dTMP into
high-molecular-weight polynucleotide was measured in the presence of an
oligo(dT)/poly(dA) primer-template. The elongated primer-template was de-
tected by phosphorimaging after microfiltration through Biodyne B membranes
FIG. 1. Structures of HCV polymerase inhibitors.
VOL. 53, 2009 PRECLINICAL CHARACTERIZATION OF PF-008685542545
(Thermo Fisher Scientific). HIV reverse transcriptase (1 nM) was assayed sim-
ilarly in the presence of 50 mM Tris-Cl (pH 8.0), 60 mM KCl, 1 mM DTT, 20 nM
template, 160 nM primer (preannealed), 8 mM MgCl2, and 20 ?M [?-32P]dTTP
for 30 min before microfiltration and phosphorimaging. HeLa cell nuclear ex-
tracts were used as a source of human nuclear RNA polymerases I, II, and III.
Activity was assayed using calf thymus DNA as template, and incorporation of
[?-32P]GMP into high-molecular-weight RNA was measured by microfiltration
onto Biodyne B membranes in the presence of 50 mM Tris-Cl (pH 8.0), 1 mM
MgCl2, 1 mM MnCl2, 1 mM DTT, 50 mM (NH4)2SO4, 5 ?g/well calf thymus
DNA, 1% (vol/vol) DMSO, 100 ?M ATP, 100 ?M UTP, 100 ?M CTP, 5 ?M
GTP, 0.2 ?Ci/well [?-32P]GTP, and 5 ?l/well nuclear extract. RNA polymerase
I activity was assessed in the presence of 230 ?g/ml ?-amanitin, RNA polymerase
(I plus II) activity was assessed in the presence of 0.77 ?g/ml ?-amanitin, and
RNA polymerase III activity was assessed differentially in the absence of
?-amanitin. The HIV protease Q7K was assayed according to published
Generation of patient-derived NS5B chimeric replicons. To eliminate the
necessity of ScaI-mediated linearization prior to in vitro transcription and to
allow transcript production from patient sequences containing ScaI sites, an
84-nucleotide hepatitis delta virus ribozyme sequence was introduced into
BB7M4hRLuc immediately downstream of the HCV sequence so that the rep-
licon RNA transcript with an exact 3? terminus of the 3? untranscribed region
(UTR) could be generated by self-cleavage. The incorporation of ribozyme was
accomplished by two rounds of PCRs, as described previously (43), using Her-
culase DNA polymerase and PCR primer pairs Ribo1 and Ribo2 for the first and
second rounds of PCR, respectively (see the supplemental material). Both
rounds of amplification were carried out for 35 cycles consisting of 95°C for 1
min, 55°C for 90 s, and 72°C for 90 s. The first-round PCR generated the fusion
between a sequence from a unique NcoI site in NS5B and the end of the 3? UTR
and the 5?-end sequence of the ribozyme. The resulting fragment was used as a
template for the second-round PCR to fuse the entire ribozyme sequence to the
HCV 3? UTR. The final product was cloned into BB7M4hRLuc at the unique
NcoI and SpeI sites to create BB7M4hRLuc.ribo. The SbfI and PacI restriction
sites were introduced into BB7M4hRLuc.ribo in the NS5A region and 3? UTR,
respectively, by site-directed mutagenesis using QuikChange mutagenesis (Strat-
agene, La Jolla, CA), according to the manufacturer’s protocol, and PCR primer
pairs SbfI and PacI (see the supplemental material). Amplification was carried
out for 16 cycles of 94°C for 30 s, 55°C for 1 min, and 68°C for 26 min. All
mutations were confirmed by sequencing.
Plasma samples from HCV-infected patients were obtained from Teragenix
(Fort Lauderdale, FL) and Cliniqa (San Diego, CA). Samples with a viral load
over 105IU/ml were randomly selected for RNA isolation using Qiagen QIAamp
viral RNA mini isolation kit (Qiagen, Valencia, CA) according to the manufac-
turer’s protocol. Purified RNA was reverse transcribed using Thermo-X reverse
transcriptase (Invitrogen) and a mixture of two poly(A) primers of 20 and 34
nucleotides in length, according to the manufacturer’s protocol.
