Book on hepatitis from page 242 to 247
242 Hepatology 2012
Figure 2. Genomic organisation of HCV.
From the initially translated HCV polyprotein the three structural and seven non-structural HCV proteins are processed by both host and viral proteases (Moradpour
2007). NS2 is a metalloproteinase that cleaves itself from the NS2/NS3 protein,
leading to its own loss of function and to the release of the NS3 protein (Lorenz
2006). NS3 provides a serine protease activity and a helicase/NTPase activity. The
serine protease domain comprises two b-barrels and four α-helices. The serine
protease catalytic triad – histidine 57, asparagine 81 and serine 139 – is located in a
small groove between the two b-barrels (Kim 1996, Kim 1998). NS3 forms a tight,
non-covalent complex with its obligatory cofactor and enhancer NS4A, which is
essential for proper protein folding (Figure 3). The NS3-4A protease cleaves the
junctions between NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B.
Besides its essential role in protein processing, NS3 is integrated into the HCV
RNA replication complex, supporting the unwinding of viral RNA by its helicase
activity. Moreover, NS3 might play an important role in HCV persistence via
blocking TRIF-mediated toll-like receptor signalling and Cardif-mediated RIG-I
signalling, subsequently resulting in impaired induction of type I interferons
(Meylan 2005). Thus, pharmacologic NS3 inhibition might support viral clearance
by restoring the innate immune response.
Hepatitis C: New Drugs 243
Figure 3. Molecular structure of the HCV NS3-4A protease.
The active site of the NS3-4A protease is located in a shallow groove between the
two b-barrels of the protease making the design of compound inhibitors relatively
difficult. Nevertheless, many NS3-4A protease inhibitors have been developed
which can be divided into two classes, the macrocyclic inhibitors and linear tetra-peptide α-ketoamide derivatives. In general, NS3-4A protease inhibitors have been
shown to strongly inhibit HCV replication during monotherapy, but also may cause
the selection of resistant mutants, which is followed by viral breakthrough. The
additional administration of pegylated interferon and ribavirin, however, was shown
to reduce the frequency of development of resistance. Future strategies aim for
combination therapies with different antiviral drugs to prevent the development of
resistance. The most advanced compounds are telaprevir and boceprevir, both
approved in 2011.
Ciluprevir (BILN 2061)
The first clinically tested NS3-4A inhibitor was ciluprevir (BILN 2061), an orally
bioavailable, peptidomimetic, macrocyclic drug binding non-covalently to the active
center of the enzyme (Lamarre 2003) (Figure 4). Ciluprevir monotherapy was
evaluated in a double-blind, placebo-controlled pilot study in treatment-naïve
genotype 1 patients with liver fibrosis and compensated liver cirrhosis (Hinrichsen
2004). Ciluprevir, administered twice daily for two days at doses ranging from 25 to
500 mg, led to a mean 2-3 log10 decrease of HCV RNA serum levels in most
patients. Importantly, the stage of disease did not affect the antiviral efficacy of
ciluprevir. The tolerability and efficacy of ciluprevir in HCV genotype 2- and 3-infected individuals was examined in an equivalent study design, but compared to
genotype 1 patients, the antiviral activity was less pronounced and more variable in
these patients (Reiser 2005). Ciluprevir development has been halted.
244 Hepatology 2012
Figure 4. Molecular structure of selected NS3-4A inhibitors.
Telaprevir (Incivek/Incivo
®
) and boceprevir (Victrelis
®
)
Telaprevir and boceprevir were approved for the treatment of chronic hepatitis C
virus genotype 1 infection by the FDA, EMA and several other countries in 2011.
