Answer questions

1. Interpret the data from the journals to determine whether each drug is in line with Lipinski’s rule of five (M4-1)
2. Analyze the journals to list the properties of each drug in the context of ADME/PK (e.g. solubility, permeability, plasma protein binding, metabolism, distribution, elimination, Cmax, half-life, etc.), and comment on any key structural modifications that were done to address ADME issues (M5-1)
3. Why is remdesivir administered as a prodrug? (M1.4, M4.1)
4. If the data is available, what is the maximum concentration (Cmax) of the drug in plasma and/or the target tissue that is observed when an animal is administered an efficacious dose? (M1-4)

Comment on the relationship of the concentration of the drug at its target tissue (or plasma) to the in vitro potency of the drug. (M1-4)

Articles for review

1. Boceprevir – George Njoroge, Chen, Neng-Yang, & Piwinski (2008)
2. Glivec – Capdeville, Buchdunger, Zimmermann, & Matter (2002), USFDA (2006), and Peng, Lloyd, & Schran (2005)
3. Brivaracetam – Klitgaard, et al. (2016) and Rogawski (2009)
4. Remdesivir – Warren, et al. (2016)and Eastman, et al. (2020)

Challenges in Modern Drug Discovery: A Case
Study of Boceprevir, an HCV Protease Inhibitor
for the Treatment of Hepatitis C Virus Infection
F. GEORGE NJOROGE,* KEVIN X. CHEN, NENG-YANG SHIH,
AND JOHN J. PIWINSKI
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Schering-Plough Research Institute, 2015 Galloping Hill Road,
Kenilworth, New Jersey 07033
RECEIVED ON MAY 4, 2007
CON SPECTUS
M
ore than 170 million people worldwide are affected
by the hepatitis C virus (HCV). The disease has been
described as a “silent epidemic” and “a serious global
health crisis”. HCV infection is a leading cause of chronic
liver disease such as cirrhosis, carcinoma, or liver failure. The current pegylated interferon and ribavirin combination therapy is effective in only 50% of patients. Its
moderate efficacy and apparent side effects underscore the
need for safer and more effective treatments. The nonstructural NS3 protease of the virus plays a vital role in
the replication of the HCV virus. The development of small
molecule inhibitors of NS3 protease as antiviral agents
has been intensively pursued as a viable strategy to eradicate HCV infection. However, it is a daunting task. The
protease has a shallow and solvent-exposed substrate
binding region, and the inhibitor binding energy is mainly
derived from weak lipophilic and electrostatic interactions.
Moreover, lack of a robust in vitro cell culture system and
the absence of a convenient small animal model have
hampered the assessment of both in vitro and in vivo efficacy of any antiviral compounds. Despite the tremendous
challenges, with access to a recently developed cell-based replicon system, major progress has been made toward a
more effective small molecule HCV drug. In our HCV program, facing no leads from our screening effort, a structurebased drug design approach was carried out. An R-ketoamide-type electrofile was designed to trap the serine hydroxyl
of the protease. Early ketoamide inhibitors mimicked the structures of the peptide substrates. With the aid of X-ray
structures, we successfully truncated the undecapeptide lead that had a molecular weight of 1265 Da stepwise to a
tripeptide with a molecular weight of 500 Da. In an attempt to depeptidize the inhibitors, various strategies such as
hydrazine urea replacement of amide bonds and P2 to P4 and P1 to P3 macrocyclizations were examined. Further
optimization of the tripeptide inhibitors led to the identification of the best moieties for each site: primary ketoamide at P′, cyclobutylalanine at P1, gem-dimethylcyclopropylproline at P2, tert-leucine at P3, and tert-butyl urea as capping agent. The combination of these led to the discovery of compound 8 (SCH 503034, boceprevir), our clinical
candidate. It is a potent inhibitor in both enzyme assay (Ki* ) 14 nM) and cell-based replicon assay (EC90 ) 0.35
µM). It is highly selective (2200×) against human neutrophil elastase (HNE). Boceprevir is well tolerated in humans
and demonstrated antiviral activity in phase I clinical trials. It is currently in phase II trials. This Account details the
complexity and challenges encountered in the drug discovery process.
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Published on the Web 01/15/2008 www.pubs.acs.org/acr
10.1021/ar700109k CCC: $40.75
© 2008 American Chemical Society
Challenges in Modern Drug Discovery Njoroge et al.
Introduction
Hepatitis C virus (HCV) infection is one of the most significant
health problems affecting humans. An estimated 170 million
individuals (3%) worldwide and more than 4 million Americans (1.3%) are infected with HCV.1 In roughly 80% of cases,
the virus leads to a chronic form of hepatitis, a condition that
is incurable in many patients. Without therapeutic intervention, it can lead to morbidity or mortality in 10 –20 years
through either cirrhosis and hepatic failure or hepatocellular
carcinoma.2 It is anticipated that a significant percentage of
those currently infected will develop cirrhosis and other associated hepatic sequelae. HCV infection is the most common
cause of liver transplantation.2
Current medical treatment options are limited. The only
drugs available are subcutaneous interferon-R (IFN-R) or pegylated-IFN-R, alone or in combination with oral ribavirin.3
IFN-R is a protein that stimulates the immune system, while
ribavirin is a nucleoside analog that works in concert with
IFN-R to control the infection. The efficacy of this combination therapy against the predominant genotype 1 affecting
North America, Europe, and Japan is moderate, with only
about 50% of the patients showing sustained virological
response. Some patients also experience significant side
effects related to the treatment. With few alternatives available, more effective agents with fewer side effects are clearly
needed.3
HCV, the etiologic agent of non-A, non-B hepatitis, was
identified in 19894 and is a member of the Flaviviridae. HCV
is an enveloped, positive-strand RNA virus of approximately
9.6 kilobases. Upon entering a suitable host cell, the HCV
genome serves as a template for cap-independent translation
through an internal ribosome entry site (IRES).5 The resulting
polyprotein undergoes both co- and post-translational proteolytic maturation by host and virally encoded proteases. The
virally encoded protease responsible for processing the nonstructural (NS) portion of the polyprotein is located in the
N-terminal third of the NS3 protein5 (Figure 1). Following autoproteolysis of the NS3-NS4A junction, the protease cleaves
the polyprotein at the NS4A-NS4B, NS4B-NS5A, and
NS5A-NS5B junctions to release the downstream NS proteins.
The mature proteins subsequently self-assemble on the endoplasmic reticulum to generate the replicative complex or replisome. The replisome, using the viral genome as a template,
generates negative-strand viral RNA intermediates, which are
then used as templates to synthesize new positive-strand
(genomic) RNAs. These are either translated to yield more
polyprotein or, later in the infection cycle, encapsulated to
FIGURE 1. Schematic representation of the HCV genome and NS3
protease polyprotein. NS3 is a bifunctional protein with protease
and helicase activities. The green box represents the NS3 protease
domain, and thin arrows depict cleavages mediated by the NS3/
NS4A protease complex.
generate progeny virions. Inhibition of the maturational activities of the NS3 protease would therefore suppress replisome
formation, RNA replication, and ultimately new virions.
The X-ray structures of NS3 protease, both as an isolated
domain and in the full-length NS3 protein, have been determined.6 The structural data has provided detailed insights to
facilitate rational inhibitor design. The NS3 protease is in
many ways a typical β-barrel serine protease, with a canonical Asp-His-Ser catalytic triad similar to the well-studied digestive enzymes trypsin and chymotrypsin. By contrast, the NS3
protease uses an extended polydentate binding cleft, with several recognition subsites to ensure specificity. It forms a heterodimeric complex with the NS4A protein, an essential
cofactor that activates the protease and assists in anchoring
the heterodimer to the endoplasmic reticulum. On the other
hand, the RNA-dependent RNA polymerase (RdRp) contained
within the NS5B protein is the catalytic component of the HCV
RNA replication machinery.5,7 This enzyme synthesizes RNA
using the RNA template. This biochemical activity is not
present in mammalian cells, offering the opportunity to identify very selective inhibitors of the viral enzyme.
Much of the recent effort has been directed toward developing drugs that inhibit viral replication. Several promising
small-molecule inhibitors of the NS3/4A protease and the
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NS5B polymerase are in clinical development.8 Early testing
has demonstrated strong antiviral activity both in vitro and in
patients. Inhibitors toward other potential targets such as IRES
and NS5A are in earlier stages of preclinical investigation.
Although efforts are ongoing to develop a vaccine,9 the
unusually rapid genetic drift of HCV makes this a daunting
task.
Challenges in Discovering
HCV Protease Inhibitors
Although HCV was characterized more than a decade ago, the
lack of a robust in vitro cell culture system capable of supporting its replication has complicated traditional approaches to
developing or evaluating antiviral compounds. Likewise, the
absence of a convenient small animal model has hampered
the assessment of in vivo efficacy. Most of our knowledge of
HCV has been derived from surrogate experimental systems
that approximate infection and often preclude definitive interpretation. Only since the development of the HCV autonomous subgenomic replicon system,10 a severe combined
immunodeficiency disease (SCID) mouse with chimeric human
liver model,11 and the chronically infected chimpanzee
model10has the preclinical evaluation of potential anti-HCV
agents become possible.
There are currently two animal models for preclinical evaluation of anti-HCV therapies; both of which suffer from limitations that make them less than ideal for preliminary studies.
HCV infects only humans and chimpanzees. The chronically
infected chimpanzee model,12 the “gold standard” for HCV
studies, is challenging and expensive because one out of three
chimpanzees spontaneously resolve their HCV infection. An
immune-deficient SCID mouse-human liver xenograft system11 was developed by researchers at the University of
Alberta. In this model, the livers of neonate SCID beige mice
are colonized with infused human hepatocytes, which rescues
them from a fatal transgene. Infection of these human liver
grafts by several genotypes of HCV and the therapeutic effects
of INF-R have been reported. Unfortunately, the animals are
fragile and scale up of the colony has been slower than
expected, thus limiting access to the system.
Owing to the fact that the NS3-NS4A protease is playing
a critical role in HCV viral replication, it has been viewed as an
ideal target for the creation of new HCV therapy.8,13 However, developing HCV protease inhibitors as drugs was no trivial task. At the onset of our work, there were no viable lead
structures from which to develop potential drug candidates.
Our screening effort of four million compounds did not generate any meaningful leads to initiate a drug discovery effort.
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Thus, early inhibitors were designed based on the
substrate-enzyme active site interactions.14 However, the
HCV protease requires an extensive peptide substrate, with
which it establishes multiple weak interactions distributed
along an extended surface. The requirement of large substrates was a major concern in the development of orally bioavailable small molecule drugs. It was feared that the enzyme
may only be inhibited by molecules large enough to mimic
the natural substrate. Indeed, early leads were long peptidic
compounds that occupied much of the substrate-binding site
to take advantage of multiple hydrogen bonding and hydrophobic interactions. The major challenge was to modify these
large molecules to the less peptidic and lower molecular
weight drug candidates with desirable pharmacokinetic (PK)
profiles, while retaining or improving potency in the enzymatic
and cellular assays. The resolution of the three-dimensional
structure6 of the enzyme damped the enthusiasm of medicinal chemists further, because the substrate-binding cleft of
NS3-NS4A protease seemed flat and featureless, lacking the
cavities, holes, and flaps, or so-called binding pockets, that had
been exploited as anchor points to design potent and selective inhibitors of other proteases.