Amplification of the HCV NS5B gene was achieved by two rounds of PCR
using AccuPrime high-fidelity Taq polymerase (Invitrogen). For both rounds, the
PCRs were carried out for 40 cycles consisting of 94°C for 30 s and 68°C for 3
min. The first-round primers (1a1 and 1b1; see the supplemental material)
amplified the full-length NS5B gene, while the second-round primers (1a2 and
1b2) incorporated the SbfI and PacI restriction sites at the 5? and 3? ends of the
products, respectively, to allow for cloning. Amplified full-length NS5B products
were cloned into the BB7M4hRLuc.ribo vector at the engineered SbfI and PacI
sites to generate chimeric replicons containing patient NS5B sequences in the 1b
Luciferase reporter assay. The wild-type replicon cells, DSR or DDR, or
resistant replicon cells were seeded at a density of 2 ? 104cells per well in 96-well
plates in the absence of selection antibiotics. In some experiments, medium was
supplemented with 50% human serum (HS) or a combination of 45 mg/ml HS
albumin (HSA) and 1 mg/ml ?1-acid glycoprotein (AAG), all of which were
obtained from Sigma. Compounds were tested at half-log serial dilutions over a
range of concentrations with appropriate solvent controls (compound free). Cells
were incubated with compounds for 3 days at 37°C. HCV RNA replication was
monitored by humanized Renilla luciferase (hRLuc) reporter activity. In DDR
cells, cytotoxicity was measured simultaneously by the FLuc reporter activity. For
DSR, hRLuc activity was determined using the RLuc assay system (Promega,
Pittsburgh, PA), according to the manufacturer’s instructions, in a Perkin Elmer
MicroBeta 1450 jet (Perkin Elmer, Wellesley, MA). For DDR, reporter activities
were determined using the dual luciferase reporter kit (Promega) according to
the manufacturer’s instructions. The 50% effective concentration (EC50) was
calculated as the concentration of compound that effected a decrease in viral
RNA replication (as measured by hRLuc activity or quantitative PCR) in com-
pound-treated cells to 50% of that produced in compound-free cells. The 50%
cytotoxicity concentration was calculated as the concentration of compound that
decreased host cell viability (as measured by FLuc activity) in compound-treated
cells to 50% of that produced in compound-free cells. The values were deter-
mined by nonlinear regression analyses.
The XTT cytotoxicity assay. Cells were seeded at a density of 2 ? 104per well
in 96-well plates. Compounds were tested at half-log serial dilutions over a range
of concentrations with appropriate solvent controls (compound free). After in-
cubation with compounds for 3 days, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide (XTT; Sigma) and phenazine methosulfate
(Sigma) were added to the cells at final concentrations of 200 ?g/ml and 1 mM,
respectively. After an additional 4-h incubation at 37°C, the amount of formazan
produced was quantified at a test reference of 450 nm and a reference wave-
length of 650 nm in a spectrophotometer (Molecular Devices, Sunnyvale, CA).
In vitro resistance selection at fixed concentrations. DSR cells were seeded at
densities of 5 ? 104, 1 ? 105, and 2.5 ? 105in 10-cm plates in medium containing
either 3?, 6?, or 12? EC90(0.96, 1.9, or 3.8 ?M, respectively) of PF-00868554,
with weekly changes of selection medium. As a positive control, DSR cells were
cultured in the presence of 3? EC90(15 ?M) of a benzimidazole polymerase
inhibitor, compound C (Fig. 1) (21). As a negative control, DSR cells were
cultured in medium without the presence of inhibitor. Colonies were isolated
after 21 days of selection and expanded to cell lines in larger culture vessels. Ten
individual resistant cell lines were randomly chosen for genotypic and phenotypic
characterizations at each selection concentration for PF-00868554 and com-
pound C. Construction of the M423T mutant replicon and enzyme was described
Population sequence analysis of the NS5B gene. Replicon RNA was extracted
using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufac-
turer’s instructions. Reverse transcription (RT)-PCR of the NS5B region was
completed using primers 5?-AAGAATTCTTCACAGAAGTGGATGGG-3? and
5?-ATTGGCCTGGAGTGTTTAGGTCC-3? and SuperScript one-step RT-PCR
with platinum Taq (Invitrogen) under the following conditions: 45°C for 30 min
and 94°C for 2 min. PCR was performed for a total of 30 cycles in a GeneAmp
9700 PCR system (PE Applied Biosystems, Foster City, CA) under the following
conditions: 94°C for 30 s, 60°C for 1.5 min, and 72°C for 3 min, followed by a
single cycle of 10 min at 72°C. The amplified products were purified and subse-
quently subjected to population sequencing.
