Download Review Nucleotide prodrugs for HCV therapy

Antiviral Chemistry & Chemotherapy 2011; 22:23–49 (doi: 10.3851/IMP1797)
Review
Nucleotide prodrugs for HCV therapy
Michael J Sofia1*
1
Pharmasset, Inc., Princeton, NJ, USA
*Corresponding author e-mail: [email protected]
HCV infection is a significant worldwide health problem
and is a major cause of hepatocellular carcinoma. The
current standard of care, interferon and ribavirin, is only
effective against a proportion of the patient population
infected with HCV. To address the shortcomings of existing therapy, the development of direct acting antiviral
agents is under investigation. The HCV RNA dependent
RNA polymerase is an essential enzyme for viral replication and is therefore a logical target against which to
develop novel anti-HCV agents. Nucleosides have been
shown to be effective as antiviral agents for other viral
diseases and therefore, have been investigated as inhibitors of HCV replication. The development of prodrugs
of nucleoside 5′-monophosphates has been pursued
to address limitations associated with poor nucleoside
phosphorylation. This is required to produce the nucleoside 5′-triphosphate which is the anabolite that is the
actual inhibitor of the polymerase enzyme. Prodrugs of
nucleoside 5′-monophosphates have been developed that
enable their delivery into cells and in vivo into the liver.
The implementation of these prodrug strategies has ultimately led to the identification of several prodrugs of
nucleoside 5′-monophosphates that are potent inhibitors of HCV replication in vitro. They have progressed into
the clinic and the early data demonstrate greatly reduced
viral load levels in HCV-infected patients. This review will
survey the state of nucleotide prodrugs for the treatment
of HCV.
Introduction
HCV is known to have infected approximately 180
million individuals worldwide [1]. It is estimated that
of those infected with HCV approximately 80% will
develop chronic liver disease and a significant proportion of those infected will eventually develop liver cirrhosis and subsequently hepatocellular carcinoma [2].
HCV is a single-stranded, positive sense RNA virus of
the Flaviviridae. Six major viral genotypes with over
100 viral subtypes have been identified for HCV. Genotypes 1a and 1b are the most prevalent genotypes in
the western world with genotypes 2 and 3 comprising
20–30% of this population. HCV genotypes 2–6 predominate in the developing world [3,4]. Because HCV
replicates in the cytoplasm of infected cells by a membrane associated replication complex and the virus has
an RNA genome with no DNA intermediate during
replication, no genomic templates are stably integrated
into the host genome. Therefore, virological cures are
possible for HCV patients. This is in contrast to other
viruses such as HIV and HBV where the viral genome is
integrated into the host DNA and a virological cure is
considered remote. However, for HCV-infected patients
virological cures are made difficult due to the high rate
of HCV viral replication and by the high spontaneous
©2011 International Medical Press 1359-6535 (print) 2040-2066 (online)
mutation rate of the virus. This high mutation rate is a
result of poor replication fidelity exhibited by the HCV
polymerase and an apparent lack of proof reading [4].
The current therapy for treating chronic HCV infection consists of regular injections of α-interferon (IFN)
with daily oral administration of ribavirin (RBV). This
standard of care (SOC) regimen does not act by directly
attacking the virus but functions by boosting the host
immune response. For genotype 1 patients regular IFN/
RBV treatments for 48 weeks result in only 40–50% of
patients achieving a sustained virological response (SVR)
indicative of a cure [5,6]. However, for genotype 2 and
3 patients the SVR rates can be as high as 75%. It is also
known that subpopulations which include individuals of
African ancestry tend to respond less well to IFN/RBV
treatments [7]. Recent genome-wide association studies
have shown that a single nucleotide polymorphism 3kb
upstream of the IL28B gene correlates with a significant
difference in response to IFN therapy [8]. IL28B which
encodes the type III interferon IFN-λ−3, is known to be
upregulated by IFNs and by RNA viral infections. It has
been shown that HCV patients who harbour a TT or TC
allele in their IL28B gene tend to respond less well to IFN/
RBV treatment than do those having the CC genotype.
23
MJ Sofia
Figure 1. Model of HCV NS5B RNA complex
RNA
template
IN
Nucleotide
IN
Thumb
Fingers
Palm
RNA duplex
OUT
Ribbon structure of the HCV NS5B RNA dependent RNA polymerase showing the
Palm, Finger and Thumb domains typical of a RNA polymerase. Also depicted is a
bound template-primer strand of RNA and the nucleotide binding site.
Patients who choose to undergo IFN/RBV therapy face
not only the possibility of not responding to treatment
but also must contend with the potential for multiple and
sometimes serious side-effects that include influenza-like
symptoms, fatigue, hemolytic anaemia and depression.
The intolerable side effects can result in a high rate of
drug discontinuations. Consequently, the modest cure
rates and subpopulation differences combined with the
side-effect profile for SOC have prompted an urgency to
develop alternative novel, safe and effective therapies.
As in the case of other viral diseases, the development
of direct acting antivirals (DAAs) has become a focus.
Development of small molecule agents to attack essential viral proteins has the potential benefit of reducing
toxicities and side effects associated with manipulating
host functions and hopefully such DAA therapies will
not have the intolerable side effects exhibited by current SOC.
The push to identify small molecule DAAs has also
prompted the discussion around the possibility of eliminating or at least reducing the use of IFN/RBV from
treatment regimens. Although clinical development of
first generation DAAs has focused on combinations
with SOC in the hope of both shortening duration of
treatment and increasing the cure rate, the long-term
desire is to completely eliminate the use of IFN/RBV
from treatment regimens. Clearly this is an aspirational
goal and a goal that can only be achieved if an immune
component of therapy is not absolutely required to
eliminate those vestiges of undetectable virus [9].
In addition, such a lofty goal can be realised if small
24 molecule DAAs either alone or more probably in combination can drive viral loads to undetectable limits and
maintain those undetectable levels over the necessary
course of therapy and after cessation of treatment without having viral breakthrough resulting from the emergence of resistant virus. As has been shown with HIV
highly active antiretroviral therapy (HAART), the HCV
treatment paradigm will likely require combinations of
anti-HCV agents [7]. The desire is to suppress virus as
rapidly and completely as possible in order to give the
body’s natural immune system the opportunity to clear
residual virus and to hold back emergence of resistant
virus. However, what will comprise those ideal combinations of DAAs is yet to be determined and studies
to clarify this question are under active discussion and
investigation.
HCV has a 9.6 kb genome of positive-stranded RNA.
This genome encodes a precursor polyprotein that is
processed into 10 functional proteins: three structural
proteins and seven non-structural proteins [10]. Several of the non-structural proteins have been the focus
of intensive efforts to identify small molecule DAA
agents as inhibitors of HCV replication. Of the seven
non-structural proteins, molecules that inhibit the functions of the NS3/4 protease, NS4A, NS4B, NS5A and
NS5B RNA dependent RNA polymerase (RdRp) have
advanced to the clinic [11–15]. The most advanced
agents are the NS3/4 protease inhibitors telaprevir and
boceprevir. Each of these compounds has completed
Phase III clinical investigation and both have been
shown to be efficacious in treating HCV infection when
given in combination with SOC. However, each of these
first generation protease inhibitors suffers from the lack
of genotype coverage, undesired side effects, that may
limit their usage, and the early emergence of resistant
virus. It is therefore not surprising that even with the
potential benefits of these first generation DAAs, warehousing of patients by physicians occurs in order to wait
for approval of more effective and tolerable agents.
The HCV RNA dependent RNA polymerase is an
~68 kd protein that has the typical palm-finger-thumb
structural motif found in many viral polymerases (Figure 1) [16,17]. HCV polymerase is an essential enzyme
involved in RNA replication. Phylogenetic analysis
shows a 65% homology of HCV RdRp across genotypes
and an 80% homology within a particular genotype [3].
The HCV polymerase active site is located in the palm
domain where the conserved aspartic acid residue-containing GDD motif is located [18]. This conserved GDD
motif is common to viral polymerases in general [18].
Through a divalent metal ion (Mg++ or Mn++) the GDD
motif functions to coordinate the binding of the ribonucleoside triphosphate. The HCV polymerase catalyzes
the addition of a single ribonucleoside triphosphate
monomer to the 3′-end of the growing RNA chain by the
©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
formation of a 3′,5′-phosphodiester linkage. To accomplish this process, the polymerase must simultaneously
bind a template RNA strand, a primer RNA strand and
a ribonucleoside triphosphate monomer [19,20]. Therefore, investigation of nucleoside analogues is a rational
choice for the development of inhibitors of the HCV
NS5B polymerase.
Of all the DAAs under clinical investigation, nucleoside/nucleotide NS5B polymerase inhibitors hold the
promise of pan-genotype coverage and a high barrier
to development of resistant virus. As in the case of HIV
infection where nucleosides have become the backbone
of therapy (for example, TRUVADA® and Combivir®),
HCV nucleosides/nucleotides are positioned to assume
a similar role. To date, only nucleosides/nucleotides
have demonstrated broad genotype coverage both in
the laboratory and in human clinical studies [21]. In
addition, to date, no pre-emergent resistant virus has
been detected in clinical studies [22]. It is for these
reasons that nucleosides/nucleotides are positioned to
play a prominent role in developing HCV treatment
paradigms.
The HCV polymerase has been shown to be a
uniquely selective polymerase as it relates to the development of nucleoside/nucleotide inhibitors. Over
the last 10 years only two broad classes of nucleosides have emerged as inhibitors of this polymerase
[23–25]. These include the 2′-methyl and the 4′-azido
classes (Figure 2). However, these classes and subgroups within these classes have clearly differentiated
themselves in preclinical and clinical studies. This
differentiation is exemplified in their viral selectivity,
viral resistance, overall safety and clinical efficacy profiles. Resistance associated with the 2′-methyl class of
nucleosides is associated with the S282T amino acid
alteration located in the finger domain of the HCV
polymerase [26–29]. This mutation has been shown to
be difficult to raise in vitro and has not been detected
as a pre-existing mutation in clinical isolates [22].