Moreover, there was another major challenge for any successful anti-HCV therapy: drug-resistant viruses emerge rapidly under the selective pressure. The fast turnover rate and
the intrinsic low fidelity of the HCV replication machinery
endowed the virus with the ability to fully explore its genome
space and quickly come up with mutations that rendered it
resistant to antiviral drugs.
As a result of all these difficulties, no HCV protease inhibitors have been approved yet for the treatment of HCV disease in spite of the fact that the virus was fully characterized
in 1989. However, efforts within the pharmaceutical industry
have resulted in several candidates in clinical development,8
including boceprevir.15
Our Approach for Inhibitor Design
Since our screening efforts failed to generate potential leads
to initiate a drug discovery effort, we embarked on a structurebased design approach. Early research for inhibitors capitalized on the observation that the enzyme is susceptible to
marked inhibition by the N-terminal peptide products released
from the substrates upon enzymatic cleavage. Learning from
experiences from others in developing potent serine-protease
inhibitors,16 incorporation of a serine trap into the molecule
was a promising approach. We envisioned that reaction of the
active site serine (Ser139)6 with conventional electrophiles
such as aldehydes, ketones, trifluoromethyl ketones, and R-ke-
Challenges in Modern Drug Discovery Njoroge et al.
FIGURE 2. Nucleophilic attack of the R-ketoamide by Ser139 led to
a covalent tetrahedron intermediate stabilized by residues His57
and Asp81.
FIGURE 3. Progress curve of peptide substrate hydrolysis by the
HCV protease domain showing the time-dependent inhibition.
toamides and subsequent trapping of the resulting transitionstate analogues by the active site triad (Ser139, His57, and
Asp81, Figure 2)6 would provide effective inhibition. After considerable research, a few large peptidic molecules, mimicking the peptide substrate structures but containing an
R-ketoamide functionality, were discovered to be potent HCV
protease inhibitors.
The R-ketoamide moiety was essential for the inhibitory
activity. When it is attacked by Ser139 of the NS3 protease,
it forms a stable, covalent, and reversible complex with the
enzyme. The time required for stable covalent adduct formation is on the order of minutes. To assess the potency of such
slowly equilibrating or “slow-binding” inhibitors accurately,
proteolytic reactions containing inhibitor are usually monitored until equilibrium is evident using progress curve analysis (Figure 3). In this continuous assay,17 the extent of
hydrolysis of chromogenic 4-phenylazophenyl (PAP) ester
from the peptide fragment Ac-DTEDVVP(Nva)-O-4-PAP was
spectrophotometrically determined. To underscore the slowbinding nature of these molecules and distinguish them from
simple, instantaneous competitive inhibitors, the equilibrium
binding constant was usually designated Ki*,18 although for
most purposes it could be considered equivalent to a traditional Ki.
FIGURE 4. Schematic representation of the RNA encoding the
bicistronic subgenomic HCV replicon.
Besides an enzymatic assay, a cell-based assay was also
essential for optimizing inhibitor potency. An HCV subgenomic
replicon system was developed by Bartenschlager and colleagues in 1999.10 The replicon cell-based assay has been
used extensively to evaluate the functional potency and subsequent antiviral efficacy of HCV protease inhibitors. The HCV
replicon is essentially a defective (i.e., noninfectious) viral
genome in which the sequences encoding the structural proteins at the 5′ end of the RNA have been replaced by a selectable marker, the neomycin resistance gene (NeoR) (Figure 4).
The NeoR marker allows selection of cells harboring functional
replicons following transfection and antibiotic treatment. Replicon constructs, including those developed to evaluate potential antiviral agents, use a bicistronic design where two
independent IRES elements are present. The HCV IRES
sequence drives expression of the neomycin resistance gene
to allow selection of replicon bearing cells and a second IRES
sequence from encephalomyocarditis virus (EMCV) initiates
translation of the RNA segment encoding HCV nonstructural
proteins from NS3 to NS5B.
Unlike a true HCV infection, cells bearing HCV replicons,
even full-length replicons expressing structural proteins, do not
generate progeny virions. At the time, the replicon system was
the only germane in vitro system for evaluating potential antiviral agents directed against the HCV nonstructural proteins
and, consequently, provided an essential and stringent system for the evaluation of potent inhibitors of HCV protease.
The EC50 and EC90 values for suppression of the bicistronic
subgenomic replicon (genotype 1b) were obtained through a
72 h assay in HuH-7 cells. At 72 h, cells were lysed, and the
replicon RNA level was determined using real-time polymerase chain reaction (PCR) analysis (Taqman) that targeted
the NS5B portion of the viral genome. Changes in replicon
RNA level were compared to an internal control, cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, in a single-tube multiplex reaction. Dose-response curves
were generated and drug concentrations resulting in a 2-fold
or a 10-fold reduction in replicon RNA were estimated using
a grid search method to give EC50 and EC90 values.
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One of the key elements contributing to the potency of an
R-ketoamide is the electrophilic ketoamide functionality, which
forms a reversible covalent bond with serine 139 of the HCV
protease. However, the ketoamide motif could also be susceptible to attack by a variety of other nucleophiles present on
other proteins. Thus, to address the selectivity issue, inhibitory activity against the human neutrophil elastase (HNE) was
measured as a gauge of selectivity (HNE/HCV) in the structure–activity-relationship (SAR) development. The active site of
HNE resembles closely the active site structure of HCV NS3
protease. Although the clinical relevance of this selectivity
parameter has not been demonstrated, it helped to serve as
a guide in designing selective inhibitors versus similar proteases, thereby potentially minimizing side effects that could
surface in the clinic.
Our early work led to the discovery of undecapeptide Rketoamide inhibitors,15 which had structures with 11 amino
acid residues that spanned from P6 to P5′.19 One of the earliest leads, compound 1 (Figure 5), exhibited excellent HCV
NS3 protease inhibition (Ki* ) 1.9 nM). It was a mixture of two
diasteromers at the epimerizable P1 R-center. However, with
a molecular weight of 1265 Da, it was not surprising that this
compound did not exhibit a desirable PK profile. Nevertheless, compound 1 and related analogs served as a starting
point for further SAR studies. Our research toward improving
the overall profile of these initial leads was greatly aided by
using X-ray structures of inhibitors bound to the HCV NS3 protease and continuous drug metabolism/pharmacokinetic
evaluation.
Truncation Effort. Starting from the lead compound 1, an
undecapeptide, a series of stepwise truncations at both the
prime and nonprime ends led to a pentapeptide inhibitor 2.20
The molecular weight was reduced to approximately half of
that of compound 1. A number of amino acids were also modified to optimize potency. Thus, P1 and P2 were replaced with
cyclopropylalanines, P3 was replaced with cyclohexylglycine,
and P2′ was substituted with phenylglycine. The compound
was capped as isobutyl carbamate at P3 and as carboxylic
acid at P2′. Inhibitor 2 was quite potent with a Ki* of 15 nM.
However, it was inactive in the cell-based HCV replicon assay
(EC90 > 5 µM), possibly due to the presence of the charged
carboxylic acid preventing it from getting into the cells.
Further optimization along the entire backbone was carried out with the aid of X-ray crystal structures. The most
important discovery occurred at P2 position. The gem-dimethylcyclopropylproline was discovered to be a superior leucine
surrogate. The tert-butylglycine was found to be a good
replacement of cyclohexylglycine at P3. The capping groups
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FIGURE 5. From undecapeptide to pentapeptide HCV NS3 protease
inhibitors.
at both ends were also changed to tert-butyl carbamate for P3
and dimethylamide for P2′, respectively. The latter would
address the cell penetration issue of compound 2. Indeed, as
a result of all these optimizations, compound 321 demonstrated a dramatic improvement over compound 2, with good
binding affinity (Ki* ) 5 nM) and replicon activity (EC90 ) 0.1
µM). Shown in the X-ray structure of 3 bound to the protease
(Figure 6), the improvement in binding potency was a result
of favorable interaction of the P2 gem-dimethylcyclopropylproline moiety with the methyl side chain of Ala156. The
cyclopropylmethyl side chain of P1 and the phenyl group of
P2′ formed a “C”-shaped clamp around the side chain of
Challenges in Modern Drug Discovery Njoroge et al.
FIGURE 6. X-ray structure of inhibitor 3 bound to the HCV
protease.
Lys136. However, the molecular weight (725 Da) of 3 was still
larger than that of a typical drug candidate, which, preferably, should be less than 500 Da.22 Thus, it was not unexpected that this translated to a poor PK profile in rats and
monkeys. Generally, an oral area-under-curve (po AUC) of
greater than 1.0 µM · h and bioavailability of at least 10% are
desired. In rats, compound 3 had a po AUC of 0.35 µM · h at
10 mg/kg and a bioavailability of 4%, while in monkeys the
AUC was 0.03 µM · h at 3 mg/kg and bioavailability was 1%.
Clearly, further reduction in the molecular weight of the inhibitors was necessary to achieve desirable PK properties.
Due to the importance of the remaining residues at nonprime side, we focused our additional truncation efforts on the
prime side. First, the P2′ phenylglycine residue was removed
with modest loss of potency. The P1′ glycine was replaced by
simple alkyl groups such as methyl, ethyl, or allyl. Unfortunately, all secondary and tertiary P1 ketoamides were significantly less potent than compound 3. However, it was
discovered that the nonsubstituted primary ketoamides had
the best potency in both enzyme and replicon assays. The representative inhibitor after this round of SAR development was
compound 4 (Figure 5). It had a Ki* of 25 nM, and a replicon
EC90 of 0.40 µM. Although both values were higher than
those of analog 3, the molecular weight was significantly
lower and fell within the preferred range for a developable
candidate.
Depeptization Studies. It is difficult to aquire desirable
pharmacokinetic properties from large peptides because they
are susceptible to hydrolysis by various peptidases. Thus, substantial efforts were devoted to depeptizing the lead molecules. Shown in Figure 7 are several examples of that effort.
FIGURE 7. Efforts toward depeptization of HCV protease inhibitors.
First, replacement of P2 amino acid residue with an aza motif
was executed. Thus, in compound 5,18 the cyclopropylalanine P2 was replaced with a substituted hydrazine urea moiety. Unfortunately, that resulted in a significant loss in binding
affinity (Ki* ) 230 nM) and a total loss of potency in replicon
assay (EC90 > 5 µM).
Efforts were also made to improve potency and PK profiles through macrocyclization of P2 and P3 residues, resulting in macrocyclic inhibitors of type 623 (Figure 7). A
phenylacetamide capping from P3 was linked to C-4 of P2
proline through a tert-alkyl ether linkage. The 16-membered
macrocyclic ring formed a donut-shaped circle over the methyl
group of Ala156 as evidenced by X-ray crystallography. The
inhibitor had excellent Ki* of 6 nM and was moderately active
in the cell-based assay (EC90 ) 0.90 µM). However, it had an
oral AUC of only 0.46 µM · h at 10 mg/kg in rats with a low
bioavailability of 2.2%. Similarly sized compounds with modificaftions along the macrocyclic ring gave equally poor PK
results. When truncated at the prime side, smaller macrocyclic inhibitors did not have the desired level of potency in replicon assay.