Transient replication assay. In vitro transcripts were generated from replicon
constructs using the MEGAscript T7 kit (Ambion, Austin, TX) according to the
manufacturer’s protocol and stored at ?80°C before use. Ten micrograms of
replicon transcripts were electroporated into 8 ? 106Huh7.5 cells in a 0.4-cm
cuvette using a Bio-Rad Gene Pulser II electroporator (Bio-Rad, Hercules, CA)
at 220 V and 950 ?F. Electroporated cells were resuspended in growth medium,
plated on 96-well black plates with clear bottoms at 6 ? 104cells per well, and
allowed to adhere for 4 h at 37°C. Compounds were tested at half-log serial
dilutions over a range of concentrations, with appropriate DMSO controls (com-
pound free). After incubation for 3 days at 37°C, hRLuc activity was determined
using the RLuc assay system, according to the manufacturer’s protocol, in the
Perkin Elmer MicroBeta 1450 jet.
Pharmacokinetic assessment with rats, dogs, and monkeys. Male Sprague-
Dawley rats were housed in individual cages with food and water provided ad
libitum. For the intravenous (i.v.) study, a single i.v. dose of 2 mg/kg PF-00868554
in a solution of 50% polyethylene glycol (PEG) 200, 10% ethanol, and 40% water
at a dosing volume of 2 ml/kg was prepared. Oral evaluation with solution for
PF-00868554 was at 2 mg/kg in a solution of 50% PEG 200, 10% ethanol, and
40% water at a dosing volume of 2 ml/kg. Oral dosing of PF-00868554 was also
prepared using a 0.5% methylcellulose suspension at a dosing volume of 2 ml/kg.
Both the solution and suspension oral gavage doses were followed by a 10-ml tap
water flush of the gavage tube. Blood samples (?0.25 ml) were collected from
the jugular vein of each rat for determination of PF-00868554 plasma concen-
trations. Blood samples for the i.v. arm of these studies were collected at pre-
dose; 2, 5, 10, 15, and 30 min; and 1, 2, 4, 6, 8, and 24 h postdose. For the oral
arm of these studies, blood samples were collected at 10, 20, and 30 min and at
1, 2, 3, 4, 6, 8, and 24 h postdose.
Male beagle dogs were housed in individual cages with water provided ad
libitum. The dogs were fasted overnight prior to dosing and during the first 4 h
of blood sampling. In the i.v. arm of the study, three dogs received an i.v. dose
of 1 mg/kg PF-00868554 in a solution of 50% PEG 200, 10% ethanol, and 40%
water at a dosing volume of 0.5 ml/kg. For the oral arm of the study, three dogs
received a single oral dose of 2 mg/kg PF-00868554 in a solution of 50% PEG
200, 10% ethanol, and 40% water and two dogs received a single oral dose of
PF-00868554 in a 0.5% methylcellulose suspension, both at a dosing volume of
1 ml/kg. The oral gavage doses were followed by a 10-ml tap water flush of the
2546 SHI ET AL.ANTIMICROB. AGENTS CHEMOTHER.
gavage tube. Blood samples (1 ml) were collected from the jugular vein of each
dog for determination of PF-00868554 plasma concentrations. Blood samples for
the i.v. portion of the study were collected at predose; 2, 5, 10, 20, 30, and 45 min;
and 1, 2, 4, 6, 8, 12, and 24 h postdose. For the oral portion of the study, blood
samples were collected at 10, 20, 30, and 45 min and at 1, 2, 3, 4, 6, 8, 12, and 24 h
Male cynomolgus monkeys were housed in individual cages with water pro-
vided ad libitum. The animals were fasted overnight, and food was also withheld
during the first 4 h of blood sampling. Monkeys received a single i.v. dose of
2-mg/kg PF-00868554 solution at a dosing volume of 1 ml/kg. Blood samples (1
ml) were collected via femoral venipuncture for each monkey for determination
of PF-00868554 plasma concentrations. Blood samples were collected at predose;
2, 5, 10, 20, 30, and 45 min; and 1, 2, 4, 6, 8, and 12 h postdose.
For all studies, each blood sample was placed in a tube containing heparin and
centrifuged to obtain plasma. The plasma was frozen immediately and stored at
?80°C until samples were analyzed by liquid chromatography-tandem mass
Pharmacokinetic parameters were estimated using the noncompartmental
analysis by WinNonlin 3.1 software (Pharsight, Mountain View, CA). Graphing
was accomplished by Microsoft Excel 5.0. Estimated parameters reported here
include the values of the area under the curve from time zero to infinity
(AUC0–?), the maximum mean plasma concentration (Cmax), the time-to-maxi-
mum-plasma concentration (Tmax), terminal half-life (t1/2), clearance (CL), and
volume of distribution at steady state (Vss). Oral bioavailability (Foral) was cal-
culated using the following equation:
Foral? (Dosei.v.? Dosep.o.)/(Dosep.o.? mean AUCi.v.)
where p.o. indicates orally.