Similarly, for the 4′-azido class, the S96T amino acid
alteration has been identified in vitro but has not been
observed in the clinic [27].
Implementation of a prodrug strategy has played
a prominent role in the development of a number of
nucleosides and nucleotides for the treatment of HCV
infection [23,30]. These prodrugs have been developed
to overcome, not only, bioavailability and stability
issues but also to address key anabolism limitations
important to nucleoside activation. Simple ester prodrugs of both the 2′-methylcytidine, 2′-α-fluoro-2′-β-Cmethylcytidine and 4′-azidocytidine nucleosides were
developed to overcome both bioavailability issues and
to curb undesirable metabolism [21,31,32]. Prodrugs of
the phosphate group of nucleoside 5′-monophosphates
were developed to address not only bioavailability
Antiviral Chemistry & Chemotherapy 22.1
Figure 2. Two major classes of nucleosides that are known to
be inhibitors of HCV polymerase.
2′-Methyl class
BASE
O
HO
HO
X
X = OH, F
4′-Azido class
HO
BASE
O
N3
HO
X
X = OH, F
These classes include the 2′-methyl and the 4′-azido ribosides.
issues but also poor in vitro and in vivo conversion of the parent nucleoside to the active nucleoside
5′-triphosphate [33]. Because nucleosides must be converted to their 5′-triphospates to be active as inhibitors
of the HCV polymerase, they need to undergo a series
of phosphorylation steps catalyzed by three separate
kinases (Figure 3). These kinases convert the nucleoside
first to the monophosphate, then to the diphosphate
and finally to the active triphosphate. However, it is
not uncommon that in the phosphorylation cascade, a
nucleoside or its corresponding mono- or diphosphate
is a poor substrate for one of the kinases. In particular,
it is the first kinase in the phosphorylation cascade that
is generally the most substrate selective. Therefore, it
is not unusual that bypassing the first kinase results in
achieving high levels of the active triphosphate. Because
nucleoside monophosphates are enzymatically dephosphorylated and negatively charged, they do not readily
enter cells and therefore are not desirable as drug candidates. To overcome the limitations of administering
a nucleoside monophosphate-containing agent, prodrugs of the 5′-monophosphate nucleoside have been
employed. Prodrugs of nucleoside monophosphates
have been known for many years and a number of
phosphate prodrug strategies have been developed to
address the need to deliver a 5′-monophosphate nucleoside into the cell [33–35]. However, there have been few
examples where a nucleotide prodrug has been shown
to deliver the corresponding 5′-monophosphate in vivo
to the desired site of action [33]. Often the prodrug
moiety decomposes prior to achieving its objective
because of either chemical or enzymatic instability in
the gastrointestinal tract and/or plasma.
The development of a nucleoside phosphate prodrug useful for the treatment of HCV faces several challenges. The nucleoside phosphate prodrug must have
sufficient chemical stability to be formulated for oral
25
MJ Sofia
Figure 3. Nucleoside kinase activation pathway
HO
O
BASE
BASE
O
Kinase 1
X
HO
O
Kinase 2
BASE
X
Y
HO
Y
O
Kinase 3
X
HO
BASE
X
HO
Y
= Phosphate
Y
Nucleoside kinase activation pathway resulting in the nucleoside triphosphate which is the active substrate for a polymerase allowing incorporation of the nucleoside
or nucleoside analogue into the growing RNA chain and thus resulting in inhibition of virus replication. Examples of kinases 1, 2, and 3 include deoxycytidine kinase
(dCk), nucleoside monophosphate kinase (YMPK) and nucleoside disphosphate kinase (NDPK), respectively.
administration. It must be stable to conditions of the
gastrointestinal tract such that the prodrug reaches the
site of absorption intact. The prodrug must have good
absorption properties and must not undergo appreciable enzymatic degradation during the absorption phase.
Once absorbed, the prodrug needs to have sufficient stability in the blood in order to reach the target organ: for
example the liver in the case of HCV. The prodrug must
then be transported into hepatocytes and release the free
5′-monophosphate nucleoside which can subsequently
be converted to the active triphosphate derivative. Since
HCV is a disease of the liver, and the liver is the first
organ the prodrug encounters after absorption, HCV is
an ideal disease for which to develop a targeted nucleotide prodrug strategy. Consequently, several of these
nucleoside monophosphate prodrugs have advanced
to the clinic and have demonstrated proof of concept
for treating HCV. Here, the application of phosphate
prodrugs in the development of nucleotide inhibitors
of HCV and the status of nucleotide prodrugs under
investigation for the treatment of HCV infection will
be reviewed.
Nucleotide phosphoramidates
Nucleotide phosphoramidates were first disclosed
by McGuigan et al. [36] as a prodrug strategy to
deliver a nucleoside 5′-monophosphate for the treatment of HIV and cancer. The structure of a nucleotide phosphoramidate typically consists of a nucleoside 5′-monophosphate where the phosphate group
is masked by appending an aryloxy group (usually a
phenol) and an α-amino acid ester (Figure 4); however, other related constructs have also appeared. The
26 phosphate group is ultimately revealed by a sequence
of enzymatic and chemical steps that requires either
carboxyesterase or cathepsin A to cleave the terminal
amino acid ester, intramolecular displacement of the
phosphate phenol and then enyzmatic cleavage of the
amino acid moiety by a phosphoramidase or histidine
triad nucleotide-binding protein 1 (HINT 1) [37–39].
It is believed that the phosphoramidate prodrug
construct increases lipophilicity of the nucleoside
5′-monophosphate and therefore increases cellular
permeability and ultimately intracellular nucleotide
concentrations. Since the phosphoramidate prodrug
moiety contains a chiral phosphorus centre, issues
arise with regard to development of a compound that
consists of a mixture of isomers with implications
arising from differential activity of each of the isomers, pharmacokinetics and manufacturing optimization, etc. In addition, the typical phosphoramidate
contains a phenolic substituent that is released during
metabolism to the free monophosphate. Successful
development also considers the metabolic release of
this phenolic substituent. Selective examples employing the phosphoramidate strategy to achieve kinase
bypass for nucleoside inhibitors of HIV reverse transcriptase (RT) inhibitors, for example, ddA, d4T and
d4A, showed that in vitro whole cell enhancement
in potency could be achieved [40–42]. Although the
phosphoramidate strategy was explored extensively to
deliver nucleotides for the treatment of HIV and colon
cancer [33,36], proof of concept in the clinic has yet
to be reported. However, the phosphoramidate prodrug approach has proven to be a valuable strategy in
the development of HCV nucleotide therapy.
©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
Figure 4. The phosphoramidate prodrug decomposition pathway that results in the release of the nucleoside 5’-monophosphate
O
O
R2
HN
R1
R2
O
O
P
O
Carboxyesterase
Nucleoside
O
or
Cathepsin A
O
HN
H
O
P
O
Nucleoside
O
Ar
Ar
Nucleotide phosphoramidate
Spontaneous
H
N
R2
O
P
O
H20
Nucleoside
O
O
O
O
R2
HN
H
O
P
O
Nucleoside
O
HINT-1
(Phosphoramidase)
O
HO
P
O
Nucleoside
OH
H
2′-C-Methyl ribonucleotide phosphoramidates
The 2′-C-methylcytidine nucleoside, NM107 (1; Figure 5), was shown to be an inhibitor of HCV in cell
culture (50% effective concentration [EC50] =1.23 µM)
and its triphosphate (3) was demonstrated to be a
potent inhibitor of the HCV polymerase enzyme (50%
inhibitory concentration [IC50] =0.09–0.18 µM) acting
as a nonobligate chain terminator [43]. NM107 also
showed broad antiviral activity against not only HCV
but also bovine virus diarrhoea virus (BVDV), yellow
fever virus, dengue virus and West Nile virus [31]. To
overcome bioavailability issues the 3′-valinate ester
prodrug, NM283 (valopicitabine; 2) [31,44], was taken
into clinical development. In a Phase I monotherapy
study, NM283 demonstrated proof of concept delivering an ~1.2 log10 IU/ml reduction in viral load at a dose
of 800 mg twice daily given over 14 days. Unfortunately, NM283 was discontinued because of significant
gastrointestinal toxicity in Phase II studies [24,30].
Subsequent work on the development of the 2′-Cmethyl class of nucleosides focused on 5′-phosphate
nucleotide prodrugs. It was observed that the 2′-Cmethylcytidine triphosphate (3) was highly active as
an inhibitor of the NS5B polymerase, yet the parent
Antiviral Chemistry & Chemotherapy 22.1
nucleoside NM107 (1) was only modestly active in the
whole cell based replicon assay. Studies had shown that
NM107-triphosphate (3) formation was inefficient,
particularly because of poor conversion of the nucleoside to its monophosphate by 2′-deoxycytidine kinase
[45]. Consequently, to circumvent this phosphorylation problem and potentially improve the therapeutic
index by increasing nucleoside triphosphate levels in
the liver, a phosphoramidate prodrug approach was
investigated [45]. This effort lead to the identification
of phosphoramidate derivative 4 (Figure 5; EC50≤0.5
µM) showing substantial increases in potency relative to NM283 [45]. The activity of compound 4 correlated with the levels of triphosphate produced in
human hepatocytes and these levels were shown to be
much higher than that seen with NM283 (2). In vivo
studies assessing liver nucleoside triphosphate levels
after oral administration in hamsters showed low triphosphate concentrations only twofold higher than
obtained with NM283. Since substantial liver triphosphate levels were seen after subcutaneous administration in vivo and the compounds were shown to
be stable in simulated gastric fluid, it was concluded
that low oral bioavailability or metabolic degradation
27
MJ Sofia
Figure 5. 2′-C-Methylcytidine nucleosides and nucleotide phosphoramidate prodrug inhibitors of HCV replication
NH2
NH2
HO
N
O
HO
N
HO
O
NH2
O
O
OH
N
N
O
OH
O
NM283
2
NM107
EC50=1.23 µM
1
NH2
O
HO
O
P
O
OH
O
P
O
OH
P
O
N
N
O
O
OH
HO
OH
IC50=0.09–0.18 µM
3
NH2
NH2
O
O
O
N
H
P
O
O
N
N
O
O
O
HO
O
O
N
H
P
O
O
O
O
HO
OH
EC50=0.22 µM, CC50=7 µM
4
N
N
OH
EC50=0.24 µM
5
NH2
NH2
O
O
O
N
H
P
O
O
N
N
O
OH
HO
O
O
O
N
H
P
O
N
N
O
OH
OH
EC50=8.2 µM, CC50=>100 µM
NTP (human hepatocytes) AUC(0–4h)=1,720 µM•h
6
O
HO
OH
NTP (human hepatocytes) AUC(0–4h)=190 µM•h
7
AUC, area under the curve; CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration.