On the other hand, macrocyclization from P1 to P3 residues through a 16-membered ring provided novel inhibitors
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FIGURE 8. HCV protease inhibitor 8.
rate the dimethylcyclopropyl-proline at P2. Compound 7 was
a single diastereomer with a nonepimerizable P1 R-center. It
had a respectable Ki* of 30 nM, and a good replicon EC90 of
0.60 µM. However, as in the other cases described above, further exploration of this series of inhibitors did not provide an
improved PK profile.
In summary, the depeptization exercises carried out neither resulted in significant improvement in potency nor provided measurable enhancement in PK properties in these HCV
protease inhibitors.
Discovery of Boceprevir (8)
Extensive SAR investigation at the P1, P3, and P3-capping
positions from the lead primary ketoamide 4 was continued.
The dimethylcyclopropylproline moiety at P2 was established
to be optimal and was retained in all subsequent analogs. Systematic variation in chain length and ring size on the P1 side
chain led to the discovery of cyclobutylalanine as the optimal choice. Rings with other sizes, such as cyclopentyl or
cyclohexyl, were found to be too large for the S1 pocket. A
large effort was also spent on exploring many P3 capping
groups. Various substituents extending from the P3 amino
group were examined; these included alkyls, aryls, amides,
carbamates, ureas, sulfonamides, and sulfonyl ureas. It was
discovered that the urea type of P3 capping gave the best
overall profile. Ultimately, the combination of various optimized moieties led to the discovery of boceprevir (8, SCH
503034, Figure 8),15 which was selected for drug safety studies and potential development in clinical trials.
The R-center of the P1 residue of 8 was racemic. The two
diastereomeric compounds could be separated by HPLC. However, when either pure isomer was treated with an organic or
inorganic base (e.g., triethyl amine or lithium hydroxide), they
underwent rapid isomerization. Fast equilibration was also
demonstrated under the conditions of biological assays. This
alleviated any need for separation of the two entities for pharmacological evaluations. The ratio of the two isomers varied
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FIGURE 9. Effect of 8 on HuH-7 cells bearing the subgenomic HCV
replicon. RNA level was measured relative to an internal control
(∆Ct). Increase in ∆Ct indicated decreasing replicon RNA levels;
each ∆Ct reflects a 2-fold change in RNA level from baseline.
significantly depending on the experimental conditions. The
isomer with an (S)-configuration at the P1 R-center was the
major isomer in most cases.
Compound 8 had an optimal overall profile. In the HCV
NS3 protease continuous assay, it had a potency of 14 nM
(Ki*) averaged over a large number of runs. In the 72-h bicistronic subgenomic cell-based replicon assay in HuH-7 cells, the
EC50 and EC90 values were determined to be 0.20 µM and
0.35 µM, respectively (Figure 9). Inhibitor 8 was also found to
be a very weak inhibitor of HNE (Ki ) 26 µM) representing a
selectivity of 2200. Additionally, the reactivity of 8 toward a
panel of other serine proteases was measured, and 8 showed
no cross-reactivity when tested up to 50 µM with trypsin, chymotrypsin, thrombin, and factor Xa. The cross-reactivity
against a broad panel of other general enzymes was also evaluated, and no major issues were identified. All these studies
indicated that compound 8 was highly selective toward the
HCV serine protease.
The pharmacokinetic profile of 8 was evaluated in several
animal species. Following oral administration, it was moderately absorbed in rats, dogs, and monkeys. Absorption was relatively rapid in dogs but slower in mice, rats, and monkeys,
as evidenced by mean absorption times (MAT) ranging from
0.5 to 1.4 h (Table 1). The AUC was good in dogs and rats,
moderate in mouse, and low in monkeys. The absolute oral
bioavailability was modest in mouse, rats, and dogs
(26 –34%) but low in monkeys (4%). There was no issue with
CYP 2D6, 2C9, 2C19, and 3A4 inhibition, either co- or preincubated. Target organ analysis in rats revealed that 8 was
Challenges in Modern Drug Discovery Njoroge et al.
TABLE 1. Mean (n ) 3) Pharmacokinetic Parameters of 8 Following
Oral Dosing
species
mouse
rat
dog
monkey
dose (mg/kg)
AUC (po, µM · h)
bioavailability (%)
MAT (h)
Cmax (µM)
10
0.93
34
1.2
2.3
10
1.5
26
1.4
0.66
3
3.1
30
0.5
2.3
3
0.12
4
1.4
0.09
both urea NH’s. Combination of a number of hydrophobic
interactions and the array of hydrogen bonds contributed
greatly to the binding potency and the selectivity of 8.
In summary, we discovered boceprevir (8) as a novel,
potent, highly selective, orally bioavailable HCV NS3 protease
inhibitor. It has been advanced to clinical trials in human
beings for the treatment of hepatitis C viral infections. It was
well tolerated and demonstrated antiviral activity in phase I
clinical trials and is currently in phase II.
Summary and Outlook
FIGURE 10. X-ray structure of inhibitor 8 bound to the HCV
protease.
highly concentrated in liver with a remarkable liver/plasma
concentration ratio of approximately 30.
The X-ray structure of compound 8 bound to NS3 protease
was solved and is shown in Figure 10. It was clear that the
diastereomer with an (S)-configuration at the P1 R-center was
the active inhibitor. The cyclobutylalanine moiety effectively
occupied most of the space available at the S1 pocket. This
group was largely responsible for the excellent selectivity
observed with 8 versus human neutrophil elastase, which has
a much smaller S1 pocket. The P2 dimethylcyclopropylproline residue adopted a bent conformation that allowed maximum overlap of the moiety with Ala156 of the enzyme. The
exo-methyl on the cyclopropane ring had favorable interaction with imidazole of His57, and the endo-methyl had contact with Ala156 and Arg155. The side chain of P3 tertbutylglycine occupied the S3 pocket, providing good
hydrophobic interaction with the enzyme. The tert-butyl group
of the P3 urea capping group had good contact in the S4
pocket, presumably also through a pure hydrophobic interaction. The ketoamide was reversibly trapped by Ser139 to form
a covalent bond and at the same time donated a hydrogen
bond to the protein backbone. In addition to van der Waals
contacts, 8 formed a series of specific hydrogen bonds with
the protein backbone, which involved P1-NH, P3-carbonyl, and
The pursuit of a potent and orally bioavailable HCV NS3 protease inhibitor as a drug candidate for the treatment of hepatitis C has been a difficult task. The shallow and featureless
nature of the enzyme’s active site presented a significant challenge for the discovery of enzyme inhibitors. Without any viable leads, a structure-based drug design approach guided by
X-ray crystal structures of the enzyme was pursued. Systematic truncations and depeptidizations on both prime and nonprime sites gave rise to smaller pentapeptides that were potent
inhibitors, but did not possess the desirable pharmacokinetic
properties. Modifications on the prime side resulted in the discovery of the primary R-ketoamide moiety, which gave excellent potency. Further SAR optimization identified P1
cyclobutylalanine, P2 dimethylcyclopropylproline, P3 tertbutylglycine, and tert-butyl urea capping group as the best
combination, which led to the discovery of boceprevir (8). It
had an in vitro potency of 14 nM (Ki*) and cell-based replicon assay potency of 350 nM (EC90). Compound 8 demonstrated good oral bioavailabilities in rats and dogs and was
found to be highly concentrated in the liver.
Across the pharmaceutical industry, several novel drug candidates have entered or will soon enter clinical evaluation to
establish their clinical effectiveness for HCV patients. Aside
from the safety and efficacy requirements common to all new
drugs, the success of HCV-targeted agents will be heavily influenced by their ability to inhibit all viral variants and prevent
the emergence of escape mutants. As is the case for HIV, combinations of several antiviral agents attacking different targets along the viral life cycle and, perhaps, the hosts
themselves will certainly be required to control infection and
prevent the emergence of drug-resistant viral variants.
The authors wish to thank the many contributors whose efforts
resulted in the successful outcome of this project. Especially
noteworthy is the successful collaborative efforts of scientists in
the Medicinal Chemistry (SPRI and Corvas), Structural Chemistry, Virology, and Drug Metabolism departments.
Vol. 41, No. 1
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ACCOUNTS OF CHEMICAL RESEARCH
57
Challenges in Modern Drug Discovery Njoroge et al.
BIOGRAPHICAL INFORMATION
George Njoroge obtained his B.S. in Chemistry at University
of Nairobi, Kenya, in 1979 and Ph.D. in Organic Chemistry at
Case Western Reserve University (CWRU) in 1985. He was a
postdoctoral fellow in the Institute of Pathology at CWRU working with Professor Vincent Monnier. He is currently director of
Chemical Research at Schering-Plough Research Institute
(SPRI).
Kevin Chen obtained his B.S. in Chemistry at University of Science and Technology of China in 1987 and Ph.D. in Organic
Chemistry in 1996 at University of WisconsinsMadison with Professor Edwin Vedejs. He was a NIH Postdoctoral Fellow until 1998
with Professor Stephen Martin at University of Texas at Austin. He
is currently a Senior Principal Scientist at SPRI.
Neng-Yang Shih is Executive Director of Chemical Research at
SPRI. He obtained his B.S. in Chemistry from Tamkang University in Taiwan and his Ph.D. in Organic Chemistry from University of Illinois at Chicago in 1981. He was a Postdoctoral Fellow
at Columbia University with Professor Thomas J. Katz and at Harvard University with Professor Elias J. Corey.
John Piwinski is Group Vice President of Chemical Research at
SPRI. He received his B.S. in Chemistry and Biochemistry from
SUNY, Stony Brook, in 1976 and his Ph.D. in Organic Chemistry
in 1980 from Yale University working with Professor Frederick
Ziegler.
REFERENCES
1 (a) Cohen, J. The Scientific Challenge of Hepatitis C. Science 1999, 285, 26–30. (b)
Alter, M. J.; Kruszon-Moran, D.; Nainan, O. V.; McQuillan, G. M.; Gao, F.; Moyer,
L. A.; Kaslow, R. A.; Margolis, H. S. The Prevalence of Hepatitis C Virus Infection in
the United States, 1988 through 1994. N. Engl. J. Med. 1999, 341, 556–562. (c)
Cuthbert, J. A. Hepatitis C: Progress and Problems. Clin. Microbiol. Rev. 1994, 7,
505–532.
2 (a) Hoofnagle, J. H. Course and Outcome of Hepatitis C. Hepatology 2002, 36, S21–
S29. (b) Alter, H. J.; Seeff, L. B. Recovery, Persistence, and Sequelae in Hepatitis C
Virus Infection: A Perspective on Long-Term Outcome. Semin. Liver Dis. 2000, 20,
17–35. (c) Brown, R. S., Jr.; Gaglio, P. J. Scope of Worldwide Hepatitis C Problem.
Liver Transplant. 2003, 9, S10–S13.