Biochemical potency and selectivity of PF-00868554 in vitro.
Previous data showed that members of the dihydropyrone class
of inhibitors are potent, noncompetitive inhibitors of HCV
polymerase 1b, binding approximately 30 Å from the active site
at the region between the thumb and finger domains of the
enzyme. Optimization of this class of compounds using com-
binatorial efforts and structure-based design has led to the
discovery of PF-00868554 (25) (Fig. 1). The IC50s against poly-
merases of the 1b Con1 and BK strains were 0.015 ? 0.010 and
0.0066 ? 0.0018 ?M, respectively (Table 1). Inhibitory potency
was clearly the greatest against the genotype 1 polymerases
tested, with IC50s in the nanomolar range, whereas the IC50s
were approximately 1 ?M against non-genotype 1 HCV poly-
merases. PF-00868554 displayed at least 1,600- to 7,500-fold
selectivity for the genotype 1 viral polymerases, compared to
the human DNA and RNA polymerases assayed (Table 1).
Because an early predecessor of the dihydropyrone series
originated from an HIV protease inhibitor program (29), the
inhibitory potency of PF-00868554 was tested against a recom-
binant HIV protease and several human proteases of physio-
logical relevance. While the molecule remains a weak inhibitor
of HIV protease, with an IC50of 2.18 ?M, it displayed no
significant inhibition of a wide variety of human aspartyl and
serine proteases, including plasma thrombin, neutrophil elas-
tase, liver cathepsin B, human caspase-2, and pancreatic chy-
motrypsin, at a concentration of 10 ?M (data not shown).
In vitro antiviral activity and cytotoxicity of PF-00868554.
The ability of PF-00868554 to inhibit HCV RNA replication
was evaluated against subgenomic replicons derived from the
1b Con1 and 1a H77 strains in replicon assays using the lucif-
erase reporter end point. PF-00868554 demonstrated strong
antiviral activity against the 1b Con1 replicon, with a mean
EC50of 0.075 ?M, and reduced activity against the 1a H77
replicon, with a mean EC50of 0.39 ?M (Table 2). The cyto-
toxicity of PF-00868554 was determined by the XTT assay with
various human cell lines, including Huh7, HepG2, HeLa, and
HEK293 cells, after 3 days of incubation. PF-00868554 dem-
onstrated little or no cytopathic effects in multiple cell lines, up
to the highest concentration of compound evaluated, with 50%
cytotoxicity concentration values of ?320 ?M.
In vitro antiviral activity of PF-00868554 against chimeric
replicons containing NS5B sequences from clinical isolates.
To evaluate the activity of PF-00868554 against different HCV
strains within genotype 1, chimeric replicons containing poly-
merase sequences derived from a panel of genotype 1 clinical
isolates in the genotype 1b Con1 backbone were generated.
The replication fitness of these chimeric replicons was first
determined in the transient replication assay with Huh7.5 cells
by use of the luciferase reporter end point (Fig. 2). Only the
replicons that displayed luciferase activities of at least 2 ? 103
relative light units (RLU) (about 20 times above the back-
ground) were included in the subsequent antiviral assays so
that the EC50s could be accurately determined. PF-00868554
demonstrated potent in vitro antiviral activity against HCV
replicons containing polymerase sequences from 23 out of 24
(95.8%) strains of genotype 1a and 1b, with a mean EC50of
0.043 ? 0.022 ?M and a range of 0.0088 to 0.087 ?M (Table 2).
The mean antiviral activity against 11 of 11 1b strains (EC50?
0.033 ? 0.023 ?M) was comparable to that demonstrated
against 12 of 13 1a strains (EC50? 0.052 ? 0.017 ?M). PF-
00868554 showed reduced activity against the H77 strain of
genotype 1a, with a mean EC50of 0.39 ? 0.42 ?M. Because
PF-00868554 has similar activities against all genotype 1 poly-
merases tested, including Con1, BK, J1 and H77 (Table 1), the
molecular basis for the reduction in activity of PF-00868554
TABLE 1. Spectrum of biochemical potency and selectivity
(at 50 ?M)
HCV NS5B polymerases
0.015 ? 0.010
0.0066 ? 0.0018a
0.030 ? 0.005
0.021 ? 0.009b
1.1 ? 0.3
1.0 ? 0.2
1.7 ? 0.3
11 ? 3
Human DNA polymerases
Human RNA polymerases
HIV reverse transcriptase0
HIV protease Q7K2.18
an ? 9.
bn ? 8.
c% inhibition at 200 ?M.