28 ©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
in the intestine was the reason for the lack of oral
efficacy [45].
Another series of 2′-C-methylcytidine phosphoramidate prodrugs having an acyloxyethylamino phosphoramidate promoiety was studied (Figure 5) [46]. The
phosphoramidate prodrug 5 provided up to a 30-fold
improvement in HCV replicon potency over NM283
(2), and it was also shown that this activity correlated
to levels of nucleoside triphosphate in rat and human
hepatocytes. However, when administered orally to rats
these phosphoramidates demonstrated no improvement
in production of triphosphate in rat liver relative to
NM283 (2). These results put into question the oral
bioavailability and conversion of these prodrugs to the
nucleoside triphosphate in the liver.
Phosphoramidate monoesters of 2′-C-methylcytidine
were also explored (Figure 5) [45]. In this case the amidate moiety was either an α-amino acid (6) or an acyloxyethylamino substituent (7). Although phosphoramidate monoester 6 was shown to have inferior replicon
potency relative to its phenolic ester counterpart (<200fold) it showed higher levels of triphosphate formation
in human hepatocytes. Formation of nucleoside triphosphate levels were observed in hepatocytes of various species for both 6 and 7 but the phosphoramidate monoester
6 was shown to be superior to 7. In both cases, liver levels
of nucleoside triphosphate 3 were achieved in vivo after
subcutaneous administration but not after oral administration suggesting a lack of oral bioavailability.
Other 2′-C-methyl nucleosides containing purine
bases were also investigated as inhibitors of HCV,
including the 7-deaza-2′-C-methyladenosine derivative
MK0608 (8) which showed potent inhibition of HCV
replication in vitro (EC50=0.25 µM) and demonstrated
SVR in an HCV-infected chimpanzee animal model
[47,48]. Although MK0608 (8) was never progressed
into clinical development, it did demonstrate the potential of purine nucleosides as inhibitors of HCV. Subsequently, phosphoramidate prodrugs of 2′-C-methyl
purine analogues were studied to determine if improvement in potency could be achieved by kinase bypass.
Although application of the phosphoramidate prodrug
strategy was not successful for the adenosine derivative
11 (Figure 6), its application to the guanosine analogue
12 resulted in an 84-fold increase in activity in the replicon assay relative to the parent nucleoside (Figure 7)
[49]. This result was rationalized by the observation
that the guanosine triphosphate (13; IC50=0.13 µM)
was more potent as an inhibitor of the HCV polymerase than the adenosine triphosphate (10; IC50=1.9
Figure 6. 2′-C-Methyladenosine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
NH2
HO
O
HO
N
N
HO
N
OH
O
HO
P
OH
O
O
P
OH
O
O
P
O
O
N
OH
HO
N
N
N
OH
EC50=0.3 µM, CC50=>50 µM
9
NH2
N
N
N
O
HO
EC50=0.25 µM, CC50=>50 µM
8
NH2
N
CH3
EtO
O
OH
IC50=1.9 µM, EC50=0.3 µM
10
N
H
O
P
NH2
N
O
O
N
O
HO
N
N
OH
EC50=0.25 µM, CC50=>50 µM
11
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration.
Antiviral Chemistry & Chemotherapy 22.1
29
MJ Sofia
Figure 7. 2′-C-Methylguanosine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
O
N
HO
N
O
HO
O
NH
N
HO
NH2
O
P
O
OH
O
P
O
P
OH
R2
R1O
OH
O
P
O
N
O
HO
N
NH
N
NH2
OH
IC50=0.13 µM
13
O
N
O
O
HO
O
HN
O
OH
EC50=3.5 µM
12
O
N
CH3
NH
N
O
NH2
O
HN
O
P
O
HO
14 R1=CH2CH2Ph, R2=Ala EC50=0.08 µM, CC50=>100 µM
15 R1=o-CIPh, R2=Val EC50=0.43 µM, CC50=>100 µM
P
N
N
N
NH2
OH
INX-08189
EC50=0.010 µM, CC50=7 µM
16
O
N
O
HN
O
O
N
O
O
O
O
OH
OCH3
N
S
HO
NH
N
NH2
OH
OH
IDX184
EC50=0.4 µM, CC50>100 µM
17
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration.
µM), yet in the whole cell replicon assay the adenosine
analogue (9; EC50=0.3 µM; Figure 6) was more potent
than the guanosine analogue (12; EC50=3.5 µM; Figure
7). Therefore, this finding coupled with the observation that low levels of triphosphate were detected in
cells for the guanosine nucleoside relative to that for
the adenosine nucleoside hinted at poor phosphorylation in the case of the guanosine nucleoside. The development of the 2′-C-methylguanosine phosphoramidate
derivative was further explored by systematically evaluating structural modifications to the phosphoramidate moiety in an effort to achieve increased HCV replicon potency, plasma stability across multiple species,
and appropriate relative stability in intestinal and liver
S9 preparations. Ultimately, these studies were able
to identify 2′-C-methylguanosine phosphoramidate
30 prodrugs 14 and 15 (Figure 7) [50]. These prodrugs
contained benzyl or alkyl l-alanine and l-valine amino
acid ester moieties and a naphthyl phosphate ester and
exhibited a 10–30-fold enhancement in HCV replicon potency with acceptable plasma and intestinal S9
stability suitable for progression into in vivo studies.
However, when mice were dosed orally in order to
assess liver levels of the nucleoside triphosphate 13, the
2′-C-methylguanosine phosphoramidates 14 and 15
(Figure 7) produced substantial liver levels of triphosphate but these levels were not significantly improved
over that observed when mice were dosed with the parent guanosine nucleoside 12.
Further investigation of the 2′-C-methylguanosine
phosphoramidate series led to the evaluation of substitution at the C-6-position of the guanosine base with the
©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
intention of increasing lipophilicity and thus improving
cellular uptake relative to the natural guanosine derivative 12. This led to the identification of the C-6-O-methyl
derivative INX-08189 (16; Figure 7) containing both a
neopentyl ester on the l-alanyl amino acid moiety and
a naphthyl ester on phosphorus of the phosphoramidate [51]. In INX-08189 (16), in addition to metabolic
conversion of the phosphoramidate pro-moiety to the
5′-monophosphate, the 6-O-methyl group of the purine
base is metabolized to the guanine base. INX-08189
(16) demonstrated exceptional potency in the HCV 1b
replicon assay (EC50=0.01 µM; CC50=7 µM), was active
against genotype 1a and 2 replicons and produced substantial levels of the guanosine triphosphate 13 in primary human hepatocytes over 48 h. The known 2′-Cmethyl nucleoside S282T mutant replicon was shown
to be moderately resistant (3- to 10-fold) to INX-08189
(16) [52]. No difference in HCV replicon potency was
observed for each of the individual diastereoisomers of
INX-08189 (16), thus INX-08189 was advanced into
clinical development as a mixture of isomers at the
phosphorus centre of the prodrug.
In a single-ascending-dose Phase Ia study in healthy
volunteers administered doses ranging from 3 mg to
100 mg, INX-08189 (16) was shown to be generally
well tolerated at all doses with no drug-related serious
adverse events and pharmacokinetics (PK) supporting
once daily oral dosing [53,54]. INX-08189 was progressed into a Phase Ib study in treatment-­naive genotype 1 HCV patients dosed once daily at either 9 mg
or 25 mg. Antiviral activity was observed with a mean
HCV RNA reduction of -0.71 and -1.03 log10 IU/ml,
respectively [54,55]. These early human data demonstrated clinical proof of concept for INX-08189 and
consequently, the compound is continuing to undergo
clinical evaluation.