3 (a) Feld, J. J.; Hoofnagle, J. H. Mechanism of Action of Interferon and Ribavirin in
Treatment of Hepatitis C. Nature 2005, 436, 967–972. (b) Neumann, A. U.; Lam,
N. P.; Dahari, H.; Gretch, D. R.; Wiley, T. E.; Layden, T. J.; Perelson, A. S. Hepatitis
C Virus Dynamics in Vivo and the Antiviral Efficacy of Interferon-R Therapy.
Science 1998, 282, 103–107. (c) Fried, M. W.; Shiffman, M. L.; Reddy,
D. K. R.; Smith, C.; Marinos, G.; Gonçales, F. L.; Häussinger, D.; Diago, M.;
Carosi, G.; Dhumeaux, D.; Craxi, A.; Lin, A.; Hoffman, J.; Yu, J. PEGinterferon
R-2a plus Ribavirin for Chronic Hepatitis C Virus Infection. New Engl. J. Med.
2002, 347, 975–982.
4 Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Isolation of a cDNA Clone Derived from a Blood-Borne Non-A, Non-B Viral Hepatitis
Genome. Science 1989, 244, 359–362.
5 (a) Lindenbach, B. D.; Rice, C. M. Unravelling Hepatitis C Virus Replication from
Genome to Function. Nature 2005, 436, 933–938. (b) Kolykhalov, A. A.; Mihalik, K.;
Feinstone, S. M.; Rice, C. M. Hepatitis C Virus-Encoded Enzymatic Activities and
Conserved RNA Elements in the 3′ Nontranslated Region Are Essential for Virus
Replication in Vivo. J.Virol. 2000, 74, 2046–2051.
6 (a) Yan, Y.; Li, Y.; Munshi, S.; Sardana, V.; Cole, J. L.; Sardana, M.; Steinkuehler, C.;
Tomei, L.; De Francesco, R.; Kuo, L. C.; Chen, Z. Complex of NS3 Protease and
NS4A Peptide of BK Strain Hepatitis C Virus: A 2.2 Å Resolution Structure in a
Hexagonal Crystal Form. Protein Sci. 1998, 7, 837–847. (b) Love, R. A.; Parge,
H. E.; Wichersham, J. A.; Hostomsky, Z.; Habuka, N.; Moomaw, E. W.; Adachi, T.;
Hostomska, Z. The Crystal Structure of Hepatitis C Virus NS3 Proteinase Reveals a
Trypsin-Like Fold and a Structural Zinc Binding Site. Cell 1996, 87, 331–342.
7 (a) Behrens, S. E.; Tomei, L.; De Francesco, R. Identification and Properties of the
RNA-Dependent RNA Polymerase of Hepatitis C Virus. EMBO J. 1996, 15, 12–22.
58
ACCOUNTS OF CHEMICAL RESEARCH
50-59
January 2008
Vol. 41, No. 1
(b) Lesburg, C. A.; Cable, M. B.; Ferrari, E.; Hong, Z.; Mannarino, A. F.; Weber, P. C.
Crystal Structure of the RNA-Dependent RNA Polymerase from Hepatitis C Virus
Reveals a Fully Encircled Active Site. Nat. Struct. Biol. 1999, 6, 937–943.
8 (a) Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.;
Bös, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A-M.; Goudreau,
N.; Kawai, S. H.; Kukolj, G.; Lagacé, L.; LaPlante, S. R.; Narjes, H.; Poupart, M-A;
Rancourt, J.; Sentjens, R. E.; George, T, S.; Simoneau, B.; Steinmann, G.; Thibeault,
D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C-L.; Llinàs-Brunet, M. An NS3 Protease
Inhibitor with Antiviral Effects in Humans Infected with Hepatitis C Virus. Nature
2003, 426, 186–189. (b) Perni, R. B.; Almquist, S. J.; Byrn, R. A.; Chandorkar, G.;
Chaturvedi, P. R.; Courtney, L. F.; Decker, C. J.; Dinehart, K.; Gates, C. A.;
Harbeson, S. L.; Heiser, A.; Kalkeri, G.; Kolaczkowski, E.; Lin, K.; Luong, Y.-P.; Rao,
B. G.; Taylor, W. P.; Thomson, J. A.; Tung, R. D.; Wei, Y.; Kwong, A. D.; Lin, C.
Preclinical Profile of VX-950, a Potent, Selective, And Orally, Bioavailable Inhibitor of
Hepatitis C Virus NS3-NS4A Serine Protease. Antimicrob. Agents Chemother.
2006, 50, 899–909. (c) Saha, A. K.; Young, C.; Del Vecchio, A. M.; Bailey, T. A.;
Reinhardt, J. A.; Kulkarni, B. A.; Faitg, T. H.; Feng, H.; Rippin, S. R.; Blackledge,
C. W.; Rys, D. J.; Lessen, T. A.; Swestock, J.; Deng, Y.; Nitz, T. J.; Chunduru, S.;
Chopra, R.; Collett, M.; Pevear, D.; Howe, A. Y. M.; O’Connell, J.; Mansour, T.;
Burns, C. J. Discovery of Hcv – 796: A Potent and Orally Bioavailable Hepatitis-C
Polymerase Inhibitor under Clinical Development. Abstracts of Papers, 233rd ACS
National Meeting, Chicago, IL, USA, March 25–29, 2007.
9 Houghton, M.; Abrignani, S. Prospects for a Vaccine against the Hepatitis C Virus.
Nature 2005, 436, 961–966.
10 Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.; Bartenschlager, R.
Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line. Science
1999, 285, 110–113.
11 Mercer, D. F.; Schiller, D. E.; Elliott, J. F.; Douglas, D. N.; Hao, C.; Rinfret, A.;
Addison, W. R.; Fischer, K. P.; Churchill, T. A.; Lakey, J. R. T.; Tyrrell, D. L. J.;
Kneteman, N. M. Hepatitis C Virus Replication in Mice with Chimeric Human Livers.
Nat. Med. 2001, 7, 927–933.
12 Walker, C. M. Comparative Features of Hepatitis C Virus Infection in Humans and
Chimpanzees. Springer Semin. Immunopathol. 1997, 19, 85–98.
13 For a review, see: De Francesco, R.; Migliaccio, G. Challenges and Successes in
Developing New Therapies for Hepatitis C. Nature 2005, 436, 953–960.
14 (a) Llinas-Brunet, M.; Bailey, M. D.; Fazal, G.; Goulet, S.; Halmos, T.; LePlante, S.;
Maurice, R.; Poirier, M.; Poupart, M.-A.; Thibeault, D.; Wernic, D.; Lamarre, D.
Peptide-Based Inhibitors of the Hepatitis C Virus Serine Protease. Bioorg. Med.
Chem. Lett. 1998, 8, 1713–1718. (b) Steinkuhler, C.; Biasiol, G.; Brunetti, M.;
Urbani, A.; Koch, U.; Cortese, R.; Pessi, A.; De Francesco, R. Product Inhibition of
the Hepatitis C Virus NS3 Protease. Biochemistry 1998, 37, 8899–8905.
15 (a) Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, J.; Liu,
Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.;
Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.; Liang,
X.; Wong, J.; Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agrawal, S.; Ingravallo, P.;
Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay, A.; Madison, V.;
Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, Y.; Brisson, J.-M.; Prelusky, D.;
Korfmacher, W.; White, R.; Bogdanowich-Knipp, S.; Pavlovsky, A.; Bradley, P.;
Saksena, A.; K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.; Njoroge, F. G.
Discovery of (1R,5S)-N-[3-Amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]-3-[2(S)[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-6,6-dimethyl3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a Selective, Potent,
Orally Bioavailable Hepatitis C Virus NS3 Protease Inhibitor: A Potential Therapeutic
Agent for the Treatment of Hepatitis C Infection. J. Med. Chem. 2006, 49, 6074–
6086. (b) Malcolm, B. A.; Liu, R.; Lahser, F.; Agrawal, S.; Belanger, B.; Butkiewicz,
N.; Chase, R.; Gheyas, F.; Hart, A.; Hesk, D.; Ingravallo, P.; Jiang, C.; Kong, R.; Lu,
J.; Pichardo, J.; Prongay, A.; Skelton, A.; Tong, X.; Venkatraman, S.; Xia, E.;
Girijavallabhan, V.; Njoroge, F. G. SCH 503034, a Mechanism-Based Inhibitor of
Hepatitis C Virus NS3 Protease, Suppresses Polyprotein Maturation and Enhances
the Antiviral Activity of Alpha Interferon in Replicon Cells. Antimicrob. Agents
Chemother. 2006, 50, 1013–1020.
16 For reviews about protease inhibitors, see: (a) Turk, B. Targeting Proteases:
Successes, Failures and Future Prospects. Nat. Rev. Drug Discovery 2006, 5, 785–
799. (b) Tyndall, J. D.; Nall, T.; Fairlie, D. P. Proteases Universally Recognize Beta
Strands in Their Active Sites. Chem. Rev. 2005, 105, 973–999. (c) Powers, J. C.;
Asgian, J. L.; Ekici, D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine, and
Threonine Proteases. Chem. Rev. 2002, 102, 4639–4643. (d) Leung, D.;
Abbenante, G.; Fairlie, D. P. Protease Inhibitors: Current Status and Future
Prospects. J. Med. Chem. 2000, 43, 305–341.
17 Zhang, R.; Beyer, B. M.; Durkin, J.; Ingram, R.; Njoroge, F. G.; Windsor, W. T.;
Malcolm, B. A. A Continuous Spectrophotometric Assay for the Hepatitis C Virus
Serine Protease. Anal. Biochem. 1999, 270, 268–275. The substrate AcDTEDVVP(Nva)-O-PAP was used in the present study..
Challenges in Modern Drug Discovery Njoroge et al.
18 For a definition of Ki* and discussions, see: Morrison, J. F.; Walsh, C. T. In Advances
in Enzymology and Related Areas of Molecular Biology, Meister, A., Ed.; John Wiley
& Sons, Inc., Hoboken, NJ, 1988; Vol 61, pp 201–301.
19 Schechter, I.; Berger, A. On the size of the active site in proteases. I. Papain.
Biochem. Biophys. Res. Commun. 1967, 27, 157–162.
20 Bogen, S.; Ruan, R.; Saksena, A. K.; Njoroge, F. G.; Girijavallabhan, V.; Agrawal, S.;
Liu, R.; Pichardo, J.; Baroudy, B.; Prongay, A. Depeptidization Efforts on P3-P2′ RKetoamide Inhibitors of HCV NS3-NS4A Serine Protease: Effect on HCV Replicon
Activity. Bioorg. Med. Chem. Lett. 2006, 16, 1621–1627.
21 Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu, Y.-T.; Lovey, R. G.;
Venkatraman, S.; Pan, W.; Parekh, T.; Pike, R. E.; Ruan, S.; Liu, R.; Baroudy, B.;
Agrawal, S.; Ingravallo, P.; Pichardo, J.; Prongay, A.; Brisson, J.-M.; Hsieh, T. Y.;
Cheng, K.-C.; Kemp, S. J.; Levy, O. E.; Lim-Wilby, M.; Tamura, S. Y.; Saksena,
A. K.; Girijavallabhan, V.; Njoroge, F. G. Discovery of SCH 446211(SCH6): A New
Ketoamide Inhibitor of the HCV NS3 Serine Protease and HCV Subgenomic RNA
Replication NS3 Serine. J. Med. Chem. 2006, 49, 2750–2757.