VOL. 53, 2009 PRECLINICAL CHARACTERIZATION OF PF-008685542547
against the H77 replicon is not known. It is possible that other
NS proteins in the replication complex may have an effect on
the replicon’s sensitivity to polymerase inhibitors. However,
even with the inclusion of the H77 replicon, the overall mean
and median EC50s of PF-00868554 against all genotype 1 rep-
licons were 0.059 and 0.042 ?M, respectively, with a range of
0.0088 to 0.39 ?M. In contrast, a benzothiadiazine polymerase
inhibitor (Fig. 1) displayed EC50s varying from ?0.001 to 2.1
?M, corresponding to a larger than 2,000-fold difference in
activity levels (Table 2). As expected, all of the chimeric rep-
licons showed similar sensitivities to an NS3 protease inhibitor
(data not shown).
The in vitro antiviral activity of PF-00868554 was also eval-
uated against the genotype 2a JFH1 replicon and chimeric
replicons, in which polymerase sequences from genotype 2b
virus isolates were substituted for the corresponding sequence
in the 1b replicon. PF-00868554 demonstrated antiviral activity
against the replicons that contain non-genotype 1 polymerase
sequences, with EC50s ranging from 11 to 17 ?M (data not
shown). No apparent structural correlations between sensitiv-
ity to PF-00868554 and polymerase sequences were identified.
The effects of protein binding on the in vitro antiviral ac-
tivity of PF-00868554. The effect of protein binding on the in
vitro antiviral activity of PF-00868554 was evaluated in the
luciferase reporter assay with DSR cells. Cell culture medium
that contained 10% FBS was supplemented with either 50%
HS or a combination of 1 mg/ml AAG and 45 mg/ml HSA in
these experiments. In the presence of 50% HS, no statistically
significant change in the antiviral activity of PF-00868554 was
observed (3.5-fold change from 0.053 to 0.19 ?M, P ? 0.05). In
the presence of 45 mg/ml HSA and 1 mg/ml AAG, PF-
00868554 demonstrated antiviral activity with a mean EC50of
0.69 ?M, which represented a 13-fold increase compared
with that observed in medium containing 10% FBS alone (P ?
Phenotypic and genotypic determination of PF-00868554-
resistant cell lines. The PF-00868554-resistant cell lines gen-
erated by exposing the DSR cell line to fixed concentrations of
the inhibitor were subjected to replication fitness analysis using
the luciferase reporter assay. The mutant replicons displayed
luciferase activities ranging from 4.9 ? 104to 1.15 ? 106RLU,
which were well above the background, to allow for accurate
EC50determinations (Table 3). Phenotypic analysis of the 30
PF-00868554-resistant cell lines showed an 8.9- to ?2,202-fold
reduction in susceptibility to PF-00868554 compared to the
wild-type cells (Table 3). After confirmation of resistance, rep-
licon RNA was isolated from PF-00868554-resistant cell lines,
amplified by RT-PCR, and subjected to population sequencing
to determine the genotypic changes in the NS5B region of the
replicon. The replicons in these cell lines were shown to harbor
one to four mutations that resulted in amino acid changes in
the polymerase (Table 3). Every resistant cell line contained at
least one change at an amino acid that was shown to be part of
the inhibitor-binding pocket, which includes M423, M426, and
I482 (29). The methionine-to-threonine change at residue 423
(M423T) in the NS5B region was identified as the most prev-
alent amino acid substitution, occurring alone in 14 of 30 cell
lines (47%) and in combination with other mutations in 8 of 30
cell lines (27%) that were selected across different concentra-
tions of PF-00868554 (Table 3). Overall, M423T was observed
at a frequency of 73% (22 of 30 cell lines), and changes at
amino acid M423, including M423V and M423I, were observed
in 87% (26 out of 30) of PF-00868554-resistant cell lines (Table
3). Changes at other amino acid residues, M426T and I482T,
FIG. 2. Replication fitness of chimeric replicons containing pa-
tient-derived NS5B sequences. The NS5B sequences from genotype 1a
(open bars) and 1b (solid bars) HCV-infected patients were cloned
into the Con1 subgenomic replicons. The NS5B sequence from the BK
strain was also cloned in the Con1 replicon to produce the BK/Con1
chimeric replicon. Huh7.5 cells were electroporated with in vitro tran-
scripts of chimeric replicons, seeded in 96-well plates at 6 ? 104/well,
and cultured for 3 days before hRLuc activity was determined. The
replication fitness of the parental Con1 replicon is designated 100%.