Another application of phosphoramidate ­prodrug
technology for delivering a 2′-C-methylguanosine
5′-monophosphate is exemplified by IDX184 (17; Figure
7) [56]. In this case the phosphoramidate prodrug moiety utilized a benzyl amine in place of an amino acid for
the amine substituent and an S-acetyl-2-thioethyl moiety
(SATE) as the phosphate ester substituent in place of the
more common aryl group. The SATE group is a known
phosphate ester prodrug construct [33,57,58]. Mechanism for release of the desired 2′-C-methylguanosine
5′-monophosphate is believed to involve both CYP450dependent and independent processes. Based on the prodrug structure one would anticipate that prodrug release
would involve cleavage of the terminal thioester followed by loss of the phosphate ester via intramolecular
attack of the free thiol group releasing the phosphate and
an equivalent of episulfide, then enzymatic cleavage of
the benzyl phosphoramide. However, a detailed mechanism for the IDX184 (17) prodrug cleavage has not been
Antiviral Chemistry & Chemotherapy 22.1
reported to date. In the whole cell HCV replicon assay
IDX184 (17) was shown to be a potent inhibitor of HCV
replication (EC50=0.4 µM; CC50>100 mM) and was also
active in the genotype 2a JFH1 replicon (EC50=0.6–11
µM) [59]. At a concentration of 2.5 µM it cleared HCV
replicon RNA after 14 days of treatment. Like other
2′-C-methyl nucleosides it was shown to select for the
NS5B S282T mutation in the HCV replicon. In combination with the protease inhibitor IDX320, IFN-α, or
RBV, IDX184 exhibited an additive or synergistic profile
[60]. When administered orally to cynomolgus monkeys,
IDX184 (17) produced high live triphosphate levels
relative to oral administration of the parent nucleoside
with what appears to be high hepatic extraction. Subsequent studies in a HCV-1-infected chimpanzee model
showed that oral administration of IDX184 (17) at 10
mg/kg over 3 days produced a median viral load decline
of approximately -2.3 log10 at day 3 and 4 [61]. Consequently, IDX184 (17) was progressed into the clinic and
in a Phase Ia single ascending dose study was shown to
be generally safe and well tolerated at oral doses from 5
mg to 100 mg [56]. PK assessment supported a liver targeting mechanism for the compound. In a Phase Ib monotherapy study in HCV genotype-1-infected patients,
IDX184 (17) was administered at doses from 25 to 100
mg once a day for 3 days. Day 4 viral load assessment
showed that at the highest dose of 100 mg, a -0.74 log10
IU/ml reduction in viral load was observed [62]. Following the positive Phase I clinical results, a Phase II
study was initiated in which IDX184 (17), at doses from
50–200 mg, was combined with pegylated IFN and RBV
for 14 days in treatment-naive HCV genotype 1 patients
[63]. At day 14 viral load reductions of -2.7 to -4.1 log10
IU/ml were achieved with no adverse events attributed
to IDX184 (17) and with no detection of resistant virus
[64]. However, in an attempt to evaluate the efficacy
of a DAA combination of IDX184 (17) with the NS3
protease inhibitor IDX320, three serious adverse events
were reported in a drug–drug interaction study in healthy
volunteers and consequently, the programme was placed
on clinical hold in September 2010, awaiting resolution
of the cause of the adverse events [65,66]. In February
2011, the US Food and Drug Administration (FDA)
removed full clinical hold for IDX184 (17) owing to
evidence that the toxicity was likely caused by IDX320,
and the programme was placed on partial clinical hold.
Initiation of a Phase IIb 12-week trial of IDX184 (17) in
combination with pegylated IFN and RBV is anticipated
in the second half of 2011 [67].
4′-Azido ribonucleotide phosphoramidates
The 4′-C-azidocytidine nucleoside R1479 (18; Figure 8)
was reported to be an inhibitor of HCV replication
(EC50=1.28 µM) and subsequently its 2′,3′,5′-tri-Oisobutryate ester prodrug R1626 (balapiravir; 20) was
31
MJ Sofia
Figure 8. 4′-Azido cytidine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
NH2
HO
O
O
N
N
NH2
HO
O
N3
HO
P
O
O
OH
P
O
O
P
OH
O
O
O
OH N3
HO
OH
EC50=1.28 µM, CC50=>100 µM
R1479
18
N
N
OH
IC50=0.32 µM
19
NH2
NH2
O
O
O
N
N
O
O
N3
O
Ph
O
O
O
N
H
P
O
O
O
O
N3
HO
O
N
N
OH
O
CH3
R1626
EC50=0.51 µM, CC50=>100 µM
20
21
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration.
taken into clinical development as a treatment for HCV
infection [32,68]. R1479 was shown to have reduced
activity against the S96T HCV polymerase mutant
[27]. In a Phase Ib monotherapy clinical study in HCVinfected patients dosed with 500, 1,500, 3,000 and
4,500 mg twice daily for 14 days, R1626 (20) demonstrated a mean decrease in viral load of -0.3, -1.2, -2.6
and -3.7 log10 IU/ml, respectively, thus providing proof
of concept for this class of nucleosides. R1626 was subsequently taken into a Phase IIa study in genotype 1
treatment-naive patients where at a dose of 1,500 twice
daily in combination with IFN/RBV a -5.2 log10 IU/ml
reduction in viral load with an 81% rapid virological
response (RVR) was observed after 28 days of therapy
[30]. However, dosing was limited by neutropenia and
in a Phase IIb study in combination with IFN/RBV, the
development of R1626 (20) was discontinued as a result
of significant haematological adverse events [69].
In an effort to increase the potency of R1479 (18),
a 5′-phosphoramidate nucleotide prodrug strategy
32 was investigated [70]. It was hoped that the increased
lipophilicity introduced by the phosphoramidate group
might result in enhanced passive diffusion and therefore, increased intracellular levels of the monophosphate and ultimately of the active triphosphate thus
leading to a significant in vitro and in vivo advantage.
In addition, if liver targeting was possible, as was
described for other nucleotide prodrugs, there existed
a possibility that the liabilities associated with R1479
(18) could be ameliorated by limiting systemic exposure. After an extensive structure–activity relationship (SAR) study, it was noted that preparation of the
5′-phosphoramidate derivative 21 (Figure 8) did not
provide any appreciable improvement in potency over
the nucleoside R1479 (18), indicating that this nucleoside is already efficiently phosphorylated and that
application of the phosphoramidate technology was
not able to boost its potency [70].
Further evaluation of the 4’-azido pyrimidine class
of nucleosides investigated the 4′-C-azidouridine
©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
Figure 9. 4′-Azido uridine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
O
O
HO
O
HO
O
N3
HO
O
NH
N
O
P
O
OH
P
O
O
P
OH
O
O
HO
EC50=>100 µM, CC50=>100 µM
22
O
OH N3
OH
NH
N
OH
IC50=0.22 µM
23
O
Ph
O
O
O
N
H
P
O
O
O
N3
HO
NH
N
O
OH
EC50=0.22 µM, CC50=>100 µM
24
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration.
derivative 22 (Figure 9) [71]. It was shown that the 4′-Cazidouridine (22) was inactive as an HCV inhibitor as
determined in the cell-based replicon assay. However its
5′-triphosphate analogue (23) demonstrated inhibition
of the HCV polymerase enzyme (IC50=0.22 µM) similar
to that of the triphosphate 19 (IC50=0.32 µM) of the
cytidine derivative 18. Speculating that poor substrate
activity for cellular kinases was contributing to the lack
of whole cell activity of the 4′-C-azidouridine (22), a
series of 5′-phosphoramidate prodrugs was prepared.
SAR around the phosphoramidate moiety showed that
the nature of the substituents on the phosphoramidate
had a dramatic effect on activity in the HCV whole cell
assay. Since the compounds were prepared as a mixture
of diastereomers at the phosphorus centre of the phosphoramidate moiety, the diastereomers were separated
chromatographically but no difference in potency was
observed. Improvements in activity of >450-fold were
reported, with the most active compound in the series
having the 1-naphthyl l-alanine benzyl ester phosphoramidate (24) substitution pattern (EC50=0.22 µM). To
date, no reports have appeared that indicate further
progression of a 4′-C-azidouridine 5′-phosphoramidate
prodrug beyond in vitro studies.
Antiviral Chemistry & Chemotherapy 22.1
In the 4′-azido riboside class of nucleosides, the
phosphoramidate prodrug strategy was also applied to
the adenosine derivative 25 (Figure 10) [72]. As in the
case of the 4′-C-azidouridine, the 4′-C-azidoadenosine
(25) was shown to be inactive in the HCV replicon
assay with the assumption that the lack of activity was
due to its inability to be phosphorylated by cellular
kinases. It was hypothesized that delivering a monophosphate prodrug derivative would both bypass the
non-productive monophosphorylation step and also
reduce the extent of intracellular metabolism by deamination. Preparation of a series of phosphoramidate
derivatives led to the identification of several compounds, like compound 27, that showed submicromolar EC50 values (0.22–0.59 µM) in the HCV replicon
assay and were devoid of cellular toxicity; however,
no additional data was provided to support protection
from deamination nor translation of the in vitro data
into an in vivo setting.
2′-α-Fluoro-2′-β-C-methyl nucleotide phosphoramidate
prodrugs
In 2005, the first disclosure of a 2′-α-fluoro-2′-βC-methylcytidine nucleoside was made with the
33
MJ Sofia
Figure 10. 4′-Azido adenosine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
NH2
N
HO
O
N
N3
HO
O
N
HO
N
O
P
O
O
P
OH
O
P
OH
O
O
HO
Ph
N
N
N
OH N3
OH
EC50=>100 µM, CC50=>100 µM
25
NH2
N
OH
IC50= Not determined
26
O
O
N
H
O
P
O
NH2
N
O
N
N
O
N
N3
HO
OH
EC50=0.22 µM, CC50=>100 µM
27
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration.
Figure 11. 2′-α-F-2′-β-C-Methylcytidine nucleoside inhibitors of HCV replication
NH2
HO
O
HO
N
NH2
O
N
HO
O
O
P
O
OH
O
P
O
OH
P
O
O
HO
N
O
OH
F
EC90=4.5 µM
PSI-6130
28
N
F
IC50=0.34 µM
29
NH2
O
O
O
O
O
N
N
O
F
RG7128
EC50=0.32–0.98 µM
30
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; EC90, 90% effective concentration; IC50, 50% inhibitory concentration.
34 ©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
publication of PSI-6130 (28; Figure 11) [73,74]. This
class of nucleosides demonstrated unique characteristics
in vitro that would eventually translate into the clinic.