Both telaprevir and boceprevir are orally bioavailable, peptidomimetic NS3-4A
protease inhibitors belonging to the class of α-ketoamid derivatives (Figure 4). Like
other NS3-4A inhibitors, telaprevir and boceprevir are characterized by a
remarkable antiviral activity against HCV genotype 1. However, monotherapy with
these agents results in the rapid selection of drug-resistant variants followed by viral
breakthrough (Reesink 2006, Sarrazin 2007). Phase II and III clinical studies have
shown that the addition of pegylated interferon α plus ribavirin leads to a
substantially reduced frequency of resistant mutants and viral breakthrough, and to
significantly higher SVR rates in both treatment-naïve and treatment-experienced
HCV genotype 1 patients compared to treatment with pegylated interferon α and
ribavirin alone (Bacon 2011, Jacobson 2011, Poordad 2011, Sherman 2011, Zeuzem
2011). Therefore, telaprevir- and boceprevir-based triple therapy can be considered
the novel standard of care for HCV genotype 1 patients. Results of the Phase III
telaprevir and boceprevir approval studies are summarized in Figure 5.
Hepatitis C: New Drugs 245
69
75
29
40
52
7
0
20
40
60
80
100
75
69
44
72
92
88
0
20
40
60
80
100
86
24
57
15
31
5
0
20
40
60
80
100
67 69
40 42
53
23
0
20
40
60
80
100
ADVANCE ILLUMINATE REALIZE
A)
B)
SPRINT-2 RESPOND-2
% SVR % SVR
% SVR % SVR
(response-guided)
relapser part. non-responder
null-responder
Caucasian Black
relapser part. non-responder
Figure 5. SVR rates in Phase III clinical trials evaluating telaprevir (A) or boceprevir
(B) in combination with PEG-IFN α and ribavirin. ADVANCE, ILLUMINATE and
SPRINT-2 enrolled treatment-naïve patients, REALIZE and RESPOND-2 enrolled
treatment-experienced patients. Telaprevir was administered for 8 or 12 weeks in
combination with PEG-IFN α-2a and ribavirin, followed by 12-40 weeks of PEG-IFN α-2a
and ribavirin alone. Boceprevir was administered over the whole treatment period of 28 or
48 weeks in combination with PEG-INF α-2b and ribavirin, except of the first 4 weeks of
lead-in therapy of PEG-IFN α-2b and ribavirin only. eRVR, extended early virologic
response; SOC, standard of care; LI, lead-in (4 weeks of PEG-INF α plus ribavirin only).
Other NS3 protease inhibitors
Other NS3 protease inhibitors are currently in various phases of development
(danoprevir (R7227/ITMN191), vaniprevir (MK-7009), BI201335, simeprevir
(TMC435350), narlaprevir (SCH900518), asunaprevir (BMS-650032), PHX1766,
ACH-1625, IDX320, ABT-450, MK-5172, GS-9256, GS-9451) and will
significantly increase treatment options for chronic hepatitis C in the near future. In
general, comparable antiviral activities to telaprevir and boceprevir in HCV
genotype 1 infected patients were observed during mono- (and triple-) therapy
studies (Brainard 2010, Manns 2011, Reesink 2010). Potential advantages of these
second and third generation protease inhibitors might be improved tolerability,
broader genotypic activity, different resistance profiles, and/or improved
pharmacokinetics for once-daily dosage (e.g., TMC435, BI201335). Different
246 Hepatology 2012
resistance profiles between linear tetrapeptide and macrocyclic inhibitors binding to
the active site of the NS3 protease have been revealed. However, R155 is the main
overlapping position for resistance and different mutations at this amino acid site
within the NS3 protease confer resistance to nearly all protease inhibitors currently
in advanced clinical development (Sarrazin 2010). An exception is MK-5172, which
exhibits potent antiviral activity against variants carrying mutations at position
R155. In addition, MK-5172 had potent antiviral activity against both HCV
genotype 1 and 3 isolates (Brainard 2010).
Resistance to NS3-4A inhibitors
Because of the high replication rate of HCV and the poor fidelity of its RNA-dependent RNA polymerase, numerous variants (quasispecies) are continuously
produced during HCV replication. Among them, variants carrying mutations
altering the conformation of the binding sites of DAA compounds can develop.