Results represent the average of triplicate values, with error bars
showing standard deviations.
TABLE 2. In vitro antiviral activity of PF-00868554 against
replicons containing genotype 1 NS5B sequencesa
0.075 ? 0.059
0.027 ? 0.018
0.022 ? 0.009
0.022 ? 0.013
0.0088 ? 0.0039
0.045 ? 0.034
0.023 ? 0.013
0.075 ? 0.036
0.025 ? 0.023
0.0037 ? 0.0017
0.062 ? 0.043
0.18 ? 0.04
0.024 ? 0.014
0.045 ? 0.042
0.021 ? 0.006
0.0029 ? 0.0007
0.049 ? 0.045
0.095 ? 0.067
0.39 ? 0.42
0.057 ? 0.045
0.049 ? 0.036
0.040 ? 0.008
0.028 ? 0.032
0.059 ? 0.043
0.042 ? 0.016
0.087 ? 0.062
0.050 ? 0.024
0.079 ? 0.012
0.042 ? 0.019
0.036 ? 0.011
0.013 ? 0.023
0.26 ? 0.14
0.061 ? 0.020
0.45 ? 0.09
1.1 ? 1.2
0.70 ? 0.17
0.93 ? 0.81
0.18 ? 0.02
0.15 ? 0.03
2.1 ? 1.4
0.97 ? 0.56
0.50 ? 0.18
aResults represent the means ? standard deviations (of three to five experi-
ments) or individual values (of one or two experiments).
bExcept where it is indicated, the NS3-to-NS5A sequence in the replicon is
derived from 1b Con1.
cThe NS3-to-NS5A sequence is derived from either 1a H77 or 1b Con1.
dn ? 3.
2548 SHI ET AL.ANTIMICROB. AGENTS CHEMOTHER.
existed at a much lower frequency (Table 3). Substitution of
proline by alanine or serine at residue 496 (P496A or P496S)
and substitution of valine by alanine at residue 499 (V499A)
were observed in cell lines resistant to a benzimidazole poly-
merase inhibitor, compound C (Fig. 1), consistent with the in
vitro resistance changes previously reported for this compound
All of the amino acid substitutions found to be associated
with resistance to PF-00868554, including M423T, M423I,
M423V, M426T, and I482T, were also observed in the in vitro
resistance study of a structurally related compound, AG-
021541 (39). The 22 resistant cell lines that contain the M423T
change alone or in combination with other amino acid changes
demonstrated a 715- to ?2,202-fold increase in the EC50when
tested for their susceptibility to PF-00868554. Consistent with
our previous findings, the M426T and I482T substitutions re-
sulted in a lower level of resistance than the changes from M
to T, V, or I at amino acid 423 of the polymerase (8.9- to
49-fold versus 706- to ?2,202-fold), confirming that M423 is a
key residue involved in the interaction between dihydropyrone
compounds and the polymerase (Table 3). M426T and I482T
were present only in replicon cell lines generated at lower
concentrations of PF-00868554 (Table 3). Regardless of their
replication fitness or level of resistance to PF-00868554, all of
the resistant replicons remained sensitive to IFN-?, with
changes in the EC50ranging from 0.1 to 6. Furthermore, the
resistance changes at the thumb base site did not significantly
affect the activity of compound C (Fig. 1), known to interact
with a different allosteric site of the polymerase protein, with
changes in the EC50ranging from 0.6 to 4.5.
The predominant resistance change, M423T, was introduced
back into the wild-type (wt) replicon, from which a stable cell
line was generated for the determination of its resistance phe-
notype. M423T conferred 761-fold resistance to PF-00868554,
but no change in susceptibility to IFN-? or compound C was
observed (Table 4). The replication fitness of the M423T rep-
licon was reduced to 1.8 ? 105RLU, which was less than 50%
of that of the wt replicon (5.2 ? 105RLU), as indicated by the
hRLuc reporter activity. However, the colony-forming effi-
ciency of the M423T replicon was comparable to that of the wt
replicon in the colony formation assay (data not shown), sug-
gesting that the mutant replicon replicates at a lower level but
is still sufficient to support colony formation. Similarly, the
purified recombinant 1b Con1 polymerase enzyme that con-
tained the M423T substitution also displayed 733-fold resis-
TABLE 3. Genotypic and phenotypic changes of PF-00868554-resistant cell lines
Amino acid change(s)a
Fold change in PF-
M423T, K535N, N590S
M423T, F572L, I585M
M423T, F572F/V, M573T, W574R
M426T, I432T, Y586C
aRNA was extracted from stable cell lines obtained at 3?, 6?, and 12? the EC90s of PF-00868554 and subjected to RT-PCR amplification and population
sequencing of the NS5B region. M423T in combination with one or more amino acid changes occurred at a frequency of 27% (n ? 8).
bResults represent the number of PF-00868554-resistant cell lines established at each concentration (?M) that contain the observed resistance mutations.
cResults represent the average of duplicate values from one experiment. The ranges of fold changes in the EC50s for benzimidazole compound C and IFN-? are
0.6 to 4.5 and 0.1 to 6, respectively.