PSI-6130 (28) was shown to be an inhibitor of HCV
replication in the cell-based replicon assay (90% effective concentration [EC90]=4.5 µM) and its triphosphate
(29) was shown to be a potent inhibitor of the HCV
NS5B polymerase (IC50=0.34 µM) with an intracellular
half-life of approximately 6 h. This class of nucleosides
demonstrated exquisite selectivity for HCV versus other
Flaviviruses, other RNA viruses as well as for HIV
and HBV. The class showed broad genotype coverage
inhibiting the HCV polymerase from genotypes 1 to 6.
Triphosphate 29 was also shown not to be an inhibitor of any of the human DNA or RNA polymerases.
PSI-6130 (28) demonstrated a safety profile that did
not exhibit any of the toxicology signals common to
nucleosides including bone marrow and mitochondrial
toxicity. Although the known 2′-C-methyl nucleoside
S282T amino acid alteration in the HCV polymerase
was shown to be resistant to PSI-6130 (28), the level of
resistance was demonstrated to be relatively minimal
(threefold) and difficult to raise in vitro [27,75]. However, in clinical studies PSI-6130 (28) delivered modest
oral bioavailability and a significant amount of the drug
was metabolized to an inactive uridine metabolite [21].
To rectify these shortcomings a prodrug was eventually
developed leading to the simple 3′,5′-di-O-isobutyrate
diester prodrug RG7128 (mericitabine; 30) [21].
RG7128 (30) demonstrated proof of concept in the
clinic when administered alone or in combination with
SOC [21,24,30]. In a Phase Ib multiple ascending dose
study in 40 genotype 1 non-responder HCV-infected
patients, RG7128 (30) was dosed at 750 mg and 1,500
mg either once daily or twice daily. The study demonstrated a dose-dependent reduction in HCV RNA levels
and also showed that twice daily dosing was preferred.
The 1,500 mg twice daily dose of RG7128 (30) resulted
in the maximum antiviral effect producing a -2.7 log10
IU/ml drop in viral load after 14 days of monotherapy.
Subsequently, two Phase IIa studies in combination
with SOC (Pegasys® and Copegus®) were initiated, one
in genotype 1 treatment-naives and another in genotype 2,3 non-responder HCV-infected patients. The
genotype 2/3 study was the first non-genotype 1 patient
study initiated with a DAA. In the Phase IIa study treating genotype 1 patients, doses of 500, 1,000 and 1,500
mg twice daily were investigated over 28 days. Study
results showed that the 1,000 and 1,500 mg doses provided the best viral load reduction and were essentially
equally effective producing RVRs of 85% and 88% and
viral load reductions of -5.05 and -5.09 log10 IU/ml,
respectively. In the genotype 2/3 patient study at a dose
of 1,500 mg twice daily over 28 days, a -5 log10 IU/ml
reduction in viral load was observed which translated
Antiviral Chemistry & Chemotherapy 22.1
into a 90% RVR. The results of the genotype 2/3 patient
study demonstrated for the first time that a DAA could
be effective in the clinic in this patient population and
corroborated the broad genotype coverage predicted by
the in vitro data. In both genotype 1 and genotype 2/3
patient studies there were no significant adverse events
reported and the safety was described as no different
than placebo with SOC.
In both the genotype 1 and genotype 2/3 Phase
IIa clinical studies where RG7128 (30) was dosed in
combination with SOC no viral breakthroughs were
observed over the course of therapy. Subsequent clonal
analyses showed that there was no identified NS5B
S282T variants in the treatment groups nor was there
any pre-existing S282T viral variants observed at baseline in the patient population studied [75]. These data
support the high barrier to resistance observed in the
preclinical studies for this nucleoside class.
RG7128 (30) progressed into a Phase IIb clinical study
evaluating 408 genotype 1 and 4 HCV patients when
dosed with 500 or 1,000 mg twice daily RG7128 (30)
in combination with SOC over 12 or 24 weeks assessing SVR as the primary end point. An interim 12-week
analysis was reported which showed an average 83%
complete early virological response (cEVR) for the 1,000
mg RG7128 12-week regimen and no evidence of drug
resistance [22,76]. RG7128 (30) is expected to complete
the Phase IIb study in the near term.
With the hope of demonstrating proof of concept
that the combination of two DAAs could lead to an
IFN-sparing clinical regimen, RG7128 (30) was combined with a protease inhibitor RG7227 (danoprevir) in
a 14-day clinical study in genotype 1 patients to assess
safety and viral kinetics [77]. The study demonstrated
that combination of the two DAAs was safe and well
tolerated and that at the highest dose levels (1,000 mg
RG7128 and 900 mg danoprevir) in both treatmentnaive and IFN-null responders, viral load declines of
-5.1 log10 IU/ml and -4.9 log10 IU/ml were observed.
This combination produced a viral decline that is quantitatively similar to that observed when each individual
agent is combined with IFN and RBV. These results
demonstrated that in the clinic the combination of a
nucleoside HCV polymerase inhibitor and a protease
inhibitor has the potential to provide a promising combination for future HCV therapy.
Metabolism studies on the cytidine nucleoside PSI6130 (28; Figure 11) showed that not only was the
nucleoside converted to its active triphosphate derivative 29, but that it was also converted to the uridine
nucleoside PSI-6206 (31; Figure 12) in vivo [78,79].
However, this uridine nucleoside was shown not to be
active as an inhibitor of HCV replication in the replicon assay. Subsequent studies indicated that the triphosphate of PSI-6206 (32; Figure 12) was a potent
35
MJ Sofia
inhibitor of the HCV NS5B polymerase with an
IC50=1.19 µM and that this triphosphate had a long
desirable intracellular half-life of 36 h [80]. Study of the
phosphorylation kinetics of the uridine nucleoside 31
showed that its lack of activity was related to the lack
of conversion to its 5′-monophosphate derivative and
that subsequent conversion of the 5′-monophosphate
to the ­corresponding 5′-diphosphate and -triphosphate derivatives was facile. In an attempt to take
advantage of the long intracellular half-life of the
uridine triphosphate 32 with the hope of potentially
developing a once-daily drug, a prodrug of the uridine
5′-monophosphate was pursued. Similar to the nucleosides discussed above, a phosphate prodrug approach
was investigated to overcome the enzymatic blockade
at the monophosphorylation step and effectively deliver
the uridine nucleoside 5′-monophosphate. A phosphoramidate prodrug strategy was implemented with the
expectation that first pass metabolism of the prodrug
moiety in the liver would achieve efficient delivery to
the liver of the uridine nucleoside 5′-monophosphate
[21,81,82]. Extensive SAR around the phosphoramidate moiety showed that the preferred substitution
to achieve submicromolar whole cell HCV replicon
potency without cellular toxicity required a small
branched alkyl ester at the terminal carboxylate ester
moiety, a small alkyl group at the alpha position of the
amino acid substitutent and a simple phenoxy or halogenated phenoxy phosphate ester group. To achieve the
required delivery of the prodrug to the liver and subsequent release of the nucleoside 5′-monophosphate in
hepatocytes, stability studies in simulated gastric fluids
(SGF) and simulated intestinal fluids (SIF) and stability on exposure to liver S9 fractions were carried out.
Several compounds provided the desired profile which
showed good stability in SGF, SIF and blood but a
short half-life in liver S9 fractions, thus supporting the
expectation that the phosphoramidate strategy could
enable liver targeting. The 2′-α-F-2′-β-C-methyluridine
5′-phosphoramidates were subsequently shown to generate high concentrations of the corresponding nucleoside triphosphate 32 in primary hepatocytes of rat,
dog, monkey and human, and eventually showed in rat
and dog PK studies that the phosphoramidates could
get to the liver and generate high levels of the uridine
nucleoside triphosphate 32 there. The lead compound
Figure 12. 2′-α-F-2′-β-C-Methyluridine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
O
HO
O
HO
O
O
NH
N
HO
O
P
OH
O
O
O
P
O
OH
P
O
O
O
OH
F
HO
NH
N
F
IC50=1.19 µM
PSI-6206
EC50=>100 µM
31
32
O
O
O
O
O
N
H
P
O
O
N
O
O
HO
NH
F
PSI-7851
EC90=0.52 µM, CC50=>100 µM,
33
O
O
O
N
H
P
O
O
N
O
O
HO
NH
F
PSI-7977
EC90=0.42 µM, CC50=>100 µM,
34
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; EC90, 90% effective concentration; IC50, 50% inhibitory concentration.
36 ©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
PSI-7851 (33) was subsequently shown to be devoid
of mitochondrial, bone marrow and general cellular
toxicities. Combination studies with either IFN and/
or RBV and other DAAs such as protease, non-nucleoside polymerase, or NS5A inhibitors showed that PSI7851 (33) was either additive or synergistic in its effect
on HCV replication in the replicon assay [83]. When
evaluated in the S282T mutant replicon known to be
resistant to 2′-C-methyl nucleosides, PSI-7851 (33)
demonstrated a 16.4-fold resistance relative to wild
type [83,84]. PSI-7851 (33), a 1:1 mixture of diastereomers at the phosphorus centre of the phosphoramidate moiety, was chosen for further development and
progressed into Phase I human clinical trials.
In a single ascending dose clinical study of healthy
volunteers receiving doses up to 800 mg once daily, PSI7851 (33) was demonstrated to be generally safe and
well-tolerated, with no dose-limiting toxicities. In addition, PK results exhibited a systemic exposure profile
that was described as consistent with rapid uptake of
the drug by the liver and low plasma exposure to the
prodrug PSI-7851 (33). PSI-7851 (33) was progressed
into a 3-day multiple ascending dose study in treatmentnaive, HCV-infected patients with the object of evaluating safety, PK and effect on viral load. HCV RNA levels
declined in a dose dependent manner with the maximal
mean change from baseline of -1.95 log10 IU/ml at the
400 mg dose. In addition, no pre-existing or treatmentemergent S282T resistant virus was detected, nor was
there evidence of viral resistance after therapy [21].