22 Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and
Computational Approaches to Estimate Solubility and Permeability in Drug Discovery
and Development Settings. Adv. Drug Delivery Rev. 1997, 23, 3–25.
23 Chen, K. X.; Njoroge, F. G.; Arasappan, A.; Venkatraman, S.; Vibulbhan, B.; Yang, W.;
Parekh, T. N.; Pichardo, J.; Prongay, A.; Cheng, K.-C.; Butkiewicz, N.; Yao, N.; Madison,
V.; Girijavallabhan, V. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors
Derived from Proline-Based Macrocycles. J. Med. Chem. 2006, 49, 995–1005.
24 Velazquez, F.; Venkatraman, S.; Wu, W.; Blackman, M.; Prongay, A.; Girijavallabhan,
V.; Shih, N.-Y.; Njoroge, F. G. Application of Ring-Closing Metathesis for the
Synthesis of Macrocyclic Peptidomimetics as Inhibitors of HCV NS3 Protease. Org.
Lett. 2007, 9, 3061–3064.
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CRITICAL REVIEW AND INVITED COMMENTARY
Brivaracetam: Rationale for discovery and preclinical
profile of a selective SV2A ligand for epilepsy treatment
Henrik Klitgaard, Alain Matagne, Jean-Marie Nicolas, Michel Gillard, Yves Lamberty, Marc De
Ryck, Rafal M. Kaminski, Karine Leclercq, Isabelle Niespodziany, Christian Wolff, Martyn Wood,
Jonas Hannestad, Sophie Kervyn, and Benoit Kenda
Epilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
SUMMARY
Dr. Henrik Klitgaard
is Vice President,
Research Fellow in
New Medicines at
UCB, Braine-l’Alleud,
Belgium.
Despite availability of effective antiepileptic drugs (AEDs), many patients with epilepsy
continue to experience refractory seizures and adverse events. Achievement of better
seizure control and fewer side effects is key to improving quality of life. This review
describes the rationale for the discovery and preclinical profile of brivaracetam (BRV),
currently under regulatory review as adjunctive therapy for adults with partial-onset
seizures. The discovery of BRV was triggered by the novel mechanism of action and
atypical properties of levetiracetam (LEV) in preclinical seizure and epilepsy models.
LEV is associated with several mechanisms that may contribute to its antiepileptic
properties and adverse effect profile. Early findings observed a moderate affinity for a
unique brain-specific LEV binding site (LBS) that correlated with anticonvulsant
effects in animal models of epilepsy. This provided a promising molecular target and
rationale for identifying selective, high-affinity ligands for LBS with potential for
improved antiepileptic properties. The later discovery that synaptic vesicle protein 2A
(SV2A) was the molecular correlate of LBS confirmed the novelty of the target. A drug
discovery program resulted in the identification of anticonvulsants, comprising two
distinct families of high-affinity SV2A ligands possessing different pharmacologic properties. Among these, BRV differed significantly from LEV by its selective, high affinity
and differential interaction with SV2A as well as a higher lipophilicity, correlating with
more potent and complete seizure suppression, as well as a more rapid brain penetration in preclinical models. Initial studies in animal models also revealed BRV had a
greater antiepileptogenic potential than LEV. These properties of BRV highlight its
promising potential as an AED that might provide broad-spectrum efficacy, associated
with a promising tolerability profile and a fast onset of action. BRV represents the first
selective SV2A ligand for epilepsy treatment and may add a significant contribution to
the existing armamentarium of AEDs.
KEY WORDS: Anticonvulsants, Epilepsy, Levetiracetam, Preclinical, Synaptic vesicle
protein 2A.
Accepted January 26, 2016; Early View publication February 26, 2016.
UCB Pharma, Braine l’Alleud, Belgium
Address correspondence to Henrik Klitgaard, UCB Biopharma SPRL,
New Medicines, Chemin du Foriest, B-1420 Braine-l’Alleud, Belgium.
E-mail: henrik.klitgaard@ucb.com
© 2016 The Authors. Epilepsia published by Wiley Periodicals, Inc. on
behalf of International League Against Epilepsy.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the
use is non-commercial and no modifications or adaptations are made.
Antiepileptic drugs (AEDs) provide relief from seizures
in many forms of epilepsy; approximately two thirds of
patients with new-onset epilepsy will achieve adequate seizure control.1,2 Despite their effectiveness in the majority of
patients, current AEDs are unable to provide effective seizure control in up to one third of patients.2,3 Moreover,
about half of all treated patients experience at least one
adverse event during first-line AED treatment.4,5 Epilepsy
has a significant impact on health-related quality of life,
affecting both social and psychological well-being6; conse-
538
539
Brivaracetam Discovery and Preclinical Profile
Key Points
• BRV is a selective, high-affinity SV2A ligand with
promising antiepileptic properties and a fast onset of
action
• BRV provides potent and complete seizure suppression in animal models of partial, generalized, and
drug-resistant seizures
• BRV is currently under regulatory review as adjunctive therapy for adults with POS
• Preclinical findings suggest BRV may provide an
effective and well-tolerated treatment and, if approved
by regulatory bodies, would make a significant contribution to the existing armamentarium of AEDs
• BRV is currently being tested in a number of longterm follow-up studies in patients with refractory epilepsy
quently, reduction of side effects and achievement of better
seizure control are key to improving patients’ quality of
life.7 Therefore, more effective, better-tolerated treatments
for epilepsy remains an important unmet need.
Brivaracetam (BRV) is one of the first AEDs in development that was discovered during a large-scale drug discovery program aimed at optimizing pharmacodynamic activity
at a novel molecular AED target. BRV is currently under
regulatory review as adjunctive therapy for adults with partial-onset (focal) seizures (POS). It has demonstrated efficacy and high tolerability in patients with POS in both phase
IIb8,9 and phase III10–12 studies with treatment periods of up
to 12 weeks. Efficacy in primary generalized seizures
remains to be established. Owing to the chronic nature of
epilepsy, treatment will be required over substantially
longer periods. To this end, BRV is currently being evaluated in a number of long-term safety and efficacy studies in
patients with POS only (NCT01728077 and NCT01339559)
and in patients with POS and primary generalized seizures
(NCT00175916).
This review describes the rationale for the discovery of
BRV and how it was derived from a major drug discovery
program, which was triggered by the novel mechanism of
action and atypical antiepileptic properties of levetiracetam
(LEV) in preclinical seizure and epilepsy models.
Discovery of SV 2 A Ligands for
Treatment of Epilepsy
During the last 50 years, chemical research at UCB has
identified several central nervous system drugs based on
modification of the pyrrolidone-acetamide scaffold.13
Piracetam was the first to be discovered, being synthesized
in 1964 in a project targeting sleep induction by c-aminobu-
tyric acid (GABA) analogs.14 However, it did not show any
effects on the GABA system or sleep but instead revealed
atypical psychotropic properties.15 In 1972, these properties
were proposed to derive from interference with higher
telencephalic integrative activity by a direct and selective
action. Thus, piracetam constitutes the founding agent in a
new class of psychotropic drugs, which were termed
“nootropics” (“noos” = mind and “tropein” = towards).15
The ensuing scientific and commercial interest in the
nootropic concept triggered attempts to identify a successor
to piracetam.16 Etiracetam, an ethyl analog of piracetam,
was synthesized in 1966; because of technical limitations at
that time, it was not separated into its S- (ucb L059, LEV)
and R-enantiomers (ucb L060) until 1977.17 During the
1980s, clinical trials failed to detect a beneficial effect of
LEV in patients with cognitive impairment. However, during the 1990s, an interest in epilepsy emerged; this was supported by the clinical activity of piracetam in the treatment
of myoclonus14 and the preclinical screening of LEV, which
revealed anticonvulsant properties in sound-sensitive
mice.17
The interpretation of these observations was complicated
by the lack of anticonvulsant activity of LEV in conventional screening models for AEDs (e.g., maximal electroshock [MES] and subcutaneous pentylenetetrazole [PTZ]
seizure tests).18,19 The validity of these tests has been supported for many decades; commonly used AEDs show anticonvulsant activity in at least one of these models, and their
activity profile is assumed to be predictive of their clinical
efficacy profile in patients with epilepsy.20 This screening
paradigm was challenged by the disparate findings with
LEV, particularly because continued testing demonstrated
LEV to possess significant broad-spectrum protection in
various animal models of both partial and primary generalized epilepsy at doses associated with only minor behavioral
changes.18,19 Further complicating these findings, electrophysiologic recordings in vitro and in vivo consistently
showed an absence of intrinsic activity of LEV on neuronal
function. This contrasted with a significant effect against
epileptiform activity, which coincided with an absence of
interaction of LEV with ion channels and receptor targets
typically involved in the action of conventional AEDs.16,17
Taken together, these findings revealed LEV to possess a
unique preclinical profile, with selective action against
abnormal patterns of neuronal activity leading to selective
seizure protection with an unusually high therapeutic index
in “epileptic” animals. These atypical antiepileptic properties led to the suggestion that LEV might represent the first
agent in a new class of AEDs.21
The unique pharmacologic profile of LEV suggested the
involvement of a novel mechanism of action different from
that of conventional AEDs.17 Indeed, binding studies
revealed a reversible, saturable, and stereoselective
brain-specific LEV binding site (LBS) in the rat.22 This
LBS was expressed ubiquitously throughout the brain, being
Epilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
540
H. Klitgaard et al.
preferentially localized on synaptic plasma membranes with
high prevalence in a number of brain structures, including
the hippocampus, cortex, and cerebellum. Known AEDs did
not bind to the LBS, and testing of a series of LEV analogs
in the audiogenic mouse model revealed a correlation
between their affinities for the LBS and their anticonvulsant
activity,22 a finding that has now been extended to other animal models of epilepsy.23 Further characterization of the
LBS identified it as synaptic vesicle protein 2A (SV2A).24
Subsequent studies have shown the anticonvulsant activity
of LEV to be reduced in SV2A knockout (+/ ) mice, partly
deficient in the SV2A protein, with a consistent reduction in
binding to SV2A.25 These findings provided further support
for the LBS as a valid and novel target for AED discovery.23,26 However, LEV possesses several additional mechanisms and has only modest affinity for SV2A.16 Therefore,
it was speculated that a selective, high-affinity ligand for
SV2A may optimize the therapeutic benefit of this novel
mechanism and may provide improved antiepileptic properties. Significant support for this assumption was provided
by the correlation between the affinity of LEV analogs for
SV2A and their anticonvulsant activity.22
Thus, a major drug discovery program was triggered at
UCB with the purpose of identifying selective, high-affinity
SV2A ligands possessing antiepileptic properties superior
to LEV (Fig. 1).13 Approximately 12,000 compounds were
screened in vitro for SV2A binding affinity; 1,200 were further screened in vivo for seizure protection in audiogenic
seizure-susceptible mice, and approximately 30 compounds
were selected and broadly characterized in a variety of animal models of seizures and epilepsy.27 This led to the identification of two distinct anticonvulsant families with high
affinity for SV2A ligands; they were named after their lead
compounds: BRV and seletracetam (SEL). In vitro models
of epilepsy showed that, while both BRV and SEL inhibited
epileptiform hypersynchronization, BRV demonstrated a
more pronounced ability to inhibit neuronal hyperexcitability than SEL.28 This likely explains BRV’s seizure protection in the MES and subcutaneous PTZ tests, albeit at
relatively high doses, and its significant protection against
the partial seizure phase in animal models of partial epilepsy,27 a profile that triggered the decision to focus on
BRV for further clinical development.