During treatment with specific antivirals, these preexisting drug-resistant variants
have a fitness advantage and can be selected to become the dominant viral
quasispecies. Many of these resistant mutants exhibit an attenuated replication with
the consequence that, after termination of exposure to specific antivirals, the wild
type may displace the resistant variants (Sarrazin 2007). Nevertheless, HCV
quasispecies resistant to NS3-4A protease inhibitors or non-nucleoside polymerase
inhibitors can be detected at low levels in some patients (approx. 1%) who have
never been treated with these specific antivirals before (Gaudieri 2009). The
clinical relevance of these pre-existing mutants is not completely understood,
although there is evidence that they may reduce the chance of achieving an SVR
with DAA-based triple therapies if the patient’s individual sensitivity to pegylated
interferon α + ribavirin is low.
More recently, the Q80R/K variant has been described as conferring low-level
resistance to simeprevir (TMC435), a macrocyclic protease-inhibitor. Interestingly,
the Q80K variant can be detected in approximately 10% of HCV genotype 1-infected patients (typically in subtype 1a isolates) and a slower viral decline during
simeprevir-based triple therapy was observed (Lenz 2011). Table 2 summarizes the
resistance profile of selected NS3-4A inhibitors. Although the resistance profiles
differ significantly, R155 is an overlapping position for resistance development and
different mutations at this position confer resistance to nearly all protease inhibitors
(not MK-5172) currently in advanced clinical development (Sarrazin 2010).
Importantly, many resistance mutations could be detected in vivo only by clonal
sequencing. For example, mutations at four positions conferring telaprevir
resistance have been characterized so far (V36A/M/L, T54A, R155K/M/S/T and
A156S/T), but only A156 could be identified initially in vitro in the replicon system
(Lin 2005). These mutations, alone or as double mutations, conferred low
(V36A/M, T54A, R155K/T, A156S) to high (A156T/V, V36M + R155K, V36M +
156T) levels of resistance to telaprevir (Sarrazin 2007). It is thought that the
resulting amino acid changes of these mutations alter the confirmation of the
catalytic pocket of the protease, which impedes binding of the protease inhibitor
(Welsch 2008).
Hepatitis C: New Drugs 247
Table 2. Resistance mutations to HCV NS3 protease inhibitors.
36 54 55 80 155 156A 156B 168 170
Telaprevir*
(linear)
Boceprevir*
(linear)
SCH900518*
(linear)
BI-201335*
(linear?)
BILN-2061 **
(macrocyclic)
Danoprevir*
(macrocyclic)
MK-7009*
(macrocyclic)
TMC435*
(macrocyclic)
BMS-650032*
(macrocyclic)
GS-9451*
(macrocyclic)
ABT450*
(macrocyclic)
IDX320**
(macrocyclic)
ACH1625**
(macrocyclic)
MK-5172***
(macrocyclic)
36: V36A/M; 54: T54S/A; 55: V55A; 80: Q80R/K; 155: R155K/T/Q; 156A: A156S; 156B:
A156T/V; 168: D168A/V/T/H; 170: V170A/T
* mutations associated with resistance in patients
** mutations associated with resistance in vitro
*** no viral break-through during 7 days monotherapy
# Q80 variants have been observed in approximately 10% of treatment-naïve patients and was
associated with slower viral decline during simeprevir (TMC435) triple therapy
As shown for other NS3-4A protease inhibitors as well (e.g., danoprevir), the
genetic barrier to telaprevir resistance differs significantly between HCV subtypes.
In all clinical studies of telaprevir alone or in combination with PEG-IFN α and
ribavirin, viral resistance and breakthrough occurred much more frequently in
patients infected with HCV genotype 1a compared to genotype 1b. This difference
was shown to result from nucleotide differences at position 155 in HCV subtype 1a
(aga, encodes R) versus 1b (cga, also encodes R). The mutation most frequently
associated with resistance to telaprevir is R155K; changing R to K at position 155
requires 1 nucleotide change in HCV subtype 1a, and 2 nucleotide changes in
subtype 1b isolates (McCown 2009).
It will be important to define whether treatment failure due to the development of
variants resistant to DAA agents has a negative impact on re-treatment with the
same or a different DAA treatment regimen. Follow-up studies of telaprevir and
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