With proof of concept demonstrated in HCV
patients, further development of the 2′-α-F-2′-β-Cmethyluridne phosphoramidate proceeded using PSI7977 (34; EC90=0.42 µM) the more active (>10-fold)
single diastereomer of PSI-7851 (33) [81]. PSI-7977
(34) was obtained by direct crystallization from the
diastereomeric mixture and it was shown to have the
Sp stereochemistry at the phosphorus atom thus representing the first example of the crystallization and
x-ray structure determination of a nucleotide phosphoramidate. In a 28-day Phase IIa clinical study in
genotype 1 treatment-naive HCV patients, in which
PSI-7977 (34) was dosed at either 100, 200 or 400 mg
once daily in combination with pegylated IFN/RBV,
RVR rates of 88%, 94% and 93%, respectively, were
observed [85,86]. In addition, no viral resistance was
detected nor were there any significant adverse events
reported. Recently, a 14-day monotherapy study with
PSI-7977 (34) showed that HCV genotype 1 treatment-naive patients achieved an average -5.0 log10
decline in viral load with 88% of patients reaching
undetectability (<15 IU/ml) after 14 days. This result
is a significant improvement from what was observed
for the diastereomeric mixture PSI-7851 (33) [87].
PSI-7977 (34) has entered Phase IIb clinical trials and
Antiviral Chemistry & Chemotherapy 22.1
it is being evaluated in combination with IFN/RBV
for its effects in genotype 1, 2 and 3 patients. In genotype 1 patients treated with PSI-7977 (34), 47 of 48
patients (98%) at 200 mg once daily and 46 of 47
patients (98%) at 400 mg once daily achieved an RVR
at week 12 with no viral breakthrough and a promising safety profile [88]. In genotype 2 and 3 HCV
patients a 400 mg once daily dose of PSI-7977 (34)
in combination with IFN/RBV achieved an SVR 12
weeks after cessation of therapy in 96% of patients
treated [88]. In addition to the continued Phase IIb
study in combination with abbreviated durations of
IFN/RBV, PSI-7977 (34) is being studied in combination with other nucleosides (PSI-352938) and with
the NS5A inhibitor BMS-790052 [15,89].
Further structure–activity relationship studies around
the 2′-α-F-2′-β-C-methyl class of nucleosides had shown
that the guanosine derivative 35 (Figure 13) was weakly
active (EC90=69.2 µM) as an inhibitor of HCV in the
replicon assay; however, the guanosine triphosphate 36
was shown to be a good inhibitor of the HCV RNA
polymerase (IC50=5.94 µM; Figure 13) [90,91]. It was
also demonstrated that the guanosine triphosphate 36
was equipotent as an inhibitor of both the wild type and
S282T mutant HCV polymerase: the first example of
such a profile for a 2′-C-methyl nucleoside. In order to
try and improve potency of this guanosine nucleoside,
speculating that the first monophosphorylation was
limiting, preparation of phosphoramidate prodrugs of
the 2′-α-F-2′-β-C-methylguanosine 5′-monophosphate
along with modifications at the C-6 position of the
guanosine base were pursued [91]. This double prodrug strategy, where in addition to the metabolism of the
phosphoramidate, the C-6-O-methyl group of the base
is metabolized to give guanine, ultimately led to a series
of HCV inhibitors having low nanomolar potency with
acceptable in vitro safety and stability profiles. Selected
compounds were shown to produce high levels of the
guanosine triphosphate 36 in primary human hepatocytes and a radiolabelled study showed that a guanosine phosphoramidate derivative provided a desirable
3.5–4.8:1 liver to plasma ratio. The single Sp phosphoramidate diastereomer, PSI-353661 (37; EC90=8 nM;
Figure 13) was obtained by crystallization from the
diastereomeric mixture and was shown to be the more
potent of the two possible diastereomers. In vitro studies with IFN or RBV and other DAAs demonstrated
that PSI-353661 (37) could be combined to produce an
additive or synergistic antiviral effect [92]. PSI-353661
(37) was selected as a clinical development candidate
for the treatment of HCV infections and is awaiting the
start of clinical evaluation.
Another 2′-α-F-2′-β-C-methyl nucleoside having
a 7-ethynyl-7-deaza adenosine base unit (38; Figure
14) was reported to be an efficient inhibitor of the
37
MJ Sofia
Figure 13. 2′-α-F-2′-β-C-Methylguanosine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
O
N
HO
O
HO
O
NH
N
N
HO
NH2
O
P
O
O
P
OH
O
P
OH
O
NH
N
O
N
OH
F
HO
EC90=69.2 µM
35
O
N
NH2
F
IC50=5.94 µM
36
O
O
N
H
O
OMe
N
P
O
N
N
O
N
O
HO
NH2
F
PSI-353661
EC90=0.008 µM
37
CC50, 50% cytotoxic concentration; EC90, 90% effective concentration; IC50, 50% inhibitory concentration.
Figure 14. 7-Deaza-7-ethynyl-2′-α-F-2′-β-C-methyladenosine nucleoside and nucleotide phosphoramidate prodrug inhibitors of
HCV replication
NH2
NH2
HO
O
HO
O
N
N
HO
N
O
P
O
OH
P
OH
O
O
P
O
O
OH
F
HO
EC50=24 µM
38
N
N
N
F
IC50=0.4 µM
39
NH2
R
O
O
O
N
H
P
O
O
O
N
N
N
Ar
HO
F
R=Me, Ph
Ar=Ph, p-Me-Ph, p-F-Ph, p-MeO-Ph, 1-naphthyl, 2-naphthyl
No observed activity
40
EC50, 50% effective concentration; IC50, 50% inhibitory concentration.
38 ©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
Figure 15. C-6-Hydrazido sulfonamide 2′-C-methyladenosine and 2′-C-methyl-2-amino-adensosine nucleoside and nucleotide
cyclic phosphate prodrug inhibitors of HCV replication
HN
N
HO
HO
HN
N
HO
R1
P
O
41 R1=H EC50=300 µM, CC50>300 µM
42 R1=NH2 EC50=92 µM, CC50>300 µM
OH
O
N
O
O
SO2CH3
N
N
P
R1
43 R1=H EC50=100 µM
44 R1=NH2 EC50=23.8 µM
HN
HO
N
OH
HO
SO2CH3
N
N
O
OH
O
N
N
O
N
N
O
SO2CH3
N
N
N
R1
OH
45 R1=H EC50=68.5 µM
46 R1=NH2 EC50=1.7 µM
HN
N
N
O
O
O
n-Pr
O
S
O
P
O
O
N
SO2CH3
N
N
R1
OH
47 R1=H EC50=0.039 µM, CC50>50 µM
48 R1=NH2 EC50=0.008 µM, CC50>50 µM
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration.
HCV NS5B polymerase (IC50=0.4 µM) yet its activity in a whole cell Huh7 replicon assay was reported
to be low (EC50=24 µM) [93]. Consequently, a small
series of phosphoramidate prodrugs (40) of 2′-α-F2′-β-C-methyl-7-ethynyl-7-deaza adenosine (38) was
reported, however, no antiviral activity was observed
for these prodrugs and the series was not progressed
further [94].
Antiviral Chemistry & Chemotherapy 22.1
3′,5′-Cyclic phosphate nucleotide prodrugs
The use of a 3′,5′-cyclic phosphate prodrug construct
for delivering a nucleoside 5′-monophosphate into cells
is a relatively uncommon prodrug motif [95–97]. Its
application has only recently been selectively applied
in the development of nucleotide inhibitors for HCV
polymerase. Little is known about the mechanism by
which these cyclic phosphates are metabolized to the
39
MJ Sofia
Figure 16. 2′-C-Methylcytidine-3′,5′-cyclic phosphoramidate
prodrug inhibitor of HCV replication
NH2
O
O
N
N
O
O
P
NH
EtO
O
OH
O
Slow eluting diastereomer
EC50=4 µM, CC50=>100 µM
49
CC50, 50% cytotoxic concentration; EC50, 50% effective concentration.
Figure 17. O-6-Ethyl-2′-α-F-2′-β-C-Methylguanosine3′,5′-cyclic phosphate prodrug inhibitor (PSI-352938) of
HCV replication
OEt
N
O
O
O
P
O
O
N
N
N
NH2
F
PSI-352938
EC90=1.37 µM, CC50=>100 µM
50
CC50, 50% cytotoxic concentration; EC90, 90% effective concentration.
desired 5′-monophosphates; however, based on the
reported HCV data and recent clinical data [98], it is
clear that this approach is an effective prodrug strategy
for delivering in vivo a nucleoside 5′-monophosphate
into cells.
2′-C-Methyl nucleotide 3′,5′-cyclic phosphate prodrugs
The first reported example of a 3′,5′-cyclic phosphate
prodrug construct for inhibiting HCV was reported
in an attempt to develop a set of 2′-C-methyl ribonucleosides with modified purine bases where the
purine base has a hydrazido sulfonamide at the C-6
40 position (Figure 15) [99]. The parent nucleosides 41,
42 were essentially inactive (EC50≥92 µM) as inhibitors of HCV replication in the replicon assay and
subsequently were shown not to be substrates for
adenosine kinase, suggesting an inability to be phosphorylated to the respective monophosphates 43 and
44. Preparation of the 3′,5′-cyclic phosphate derivatives 45, 46 resulted in significant improvement in
replicon potency (EC50s=68.5 and 1.7 µM) relative
to both the 5′-monophosphate derivatives 43 and
44 (EC50s=100 and 23.8 µM) and the parent nucleosides 41 and 42. This suggests that even though 44
and 45 retain a phosphate monoacid, the cyclic phosphate construct sufficiently reduces the polarity relative to the 5′-monophosphates 43 and 44 to improve
cell penetration. To further enhance cell permeability characteristics, the acid of the cyclic phosphates
was converted to the SATE 47 and 48 resulting in
substantial improvement in HCV replicon potency
[99]. The SATE cyclic phosphate prodrugs 47 and
48 demonstrated a 7,000- to 11,000-fold improvement in potency (EC50s=0.463 to 0.008 µM) relative
the parent nucleosides with no observed cytotoxicity.