TABLE 4. Susceptibility of the M423T mutant replicon and polymerase to PF-00868554a
FC in EC50
FC in IC50
wtM423TNS5B?21 wt NS5B?21M423T
Benzimidazole compound C
0.035 ? 0.031
1.6 ? 0.9
0.97 ? 0.09
27 ? 9
2.4 ? 0.7
1.3 ? 0.2
7610.015 ? 0.010
0.20 ? 0.01b
11 ? 3
0.38 ? 0.03
aThe susceptibility of the 1b Con1 wt and M423T replicon cell line to inhibitors was evaluated in the reporter replicon assay. Cells were exposed to compounds for
3 days before RLuc activity was determined. The susceptibility of the 1b Con1 wt and M423T mutant polymerase to inhibitors was evaluated in the HCV polymerase
biochemical assay as described in Materials and Methods. Results represent the means ? standard deviations (three experiments) or individual values (one to two
experiments of duplicates). FC, fold change; ND, not determined.
bThe result was generated against the 1b BK polymerase.
cThe replicon EC50s for IFN-? are given as IU/ml.
VOL. 53, 2009PRECLINICAL CHARACTERIZATION OF PF-00868554 2549
tance to PF-00868554 but no change in susceptibility to com-
pound C in the enzymatic assay in vitro (Table 4).
Pharmacokinetic properties of PF-00868554 in preclinical
species. In both rats and monkeys, PF-0868554 exhibited mod-
erate plasma CL and V (Table 5). In dogs, however, it exhib-
ited both low plasma CL (4.4 ? 1.1 ml/min/kg) and V (0.25
liter/kg). The t1/2of PF-00868554 in rats, monkeys, and dogs
were between 2 and 4.4 h, but the calculated effective half-life
(0.693 ? V/CL) was substantially shorter (0.5 h in both rats and
monkeys and 3 h in dogs). The preclinical results suggest that
PF-00868554 would be amenable for a twice-daily dosing reg-
imen. PF-00868554 was rapidly absorbed after an oral dose in
both species (with a Tmaxof 0.94 ? 0.9 h and 1 h for rats and
dogs, respectively) with acceptable F (75% and 49% for rats
and dogs, respectively) (Table 5). In summary, the absorption
of PF-00868554 in preclinical species was rapid, resulting in a
high predicted human absorption rate constant (Ka) of 1.1 h?1.
Current therapy for HCV is poorly tolerated and of limited
efficacy, such that patients infected with the 1a and 1b geno-
types have more severe liver disease and lower response rates
to current IFN therapy (54). In the United States, infection by
the 1a and 1b genotypes is the most prevalent, whereas in
Europe and Japan, the prevalence of infection by the 1b ge-
notype significantly exceeds that of any other HCV genotypes
(53). Thus, there remains a significant unmet need for tar-
geted, efficacious chemotherapy against HCV genotypes 1a
We have demonstrated that PF-00868554 displays both po-
tency and selectivity as a promising inhibitor of the 1a and 1b
genotypes of HCV RNA-dependent RNA polymerase. With
low nanomolar inhibitory potency against several recombinant
genotype 1 NS5B constructs in vitro, the molecule displays no
detectable inhibition of several human polymerases and no
detectable inhibition of several human proteases, in spite of its
weak inhibitory activity against recombinant HIV protease.
This biochemical selectivity likely translates into cellular selec-
tivity and a favorable therapeutic index, in that no detectable
cytotoxicity was detected with several immortalized human
cell lines at concentrations more than 5,400-fold greater than
the potent activity against the genotype 1 HCV replicons.
One obstacle to developing effective HCV therapies is the
heterogeneity of the viral genomes present in infections. The
RNA-dependent RNA polymerase that synthesizes the viral
genome lacks a proof-reading mechanism. The error rate of
the polymerase is estimated at 10?4(35), which results in
significant genetic diversity among the viral population within
each patient. This heterogeneity has been associated with re-
ductions in activity of HCV polymerase inhibitors up to 25-fold
when tested in a chimeric replicon system containing NS5B
sequences derived from genotype 1a and 1b patients (31, 48).