Mechanism of Action
SV2A
SV2A is ubiquitously expressed in the brain, and studies
from knockout mice implicate SV2A in the modulation of
synaptic vesicle exocytosis and neurotransmitter
release.29,30 Characterization of the binding properties of
BRV in rat and human cerebral cortex indicated that tritiated BRV ([3H]BRV) binds to a homogenous population of
binding sites with the characteristics of SV2A.31 Further
characterization revealed BRV to be a selective, high-affinity SV2A ligand.
The in vitro binding of [3H]BRV in rat and human brain
showed that, like LEV,32 BRV displays identical affinity for
rat and human SV2A31; however, it has a 15- to 30-fold
increased affinity for SV2A compared with LEV
(Table 1).31 Furthermore, results from SV2A / knock-out
mice clearly show that up to concentrations of 600 nM, [3H]
BRV specifically labels SV2A proteins: no binding could
be observed in the brains of these knockout mice.31 Given
the sensitivity of the binding assay, the authors suggest that
should BRV bind to other targets, it would probably occur
Table 1. SV2A affinity and effects of BRV and LEV on
HVA Ca2+ channels and AMPA receptors
Property
SV2A affinity
Human cortex; Ki
HVA Ca2+ channel current
IC50 value
AMPA gated current
IC50 value
Figure 1.
Overview of drug discovery efforts at UCB based on modulation
of the pyrrolidone-acetamide scaffold. MES, maximal electroshock
seizure test; PTZ, subcutaneous pentylenetetrazole seizure test;
SV2A, synaptic vesicle protein 2A.
Epilepsia ILAE
Epilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
BRV
0.05 lM
31
LEV
1.6 lM31
Inactive up to 1,000 lM43
13.9 lM83
Inactive up to 100 lM47
268 lM16
AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor;
BRV, brivaracetam; HVA Ca2+ channel, high-voltage-activated calcium channel; IC50, half maximal inhibitory concentration; Ki, inhibitory constant; LEV,
levetiracetam; SV2A, synaptic vesicle protein 2A.
Republished with permission of Wiley-Blackwell from Simon D. Shorvon
(Editor), Emilio Perucca (Editor), Jerome Engel, Jr. (Editor). The Treatment of
Epilepsy, 4th Edition. Wiley Books. Copyright© 1999–2015 John Wiley & Sons,
Inc. All Rights Reserved.
541
Brivaracetam Discovery and Preclinical Profile
in the high micromolar range at concentrations that are most
likely not relevant to its overall mechanism of action.
Indeed, at a concentration of 10 lM (>100-fold higher than
its affinity for SV2A), BRV did not bind, activate, or inhibit
a panel of 55 other receptors, channels, and enzymes.31
Attempts to elucidate the functional consequence(s) of
ligand binding to SV2A involved the electrophysiologic
investigation of LEV on synaptic transmission in rat hippocampal slices. These initial studies showed that LEV
decreased vesicular release in a dose-, time-, and stimulation-dependent manner, suggesting that repetitive stimulation is required to allow LEV entry into recycling synaptic
vesicles and intravesicular LEV binding to SV2A.33 Further
studies comparing BRV and LEV using high-frequency
neuronal stimulation, indicated that BRV augments synaptic depression and thereby decreases synaptic transmission
at 100-fold lower concentrations than LEV (Fig. 2A).34 In
studies with dye-loaded vesicles, BRV slowed stimulationinduced destaining (i.e., slowed vesicle release) more than
LEV (Fig. 2B),34 indicating that BRV reduces vesicle
mobilization more effectively than LEV, which may explain
their distinct pharmacodynamic properties. Of interest, a
recent study has shown LEV to decrease vesicular release
during incipient epileptic activity by accelerating the induction of supply rate depression at excitatory synapses.35 The
potential effect of BRV on synaptic supply rate depression
remains to be determined.
The recent identification of an SV2A allosteric modulator
has allowed the interaction of LEV and BRV with human
SV2A to be compared in recombinant cells expressing
SV2A, and in human cortex.36,37 For [3H]LEV, the modulator produced similar increases in affinity for SV2A (2- to
3-fold) and in maximum binding capacity (2-fold). In
contrast, the modulator increased the affinity of [3H]BRV to
a much greater degree (10-fold) than the increase observed
in maximum binding capacity (1.3-fold) (Fig. 3). The differential effect of the SV2A modulator on the binding of
LEV and BRV suggests that they induce or stabilize different conformations of the SV2A protein, which may provide
the molecular correlate to their distinct pharmacodynamic
properties.
Other mechanisms of action studied
Conventional AEDs modify excitatory neurotransmission by: (1) blocking sodium and/or calcium channels, (2)
antagonizing glutamate receptors, (3) modifying inhibitory
neurotransmission through increases in GABA, or (4)
enhancing GABA receptor function.20 In addition to acting
as an SV2A ligand, it was important to assess whether BRV
may also have an impact on any of these conventional AED
mechanisms.
Figure 2.
Effect of BRV and LEV on synaptic transmission. Repetitive stimulation in hippocampal slices pretreated for 3 h with BRV or LEV
shows a dose-dependent increase in synaptic depression at 40 Hz,
with 100-fold greater potency for BRV (A). De-staining curves
show a decrease in intensity of the vesicle marker dye, FM1-43,
under continual stimulation (1 Hz) in the absence (control) and
presence of maximal concentrations of BRV or LEV (B). BRV, brivaracetam; fEPSP, electrophysiologic field excitatory postsynaptic
potential; LEV, levetiracetam. Republished with permission of
Wiley-Blackwell from Yang X, et al.34 Copyright© 1999–2015
John Wiley & Sons, Inc. All Rights Reserved.
Epilepsia ILAE
Figure 3.
Differential interaction of BRV and LEV with SV2A. Combination
experiments assessing the impact of the allosteric SV2A modulator
UCB 1244283 on [3H]-BRV and [3H]-LEV binding in recombinant
cells expressing SV2A. BRV, brivaracetam; Bmax, maximum number
of binding sites; Kd, dissociation constant; LEV, levetiracetam;
SV2A, synaptic vesicle protein 2A
Epilepsia ILAE
Epilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
542
H. Klitgaard et al.
Voltage-gated ion channels
An initial report suggested that inhibition of voltagegated sodium channels may contribute to the antiepileptic
activity of BRV (Table 2).38 At low (micromolar) concentrations, BRV was shown to inhibit voltage-gated sodium
currents by 65% in rat primary cortical neurons.38 However,
subsequent studies indicated weaker effects of BRV on voltage-gated sodium currents (~20%, Table 2) in primary cortical neurons, which triggered experiments to determine
whether this minor inhibition of voltage-gated sodium channels is sufficient to modulate the intrinsic firing properties
of neurons. Indeed, a well-known feature of AEDs with a
primary mechanism of action on voltage-gated sodium
channels is an efficient inhibition of sustained repetitive firing. However, additional studies with BRV in this experimental paradigm, even at supratherapeutic concentrations,
showed BRV to be ineffective in reducing the neuronal firing (Table 2).39 This later study confirmed that minor inhibition of the transient currents of voltage-gated sodium
channels by BRV does not appear to translate into physiologically relevant effects on the intrinsic firing properties of
neurons.40 Another study established the lack of effect of
BRV on the amplitude of the persistent voltage-gated
sodium currents in CA1 neurons.41 Altogether, these findings suggest that the antiepileptic mechanism of BRV is
unrelated to inhibition of voltage-gated sodium channels.
Further studies on other potential AED mechanisms,
including voltage-gated potassium channels41,42 and both
high and low voltage-gated calcium channels (Table 1),41,43
have shown no effect of BRV at therapeutically relevant
concentrations. In contrast, LEV inhibits neuronal high voltage-gated calcium currents in rat CA1 hippocampal neurons44 and superior cervical ganglia neurons45 at clinically
relevant concentrations; an effect that appears to be selective to the N-type calcium channel.46
Table 2. Effects of BRV on voltage-gated sodium
currents and on sustained repetitive firing in neurons
Effect
INa
65% inhibition (inactivated and resting states) at 200 lM
(0.2–500 lM) in embryonic rat primary cortical neurons38
30% inhibition at 100 lM (1–100 lM) in mouse neuroblastoma
N1E-115 cell line
20% inhibition (inactivated state only) at 300 lM in embryonic
rat primary cortical neurons
No effect at 300 lM in adult mouse CA1 neurons39
No effect at 300 lM in entorhinal cortex neurons from sham and
pilocarpine mice40
INaP
No effect at 20 lM in adult rat CA1 neurons41
SRF
No effect at 300 lM in entorhinal cortex neurons from sham and
pilocarpine mice40
No effect at 300 lM in embryonic rat primary cortical neurons
No effect at 100 lM in adult mouse CA1 neurons39
BRV, brivaracetam; INa, fast transient voltage-gated sodium current;
INaP, persistent voltage-gated sodium current; SRF, sustained repetitive firing.
Epilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
Excitatory and inhibitory receptors
BRV is devoid of direct action on inhibitory and excitatory receptors including a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA), glycine, and GABAA,
with the exception of a weak minor inhibition of the Nmethyl-D-aspartate receptor current at supratherapeutic
concentrations.47 Although it shares with LEV an ability to
oppose the action of negative modulators on the two main
inhibitory receptors, GABAA and glycine, the clinical relevance of this finding remains elusive.47 Unlike LEV,48
BRV does not inhibit AMPA receptors (Table 1).47
Taken together, these studies support the absence of any
relevant contribution of conventional AED mechanisms to
the antiepileptic properties of BRV and suggest it represents
the first, selective SV2A ligand for epilepsy treatment. This
contrasts with LEV, which possesses several other pharmacologic mechanisms (Table 1). The selectivity of BRV may
be associated with fewer clinical adverse effects. This is
supported by findings obtained from a small, open-label
study showing that epilepsy patients experiencing nonpsychotic behavioral adverse events associated with LEV did
benefit from switching to BRV.49
Blood–Brain Barrier
Permeability and
Pharmacokinetics
In vitro investigations revealed that BRV has a high passive diffusion permeability across cell membranes, superior
to that of LEV, with no evidence of transporter-mediated
extrusion from the brain;50 properties suggesting fast and
unrestricted entry of BRV across the blood–brain barrier.