Further development of these novel inhibitors has not
been reported.
Another application of the 3′,5′-cyclic phosphate prodrug approach focused on the study of 3′,5′-cyclic phosphoramidates in an attempted to improve the activity
of 2′-C-methylcytidine (Figure 16) [100]. It was postulated that relative to the 5′-phosphoramidate prodrug,
the 3′,5′-cyclic version would reduce the molecules
rotational degrees of freedom and potentially improve
entry into cells. In addition, use of a cyclic phosphate
would remove the need for a phenolic substituent on
the phosphoramidate moiety. Preparation of 2′-Cmethycytidine 3′,5′-cyclic phosphoramidates like 49
(Figure 16) lead to prodrugs which were a mixture of
diastereomers that were ultimately separated chromatographically. In some cases there was a substantial difference in activity between the prodrug diastereomers,
however, in no case did any of the prodrugs demonstrate
significant improvement in anti-HCV activity over 2′-Cmethylcytidine itself. Contrary to the results from the
HCV replicon assay, several cyclic phosphoramidate
analogues did produce enhanced levels of nucleoside triphosphate when incubated with primary human hepatocytes (two- to sevenfold). Subsequent in vivo studies
in the hamster showed a lack of oral bioavailability as
measured by the levels of nucleoside triphosphate in the
liver, possibly due to poor absorption or gastrointestinal instability. However, subcutaneous administration
did show a 10- to 50-fold improvement in the levels of
nucleoside triphosphate relative to that produced with
2′-C-methylcytidine. No further developments on this
class of nucleotide prodrugs have been reported.
©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
2′-α-F-2′-β-C-Methyl nucleotide 3′,5′-cyclic phosphate
prodrugs
The 3′,5′-cyclic phosphate prodrug strategy was also
applied to the 2′-α-F-2′-β-C-methyl class of nucleosides
and ultimately resulted in the clinical development candidate PSI-352938 (50; Figure 17) [101]. PSI-352938
(50) demonstrated for the first time human clinical
proof of concept for use of the cyclic phosphate prodrug approach to deliver a 5′-monophosphate [98].
In the case of the 2′-α-F-2′-β-C-methyl class of nucleosides the 3′,5′-cyclic phosphate prodrug approach
was explored in an attempt to deliver 6-substituted
guanosine derivatives [101]. This approach was taken
because the parent 2′-α-F-2′-β-C-methylguanosine
nucleoside 35 was a weak inhibitor in the HCV replicon assay even though its corresponding 2′-α-F-2′β-C-methylguanosine triphosphate (36) was a good
inhibitor of the HCV polymerase (IC50=5.94 µM)
and equipotent against both the wild type and S282T
mutant polymerases. In the case of the 2′-α-F-2′-βC-methylguanosine series, unlike in the case of other
2′-C-methyl nucleosides, simple alkyl and aryl esters
of the 3′,5′-cyclic phosphate were explored and SAR
optimization focused around both the 6-position of the
guanosine base and the phosphate ester moiety. These
2′-α-F-2-β-C-methyl 6-substituted guanosine 3′,5′-
cyclic phosphate esters were shown to be metabolized
to the 2′-α-F-2-β-C-methylguanosine triphosphate (36);
however, the mechanistic details of the metabolism
have yet to be reported. It was ultimately determined
based on potency, cytotoxicity profile, gastrointestinal
stability characteristics, and triphosphate production in
vitro and in vivo that the Rp diastereomer PSI-352938
was the optimal candidate for clinical development.
PSI-352938 was shown to have an EC90=1.37 µM with
no cytotoxicity in multiple cell lines nor any observed
mitochondrial toxicity or bone marrow toxicity when
tested up to 100 µM in vitro. When tested against the
HCV NS5B polymerase isolated from genotypes 1 to
4, the triphosphate 36 of PSI-352938 (50) was shown
to be equipotent against all genotypes and PSI-352938
demonstrated in the replicon assay to be additive or
synergistic in combination with IFN and RBV and with
other DAAs [83,102]. In addition, the combination of
PSI-352938 and the uridine nucleotide phosphoramidate PSI-7977 (34; Figure 12) was shown to effectively
clear both the wild type and S282T mutant replicons
[102,103]. In vitro and in vivo studies showed that high
triphosphate levels were produced in primary human
hepatocytes and in rat liver (area-under-the-curve
[AUC0-24]=41,893 ng h/g) when PSI-352938 (50) was
administered orally at a 50 mg/kg dose. PSI-352938 (50)
Table 1. Nucleoside/nucleotide prodrugs: comparison of Phase I and Phase IIa human clinical trial resultsa
Drug (compound
number)
Class
Inhibition HCV
replicon, µM CC50, µM
Monotherapy
Combination with
GT 1 HCV patients
SOC GT 1 HCV patients
Maximum effect Log10 IU/ml, Maximum effect Log10 IU/ml, Status of
dose, mg
daysb
dose, mg
daysb
program
Nucleoside
prodrugs
RG7128
2′-F,2′-methyl EC90=2.5
>100
1,500
-2.7 (14)
1,000
-5.05 (28)
(30)
cytidine
(twice daily)
(twice daily)
NM283
2′-OH,2′-methyl EC50=1.23
>100
800
-1.2 (14)
800c
>-4 (28)
(2)
cytidine
(twice daily)
(twice daily)
>100
4,500
-3.7 (14)
1,500
-5.2 (28)
R1626
4′-azido
EC50=1.28d
(20)
cytidine
(twice daily)
(twice daily)
Nucleotide prodrugs
PSI-7977
2′-F,2′-methyl EC90=0.42
>100
400e
-1.95e (3)
400
-5.3 (28)
(34)
uridine PA
(once daily)
(once daily)
PSI-352938
2′-F,2′-methyl EC90=1.37
>100
200
-4.64 (7)
ND
ND
(50)
guanosine 3′,5′-
(once daily)
cyclic phosphate
IDX184
2′-OH,2′-methyl EC50=0.4
>100
100
-0.74 (3)
150
-4.1 (28)
(17)
guanosine PA
(once daily)
(once daily)
INX-08189
2′-OH,2′-methyl ED50=0.01
7
25
-1.03 (7)
ND
ND
(16)
guanosine PA
(once daily)
Completed
Phase II
Discontinued GI
toxicity
Discontinued
haematological
toxicity
Ongoing Phase IIb
study
Ongoing Phase Ib
study
Full clinical hold
changed to partial
clinical hold
Ongoing Phase Ib
study
Comparison across clinical studies. bNumbers in brackets represent number of days on the drug. cCombination with interferon only no ribavirin present. dNo direct
50% effective concentration (EC50) available, represents EC50 of the parent nucleoside R1479. eResult from PSI-7851 the diastereomeric mixture. CC50, 50% cytotoxic
concentration; EC90, 90% effective concentration; GI, gastrointestinal; GT, genotype; ND, no data available; PA, 5′-Phosphoramidate; SOC, standard of care.
a
Antiviral Chemistry & Chemotherapy 22.1
41
MJ Sofia
Figure 18. HepDirect prodrug technology and the prodrug decomposition pathway.
Ar
O
O
P
O
Cytochrome P450
Nucleoside
Ar
OH
O
O
P
O
Nucleoside
O
O
HepDirect Prodrug
HO
Spontaneous
Ar
O
P
O
Nucleoside
Spontaneous
O
O
O
Ar
O
HO
P
O
Nucleoside
OH
was taken into a Phase I clinical study and was shown
to be generally safe and well-tolerated when dosed up
to 1,600 mg in healthy volunteers [104]. In a multiple ascending dose study in HCV genotype-1-infected
patients, PSI-352938 was administered at doses of 100,
200 and 300 mg once daily and 100 mg twice daily
over 7 days to assess safety, tolerability and antiviral
response [98]. In the 40 patients studied HCV RNA
levels declined consistently through the 7-day treatment
period with a mean HCV viral load change from baseline of -4.31 log10 IU/ml, -4.65 log10 IU/ml, -3.94 log10
IU/ml and -4.59 log10 IU/ml for patients receiving 100
mg once daily, 200 mg once daily, 300 mg once daily
and 100 mg twice daily. For those patients receiving the
200 and 300 mg doses 11 of 16 patients achieved HCV
viral load below the limit of quantification. In addition,
there were no reported adverse events in this study [98].
The early potency achieved by PSI-352938 (50) is comparable to that observed with NS3 protease inhibitors
and PK parameters supported once-daily dosing. These
results represent the first demonstration of clinical efficacy for a cyclic phosphate prodrug and represent the
most potent nucleoside evaluated to date in a clinical
setting (Table 1).
PSI-352938 (50) was further evaluated in a dual
nucleotide DAA combination study with the pyrimidine nucleotide prodrug PSI-7977 (34) to evaluate the
need for IFN in the treatment of HCV infection [87]. In
this proof of concept study, no significant PK interactions between PSI-352938 (50) and PSI-7977 (34) were
42 observed and no viral breakthrough was detected during therapy. This dual nucleotide combination demonstrated at day 14 a -4.6–5.5 log10 decline in viral load
with an average of 94% of patients having viral loads
below the limit of detection (<15 IU/ml). This study is
the first proof of concept that two nucleosides/nucleotides can be combined as a possible DAA treatment
regimen. A Phase II study is being planned for this dual
nucleotide DAA combination [87].