Therefore, an important part of preclinical development for
HCV inhibitors is the assessment of the antiviral activity
against a variety of clinical isolates. PF-00868554 demonstrated
potent antiviral activity against polymerases derived from a
majority (95.8%) of HCV strains, including primary clinical
isolates and laboratory strains. The overall mean and median
EC50s of PF-00868554 against all genotype 1 replicons were
0.059 and 0.042 ?M, respectively, with a range of 0.0088 to 0.39
?M. The presence of 50% HS only modestly attenuated the
antiviral activity of PF-00868554. These studies demonstrate
that PF-00868554 has potent in vitro antiviral activity in the
HCV replicon system against a variety of genotype 1 poly-
merases and support its potential use as an antiviral agent with
patients infected with genotype 1 HCV.
The emergence of drug-resistance variants has been a major
factor limiting the efficacy of virus-specific inhibitors against
retroviruses and many other RNA viruses. In vitro resistance
studies of various HCV inhibitors, including NS3 protease (26,
27, 30, 47, 52) and NS5B polymerase inhibitors (17, 21, 22, 33,
34, 45, 46, 50), identified resistance mutations in the corre-
sponding viral target regions, some of which have also been
observed in subsequent clinical studies. A recent report indi-
cated that resistance mutations observed in vitro were also
developed in vivo after a 14-day monotherapy treatment with
an NS3 protease inhibitor, VX-950, and correlated strongly
with clinical outcome (37). A nonnucleoside polymerase inhib-
itor from Viropharma, HCV-796, achieved a peak reduction in
viral load of ?1 log on day 4, but the reduction dropped to
approximately 0.7 log on day 14 (6) as a result of the emer-
gence of resistance (49). These results highlight the impor-
tance of conducting in vitro resistance studies, which could
provide important insights into resistance development in fu-
ture clinical trials.
TABLE 5. In vivo pharmacokinetics of PF-00868554 in rats, dogs, and monkeysa
No. of animals
AUC0–?(?g ? h/ml)
21 ? 4.8
1 ? 0.4
2.9 ? 0.7
2.5 ? 1
0.93 ? 0.1
18 ? 2.3
4.4 ? 1.1
21 ? 5
0.8 ? 0.05
2.6 ? 1.0
2.1 ? 0.4
0.93 ? 0.2
2.8 ? 1.2
0.64 ? 0.28
0.94 ? 0.9
0.93 ? 0.2
2.85 ? 0.2
0.7 ? 0.1
17.7 ? 1.8
3.6 ? 0.8
4.7 ? 1.1
ai.v. and oral solution formulation was as follows: 50% PEG 200, 10% ethanol, 40% water. Oral suspension formulation was as follows: 0.5% methylcellulose.
bCLblood, blood clearance; Vss, volume of distribution at steady state.
2550 SHI ET AL.ANTIMICROB. AGENTS CHEMOTHER.
Our in vitro resistance study of PF-00868554 in the HCV
subgenomic replicon cells identified amino acid changes at the
thumb base allosteric site of the polymerase, including M423T/
V/I, M426T, and I482T, all of which were observed in our
previous study of a structurally related compound, AG-021541
(39). Similar to our previous findings, amino acid substitutions
at M423 resulted in a significantly higher level of resistance
than those of substitutions at M426 and I482 (Table 3). More
importantly, there was no cross-resistance of replicons contain-
ing these resistance changes to IFN-? and a number of other
polymerase inhibitors (39), supporting the use of PF-00868554
in combination therapies with other inhibitors targeting differ-
ent regions of the polymerase for the treatment of HCV.
In conclusion, we have presented a summary of preclinical in
vitro and in vivo data demonstrating that PF-00868554 has
potent and broad-spectrum activity against HCV polymerase
1a and 1b viral strains, while displaying a selectivity profile that
is consistent with a significant margin of safety. Furthermore,
the pharmacokinetic properties of PF-00868554 were deter-
mined by in vivo studies to be favorable in preclinical species,
suggesting that a twice-daily dosing regimen for human pa-
tients will be feasible.
We acknowledge all members of the HCV polymerase project team
at Pfizer Global Research and Development, La Jolla Laboratories,
and Kalamazoo Laboratories (formerly Pharmacia and Upjohn, Inc.).
In addition, we thank Karen Maegley and Laura Lingardo for con-
ducting protease selectivity assays and Robert Hunter for conducting
the in vivo rat pharmacokinetic studies.
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