These in vitro data were confirmed in subsequent pharmacokinetic/pharmacodynamic experiments in audiogenic
mice.51 After a single oral dose of BRV, maximal pharmacologic activity was simultaneous with maximal plasma
concentrations. In contrast, LEV peak activity followed
peak plasma levels by almost 1 h. The faster onset of action
of BRV was found to originate from its immediate distribution into brain tissues, a property not entirely shared by
LEV. Similarly, ex vivo binding experiments in the same
audiogenic mouse model showed maximal SV2A occupancy and maximal activity within 5–15 min following
intraperitoneal (i.p.) dosing with BRV, compared with
30–60 min for LEV.31 Positron emission tomography
(PET) imaging in rhesus monkeys has also confirmed the
faster brain entry of BRV.51 BRV and LEV were compared
for their ability to displace the SV2A PET tracer [11C]UCB-J
after a single intravenous (i.v.) dose. Estimated half-times,
describing brain entry for BRV and LEV after correction for
the half-time of tracer clearance, were approximately 3 min
and 27 min, respectively (Fig. 4).51
Lipophilicity is a key attribute for drug penetration into
the brain, with optimal permeability at LogD values
543
Brivaracetam Discovery and Preclinical Profile
Figure 4.
Displacement of the SV2A PET tracer [11C]UCB-J after injection of
5 mg/kg BRV or 30 mg/kg LEV (administered i.v. 45 min after the
PET tracer). The displacement graph shows the amount of radioactivity in the brain over time. The baseline curve (black squares)
shows that the tracer by itself leaves the brain very slowly (there is
a gradual reduction in radioactivity over time). When BRV or LEV
is administered, the radioactivity in the brain is quickly reduced
(dark blue circles and light blue triangles, respectively). This is
because BRV and LEV displace the tracer. The BRV curve is steeper
than the LEV curve, which means that BRV displaces the tracer faster than LEV does; therefore, it enters the brain faster than LEV.
The steepness of these curves was used to estimate the speed of
entry of LEV and BRV. BRV, brivaracetam; i.v., intravenous; LEV,
levetiracetam; PET, positron emission tomography; SV2A, synaptic
vesicle protein 2A; TAC, time-activity curve. Republished with permission of Wiley-Blackwell from Jean-Marie Nicolas, et al.51 Copyright© 1999–2015 John Wiley & Sons, Inc. All Rights Reserved.
Epilepsia ILAE
between one and three.52 It is thought that BRV has an optimal lipophilicity (logDpH7.4 = 1.04 vs. 0.64 for LEV),51
that is, high enough to favor cell membrane penetration but
below levels associated with water solubility and formulation issues (as observed with phenytoin and most benzodiazepines). This appears to be supported by the in vivo
blood–brain barrier permeability measured for BRV in
rodents, which not only exceeds that of LEV (0.315 vs.
0.015 mL/min/g brain, respectively) but, more importantly,
approaches that measured for fast-acting AEDs used in the
clinical management of acute seizures (Fig. S1).51
Physiologically based pharmacokinetic modeling was
used to predict brain kinetics in humans. Following intravenous administration of 100 mg BRV to patients, brain
concentrations should peak within minutes, compared with
approximately 1 h after 1,500 mg LEV.51 The rapid brain
entry of BRV consistently observed across in silico, in vitro,
and in vivo preclinical studies remains to be confirmed in
the clinical setting.
BRV is rapidly and completely absorbed after oral dosing, has low plasma protein binding, and is associated with
low clearance across species.13,53,54 BRV is eliminated
primarily by metabolism: the major metabolic pathway
involves non-cytochrome P450 (CYP)–dependent hydrolysis of the acetamide group resulting in formation of an acid
metabolite.53 The acid metabolite subsequently undergoes
CYP2C9-mediated hydroxylation leading to the formation
of a hydroxyacid metabolite.55 A secondary pathway, mediated mainly by CYP2C19,56 involves b-oxidation of the
propyl sidechain to form a hydroxy metabolite.53 From the
in vitro metabolism data, BRV is not expected to show clinically relevant variability in its disposition (between individuals, sexes, ethnic groups, or disease states). All three
metabolites of BRV are pharmacologically inactive.
BRV was found to be devoid of relevant drug–drug interaction risks,50 an important factor when considering the
potentially complex drug regimens of patients with chronic
epilepsy and other concomitant conditions. BRV neither
inhibits nor induces CYPs in a clinically relevant manner,
nor does it inhibit drug transporters, and its metabolism is
not affected by reference AEDs including felbamate, phenytoin, valproate, lamotrigine, zonisamide, and phenobarbital.50 The only noticeable finding is a modest increase in
carbamazepine-epoxide when BRV is coadministered with
carbamazepine.57,58 This interaction is without safety consequences and does not require dose adjustment.
Pharmacodynamic Effects
Anticonvulsant efficacy in animal models of seizures and
epilepsy
The increased affinity for SV2A of BRV over LEV
shown in vitro correlates well with observations from various in vivo models of epilepsy that show BRV to have a
markedly higher potency over LEV for seizure protection
(Table S1). Furthermore, in contrast to LEV, BRV is also
effective against seizures induced in the classical MES and
PTZ seizure tests, albeit at relatively high doses.
BRV is more potent than LEV in protecting against secondary generalized seizures in animal models of partial epilepsy. In both corneal-kindled mice and hippocampal-kindled
rats, BRV provided more potent protection than LEV against
secondary generalized seizures.59 BRV displayed dosedependent protection against 6 Hz seizures in mice with a
potency several-fold higher than that of LEV.60 Studies using
a newly developed 6 Hz corneal-kindling model in mice confirmed the high potency and efficacy of BRV against both
partial and secondarily generalized seizures.61
Both BRV and LEV have been shown to reduce epileptiform responses in rat hippocampal slices; however, BRV
possessed both higher potency and higher efficacy than
LEV.59 BRV has also shown more complete seizure suppression than LEV in drug-resistant seizure models. In
amygdala-kindled mice, BRV (6.8–210 mg/kg) doseEpilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
544
H. Klitgaard et al.
Figure 5.
Seizure suppression by BRV and LEV
in mouse (A) and rat (B) amygdalakindling models (suprathreshold
stimulation). Effect of BRV and LEV
on seizure parameters in fully
amygdala-kindled mouse (A) and rat
(B). The behavioral effect of
stimulation was measured by a
seizure severity score, according to
the scale of Racine, and the
electroencephalographic effect was
determined by measuring the
duration of the stimulation-induced
afterdischarge. BRV, brivaracetam;
i.p., intraperitoneal; LEV,
levetiracetam. Panel B only: Matagne
A, et al.59 © 2008 Nature Publishing
Group. All rights reserved 0007–
1188/08.
Epilepsia ILAE
dependently reduced seizure severity, with nearly complete
suppression of seizures observed at the highest tested dose
(Fig. 5A).62 In contrast, LEV (17–540 mg/kg) provided
only limited protection at the highest dose tested
(Fig. 5A).62 Furthermore, BRV produced a very strong
reduction in the duration of the afterdischarge, whereas
LEV was inactive on this parameter (Fig. 5A). BRV also
contrasted with LEV by inducing a nearly complete suppression of motor seizure severity and significantly reducing the afterdischarge duration in fully amygdala-kindled
rats (Fig. 5B).59
Experiments in genetic models of epilepsy have also
shown significant anticonvulsant effects of BRV. Potent
and complete suppression of seizure activity was observed
in audiogenic seizure-susceptible mice.13,59 In contrast to
LEV, BRV was also found to completely suppress spontaneous spike-and-wave discharges in the Genetic Absence
Epilepsy Rat from Strasbourg, a model thought to be predictive of activity against absence epilepsy.63 In addition, in a
rat model of cardiac arrest induced posthypoxic myoclonus,
BRV displayed a 10-fold higher potency than LEV against
myoclonus and generalized seizures.64
BRV has also shown potent anticonvulsant effects in animal models of self-sustaining status epilepticus (SSSE)
(Fig. S2). In adult rats, SSSE can be induced by intermittent
stimulation of the perforant path through chronically
implanted electrodes. Intravenous BRV shortened the
cumulative duration of active seizures in this model to 11%
and 0.8% of control at doses of 20 and 300 mg/kg, respectively, compared with a reduction to 35% of control with
LEV 200 mg/kg.65 The greater efficacy of BRV compared
Epilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
with LEV is likely to be related, at least in part, to its more
rapid entry into the brain and was markedly enhanced when
BRV was combined with low-dose diazepam.66 Further
studies demonstrated that, in addition to short-term effects
against SSSE, BRV also reduced the number of chronic
spontaneous recurrent seizures occurring both 6 weeks and
1 year after treatment.66 This suggests that BRV has both
anticonvulsant and disease-modifying effects against
refractory SSSE in rats.
The comparative studies between BRV and LEV
reviewed earlier were conducted by different laboratories
using animal models that express different seizure types and
involving different species and varying pretreatment times.
This resulted in some variability for the potency difference
obtained between BRV and LEV. However, the overall pattern of the findings is suggestive of a broad-spectrum activity profile of BRV after systemic administration with more
potent protection than LEV against both primary and secondary generalized seizure types and more complete suppression than LEV, in particular, against POS. This likely
reflects the more potent inhibition of synaptic transmission
and the more effective reduction in vesicle mobilization and
release by BRV, due to its higher affinity and differential
SV2A interaction compared with LEV. Taken together,
these findings suggest that BRV may have potential to benefit epilepsy patients with uncontrolled seizures despite treatment with current AEDs. This seems supported by recent
data from a major clinical study with adjunctive BRV in
adult epilepsy patients with uncontrolled POS.12 Although
the findings will need to be confirmed in future clinical trials, a post hoc analysis on the impact of previous LEV expo-
545
Brivaracetam Discovery and Preclinical Profile
sure showed statistically significant seizure reduction in
patients both previously exposed and never exposed to
LEV, suggesting that BRV may confer a benefit to seizure
control in patients who have previously failed to respond to
LEV.
As a result of its fast brain entry and pharmacodynamic
properties, intravenous bolus administration of BRV provides rapid, potent, and complete seizure protection in animal models of status epilepticus. This suggests that BRV
has promising potential for the clinical management of
acute seizures; however, this remains to be confirmed by
clinical studies.51
Antiepileptogenic activity
Current AEDs provide symptomatic benefit by preventing seizures in the majority of patients with epilepsy; most
have also been assessed preclinically for potential
antiepileptogenic properties.67 This research has been facilitated by the availability of various animal models of
acquired and genetic epilepsy.63 LEV has revealed promising antiepileptogenic properties in several of these models68
and in kindling models in particular.67,69 This observation
triggered the first comparative study between BRV and
LEV to assess their ability to inhibit kindling acquisition
during corneal kindling of mice.59
Pretreatment with BRV provided a similar reduction in
the incidence of generalized motor seizures at doses 10
times lower than those of LEV (Fig. S3A & B). The animals
pretreated with the highest dose of BRV (6.8 mg/kg)
showed a persistent reduction in the incidence of generalized motor seizures after cessation of treatment (Fig. S3A);
an effect that was not observed in any animals pretreated
with LEV.59 Evidence from this initial study, and the potential disease-modifying effects observed in the rat SSSE
model, support the continued evaluation of the antiepileptogenic potential of BRV.
Other pharmacologic actions
Pharmacologic studies have also shown that, in contrast
to LEV, BRV shows significant activity in animal models of
experimental pain, tremor, and cortical spreading depression (CSD) (Table S2). Due to its selective SV2A mechanism, this likely reflects the improved ability of BRV to
inhibit synaptic transmission and vesicle release.