Cyclic 1-aryl-1,3-propanyl nucleotide phosphate
esters: HepDirect prodrugs
Preparation of a nucleoside 5′-monophosphate cyclic
diester using a 1-aryl-1,3-propanyl prodrug moiety to
accomplish liver targeting has been called HepDirect
prodrug technology [33,105]. HepDirect technology
was developed specifically to deliver drugs to the liver
by taking advantage of liver-specific enzymes which
would cleave the prodrug moiety thus revealing the
active drug in hepatocytes. HepDirect prodrugs have
been reported to be very stable in aqueous solutions,
blood and non-hepatic tissues other than that of the
gastrointestinal tract. For HepDirect technology, activation is achieved via a cytochrome P450 mediated
oxidation (Figure 18). The described mechanism for
nucleoside prodrug cleavage starts with hydroxylation of the C-4 tertiary carbon of the pro-moiety by
CYP3A4 followed by rapid ring opening and then
β-elimination to reveal the nucleoside monophosphate and an aryl vinyl ketone byproduct. Although
©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
Figure 19. HepDirect prodrug analogues of 2′-C-methyladenosine nucleoside monophosphates
O
P
O
NH2
N
O
O
O
HO
Cl
N
N
N
OH
NTP levels in rat hepatocytes =389 nmol/g
51
O
Cl
P
O
NH2
N
O
O
O
H2N
O
N
N
OH
O
NTP levels in rat hepatocytes =117 nmol/g
52
release of aryl vinyl ketone as a byproduct of HepDirect cleavage raises toxicological concerns, it is
hypothesized that this byproduct would be rapidly
scavenged by high levels of glutathione in the liver,
thus minimizing the associated risks. The HepDirect
liver targeting prodrug approach was demonstrated
in the clinic with pradefovir, a HepDirect prodrug
version of adefovir [33].
2′-C-Methyladenosine HepDirect prodrugs
2′-C-Methyladenosine (9; Figure 6; EC50=0.3 µM) was
shown to be active as an inhibitor of HCV replication in
the subgenomic replicon assay, however it was also determined to be an efficient substrate for adenosine deaminase, thus limiting its potential in vivo [106,107]. Since it
was hypothesized that preparation of a 5′-monophosphate
prodrug would block deamination and increase therapeutic levels of 2′-C-methyladenosine triphosphate (10) in the
liver, a HepDirect prodrug approach was investigated.
An SAR study evaluated activation in rat hepatocytes
by modifications to the aryl substituent of the HepDirect
prodrug moiety [108]. This assessment showed that the
prodrugs were generally efficiently cleaved and converted
to the nucleoside triphosphate 10 (Figure 6) particularly
those with halogen substituted phenyl 51 or pyridyl
groups (Figure 19). In vivo intravenous administration
Antiviral Chemistry & Chemotherapy 22.1
O
N
Cl
P
O
NH2
N
O
N
O
O
O
N
N
O
O
NTP levels in rat hepatocytes =296 nmol/g
53
was able to achieve substantial levels of nucleoside triphosphate in rat liver, but oral administration resulted in
a > fivefold lower level of liver triphosphate and only a
5% oral bioavailability. To circumvent this bioavailability issue the HepDirect prodrug approach was combined
with other prodrug groups that would protect the 2′- and/
or the 3′-hydroxyl groups or the N-6 amino group of the
base (Figure 19). The combination of HepDirect with a
2′,3′-cyclic carbonate 53 appeared to be the most fruitful in that it provided a 2- to 10-fold improvement in
the amount of nucleoside triphosphate observed in the
liver compared to compounds incorporating only the
HepDirect moiety. Unfortunately, no inhibition of HCV
replicon activity was provided for any of the HepDirect
derivatives to compare their potency to other prodrug
approaches and no additional data have been forthcoming on the status of these agents.
Bis(S-acyl-2-thioethyl) nucleotide phosphate
prodrugs: SATE prodrugs
The bis(S-acyl-2-thioethyl) phosphate strategy (SATE)
has been applied widely in the field of nucleoside
5′-phosphate prodrugs [33,34]. As described above in
the case of IDX184 which contains a SATE monoester,
an acid and an equivalent of episulfide is liberated as a
result of each thioester cleavage (Figure 20). Episulfide
43
MJ Sofia
Figure 20. SATE prodrug decomposition pathway
O
O
R
S
R
S
O
O
O P
O
R
Esterase
Nucleoside
S
HS
O
O
O P
O
Nucleoside
O
SATE Prodrug
O
R
O
O
HO P
S
O
Esterase
Nucleoside
S
S
O
HO
HO P
O
Nucleoside
Figure 21. SATE prodrug of the 5′-monophosphate of
7-deaza-7-ethynyl-2′-α-F-2′-β-C-methyladenosine
O
NH2
S
O
O
O
P
S
N
O
O
HO
N
N
F
O
EC50=8 µM
54
EC50, 50% effective concentration.
is highly reactive and is a known mutagen, consequently it has been assumed that release of such an
44 agent from a pronucleotide compound would be of
some concern [109].
The only reported application of the bis-SATE prodrug method to nucleotide HCV inhibitors was toward
2′-α-F-2′-β-C-methyl 7-ethynyl-7-deaza adenosine 38
[94]. In this case the activity of the SATE prodrug 54
(EC50=8 µM) was threefold better than the parent
nucleoside (Figure 21). However, no additional data
or progress has been reported for this compound or
on the use of bis-SATE prodrugs for delivering nucleotides for inhibiting HCV.
Discussion
Never before has the use of phosphate prodrugs played
such a significant role in the discovery and development
of nucleoside-based drugs as has been the case in the
area of inhibitors of HCV. The translation of multiple
examples of phosphate prodrugs of nucleotides into the
clinic and the demonstration of clinical proof of concept and beyond are equally unprecedented. HCV has
offered a unique opportunity to apply phosphate prodrug technology. Since HCV is a disease of the liver, drug
hunters are able to leverage the robust characteristics
©2011 International Medical Press
Nucleotide prodrugs for HCV therapy
of liver metabolism and first pass metabolism as the
means for achieving target organ drug levels and obviating the need for maintaining high circulating levels
of a potentially unstable prodrug. However, because of
liver targeting, decomposition of the phosphate prodrug to the desired nucleoside 5′-monophosphate does
release prodrug byproducts into the liver at potentially
high concentrations. This release of sometimes potentially reactive or toxic metabolites does pose a concern
in developing prodrugs of nucleoside monophosphates.
This concern is particularly relevant to HCV patients
with advanced liver disease where liver function is compromised and therefore, may have a lower capacity to
clear or process metabolites. Some of the prodrug strategies implemented for delivering nucleoside monophosphates such as the SATE or HepDirect strategies are
known to release reactive metabolites that should be
of concern in assessing their long term safety potential.
Other prodrug strategies such as the phosphoramidate
release different metabolites such as phenol or naphthol. Although phenol is readily cleared by the liver, the
toxicity of substituted phenols may be of concern in
deciding to progress a nucleotide prodrug forward into
development [110,111]. Each of these liability concerns
must be put into context of dose and PK. It is possible to circumvent any toxicity concerns if the dose of
drug administered is sufficiently small to not warrant
concern or if the PK profile of the metabolites limits
exposure. Clearly the increased potency of these nucleotide prodrugs relative to the first generation nucleoside HCV inhibitors increases the therapeutic index
and reduces the chances of observing undesired side
effects. However, long term animal toxicity studies and
clinical safety assessment over extended dosing periods
will ultimately address the concerns over any potential
human toxicity.
The nucleotide phosphate prodrugs which have
entered clinical study have demonstrated superiority
over the first generation nucleoside inhibitors developed
for treating HCV (Table 1). The use of the nucleoside
5′-monophosphate prodrugs has demonstrated dramatic potency enhancements relative to first generation
nucleoside inhibitors in the cellular subgenomic replicon assay, higher levels of triphosphate in whole cells
and in livers of animals when dosed orally. To a large
extent this improved in vitro and whole animal profile
has translated into the clinical setting. In the clinic, dosing frequency has been reduced from twice daily to once
daily, drug load has been greatly reduced and impressive
viral load declines and RVR rates have been achieved.
However, in vitro HCV replicon potency alone can not
itself justify the robust initial clinical results observed
for these phosphate prodrugs. The least potent inhibitor in the subgenomic replicon assay, PSI-352938 (50;
Figure 17), produced the greatest viral load decline in
Antiviral Chemistry & Chemotherapy 22.1
human monotherapy studies, therefore issues such as
PK must be playing a critical role in the overall human
clinical response. How the human body handles these
prodrugs is yet to be determined. A question that
remains unanswered is whether there are specific transporters that play a clinically meaningful role in prodrug
uptake into the liver or transport through the gut.
It has been suggested that nucleosides/nucleotides
will eventually become the backbone of anti-HCV
therapy, as they have for HIV HAART. Their broad
genotype coverage and resistance profile put them in
a unique position and make them the drug class of
choice to combine with DAAs of different mechanisms
of action. With the identification of both purine and
pyrimidine nucleotide prodrugs with different resistance profiles and metabolic pathways, the possibility
also exists for combining two nucleosides/nucleotides
into one regimen as has been done in the HIV field.
Studies to explore different combinations of DAAs
including dual nucleotide combinations have begun.
Results in a 14-day dual nucleotide proof of concept
study with PSI-7977 (34) and PSI-352938 (50) appear
promising. It is the hope that these studies will pave
the way forward for potentially new therapeutic paradigms in the treatment of HCV which include the
elimination of IFN. However, issues such as safety,
tolerability and the extent of a SVR after 12 or 24
weeks of therapy will help determine which of the current and future nucleotide produgs will successfully
reach the market. At this stage long term safety is still
unknown and only PSI-7977 (34) has demonstrated
an SVR in the clinic, but only on a small patient population. Longer term studies are underway to better
define the risk:benefit profile of these agents. However,
these agents provide great hope for patients suffering
from HCV.
Disclosure statement
MJS is an employee of Pharmasset, Inc. and receives
salaried compensation in his role as Vice President of
Chemistry.
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