As shown for LEV,70 BRV (21–210 mg/kg) did not modify the acute pain response in the hot-plate test in rats. However, in two rat models of neuropathic pain, BRV doses
≥21 mg/kg significantly increased vocalization thresholds
and reversed hyperalgesia in mononeuropathic and diabetic
rats.71 While LEV has been shown to produce a similar
effect, much higher doses (540 mg/kg) are required.70 In a
rat L5/L6 spinal nerve ligation model of neuropathic pain,
BRV displayed slightly more potent antiallodynic activity
compared with gabapentin (GBP).72 Within the dose-range
used (7–68 mg/kg, i.p.), BRV did not produce sedation, as
indicated by an effect restricted to the neuropathic paw. In
contrast, the bilateral effect of GBP 120 mg/kg, like that of
morphine 4 mg/kg, i.v., was most likely due to sedation.72
In addition, in harmaline-induced tremor in rats, BRV
reduced the elicited tremor index (ET) by 25%, 53%, and
66% at doses of 38, 70, and 120 mg/kg, respectively, compared with vehicle-controlled animals.73 In contrast, LEV
(54–540 mg/kg) produced only small reductions in ET
(3–25%). Furthermore, BRV dose-dependently reduced
both ET and spontaneous tremor index (maximal effects:
53% and 43%, respectively) at doses (21–70 mg/kg)
that did not significantly alter sedation.74 In addition, BRV
was shown to prevent harmaline-induced electrophysiologic
changes in oscillating inferior olivary neurons, which might
be involved in its anti-tremorogenic effect.75
Studies were conducted in a rat neocortical slice model to
compare the effects of LEV and BRV on CSD, which is considered to be a key event in migraine and stroke.76 BRV (10
and 32 lM) reduced the amplitude of elicited CSD and transiently reduced the duration at half-amplitude, whereas
LEV (32 and 100 lM) had no effect on either parameter.
Adverse Effects
Impairment of motor function
The therapeutic dose-range of AEDs, between doses
inducing seizure control and those causing adverse effects,
markedly impact their clinical utility. Furthermore, patients
with temporal lobe epilepsy have shown greater sensitivity
to drug-induced adverse effects,77 which appears to be compatible with the enhanced behavioral reactions to drugs of
corneal-kindled78 and amygdala-kindled79 animals. This
finding emphasizes the importance of assessing the potential of new AEDs to induce adverse effects in kindled animals in order to predict their potential impact on patients.
Testing of BRV in corneal-kindled mice and amygdalakindled rats showed minimal behavioral effects and did not
reveal any unwanted reactions, including an absence of psychomimetic effects.27,59 A comparison of the ability to provide seizure suppression against the impact on motor
function in the rotarod test showed BRV to have a lower
therapeutic index in corneal-kindled mice than LEV (46 vs.
148, respectively) (Fig. S4A); however, it was markedly
higher than that observed for classical and other newer
AEDs (therapeutic indexes 2–21).19 In contrast, rotarod testing in amygdala-kindled rats showed BRV to have a higher
therapeutic index than LEV (4 vs. 2, respectively)
(Fig. S4B).59 By extrapolation, this would suggest that BRV
might offer a promising tolerability profile in patients with
epilepsy.
Impact on cognitive function
Cognitive disorders are common in patients with epilepsy. Their etiology is multifactorial and depends on a
number of factors, including the specific epilepsy synEpilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
546
H. Klitgaard et al.
drome, location, and type of epileptic lesion, seizure frequency, and age of onset.80 In addition, some AEDs may
induce cognitive side effects or exacerbate an existing cognitive deficit.80 Therefore, evaluation of the impact of AEDs
on cognitive function is important for the quality of life of
patients with epilepsy.
Amygdala-kindled rats are more sensitive than normal
rats to the cognitive adverse effects of certain AEDs.77,81
However, in an amygdala-kindling model of temporal lobe
epilepsy, BRV, administered at doses that exerted pronounced anticonvulsant effects in fully kindled rats, did not
negatively impact hippocampal-dependent spatial learning
and memory performance in the Morris water-maze test; an
effect that was also demonstrated in normal rats (Fig. S5).82
This result was further substantiated by the lack of impact
of BRV on long-term potentiation (LTP) in hippocampal
slices.82 LTP is a widely used paradigm to investigate methods of synaptic plasticity, memory formation, and consolidation. Application of BRV at concentrations that decreased
epileptiform activity did not modify the normal synaptic
activity or LTP recorded in the CA1 region of rat hippocampal slices. Taken together, these findings suggest that BRV
would not be expected to alter hippocampal-dependent cognitive function in patients with epilepsy.82
Conclusion
Existing preclinical data show that BRV is a selective
SV2A ligand with an affinity and ability to inhibit synaptic
transmission and vesicle release that is superior to LEV.
BRV provides immediate, potent, and complete seizure suppression in animal models of partial, generalized, and drugresistant seizures, at doses that are devoid of psychomimetic
effects and show no impact on motor or cognitive function.
These data are supportive that BRV may benefit patients
with uncontrolled seizures or experiencing significant
adverse events. BRV is currently under regulatory review as
adjunctive therapy for adults with POS and is being tested in
a number of long-term follow-up studies in patients with
refractory epilepsy, mostly with POS, with or without secondary generalization. Thus, the preclinical findings suggest that BRV represents the first selective SV2A ligand for
epilepsy treatment and may provide an effective and welltolerated therapy that contributes significantly to the existing armamentarium of AEDs.
Acknowledgments
For their committed and skillful contributions to the experiments
reviewed in this paper, the authors are grateful to Zita Povegliano, Michel
Neveux, Marie-Christine Tordeur, Antonella Gallisz, Colette Chauss
ee,
Hugues Chanteux, Dominique Tytgat, Ludo Staelens, David Urbain,
Bernadette Wantier, Christy Van Der Perren, Murielle Martini, Patricia
Piette, Fabienne Coddens, Brice Mullier, Nathalie Leclere, Sabrina Tempesta,
Val
ery Crowet, Yves Evrard, Benedicte Lallemand, Patrick Pasau, Stephane
Carre, Marie-Christine Vandergeten, Natalie Price, Florian Montel, and
Jo€el Mercier (all UCB Pharma). The authors also acknowledge C
edric
Epilepsia, 57(4):538–548, 2016
doi: 10.1111/epi.13340
Laloyaux (UCB Pharma, Brussels, Belgium) for critical review and coordination of the manuscript preparation, and Rachel Bell PhD and Jennifer
Stewart MSc (QXV Communications, an Ashfield Business, Macclesfield,
United Kingdom) for writing assistance, which was funded by UCB
Pharma.
Disclosure of Conflicts of
Interest
Henrik Klitgaard, Alain Matagne, Jean-Marie Nicolas, Michel Gillard,
Yves Lamberty, Marc De Ryck, Rafal M. Kaminski, Karine Leclercq,
Isabelle Niespodziany, Christian Wolff, Martyn Wood, Jonas Hannestad,
Sophie Kervyn, and Benoit Kenda are current employees of UCB Pharma,
Braine l’Alleud, Belgium. HK, MG, CW and JH own UCB stocks. MW
owns UCB and GSK stocks. We confirm that we have read the journal’s
position on issues involved in ethical publication and affirm that this report
is consistent with those guidelines.
References
1. Cockerell OC, Johnson AL, Sander JW, et al. Prognosis of epilepsy: a
review and further analysis of the first nine years of the British National
General Practice Study of Epilepsy, a prospective population-based
study. Epilepsia 1997;38:31–46.
2. Kwan P, Brodie MJ. Early identification of refractory epilepsy. N Engl
J Med 2000;342:314–319.
3. Brodie MJ, Barry SJ, Bamagous GA, et al. Patterns of treatment
response in newly diagnosed epilepsy. Neurology 2012;78:1548–
1554.
4. Marson AG, Al-Kharusi AM, Alwaidh M, et al. The SANAD study of
effectiveness of carbamazepine, gabapentin, lamotrigine, oxcarbazepine, or topiramate for treatment of partial epilepsy: an unblinded
randomised controlled trial. Lancet 2007;369:1000–1015.
5. Marson AG, Al-Kharusi AM, Alwaidh M, et al. The SANAD study of
effectiveness of valproate, lamotrigine, or topiramate for generalised
and unclassifiable epilepsy: an unblinded randomised controlled trial.
Lancet 2007;369:1016–1026.
6. Taylor RS, Sander JW, Taylor RJ, et al. Predictors of health-related
quality of life and costs in adults with epilepsy: a systematic review.
Epilepsia 2011;52:2168–2180.
7. Baker GA, Jacoby A, Buck D, et al. Quality of life of people with epilepsy: a European study. Epilepsia 1997;38:353–362.
8. French JA, Costantini C, Brodsky A, et al. Adjunctive brivaracetam
for refractory partial-onset seizures. A randomized, controlled trial.
Neurology 2010;75:519–525.
9. Van Paesschen W, Hirsch E, Johnson M, et al. Efficacy and tolerability
of adjunctive brivaracetam in adults with uncontrolled partial-onset
seizures: a phase IIb, randomized, controlled trial. Epilepsia
2013;54:89–97.
10. Ryvlin P, Werhahn KJ, Blaszczyk B, et al. Adjunctive brivaracetam in
adults with uncontrolled focal epilepsy: results from a double-blind,
randomized, placebo-controlled trial. Epilepsia 2014;55:47–56.
11. Biton V, Berkovic SF, Abou-Khalil B, et al. Brivaracetam as adjunctive treatment for uncontrolled partial epilepsy in adults: a phase III
randomized, double-blind, placebo-controlled trial. Epilepsia
2014;55:57–66.
12. Klein P, Schiemann J, Sperling MR, et al. A randomized, double-blind,
placebo-controlled, multicenter, parallel-group study to evaluate the
efficacy and safety of adjunctive brivaracetam in adult patients with
uncontrolled partial-onset seizures. Epilepsia 2015;56:1890–1898.
13. Kenda BM, Matagne AC, Talaga PE, et al. Discovery of 4-substituted
pyrrolidone butanamides as new agents with significant antiepileptic
activity. J Med Chem 2004;47:530–549.
14. Winblad B. Piracetam: a review of pharmacological properties and
clinical uses. CNS Drug Rev 2005;11:169–182.
15. Giurgea C, Salama M. Nootropic drugs. Progress Neuropsychopharmacol 1977;1:235–247.
16. Margineanu D, Klitgaard H. Levetiracetam mechanisms of action. In
Levy RH, Mattson RH, Meldrum BS, et al. (Eds) Antiepileptic drugs.
Philadelphia: Lippincott Williams & Wilkins; 2002:419–427.
547
Brivaracetam Discovery and Preclinical Profile
17. Klitgaard H, Verdru P. Levetiracetam: the first SV2A ligand for the
treatment of epilepsy. Expert Opin Drug Discov 2007;2:1537–1545.
18. L€
oscher W, Honack D. Profile of ucb L059, a novel anticonvulsant
drug, in models of partial and generalized epilepsy in mice and rats.
Eur J Pharmacol 1993;232:147–158.
19. Klitgaard H, Matagne A, Gobert J, et al. Evidence for a unique profile
of levetiracetam in rodent models of seizures and epilepsy. Eur J Pharmacol 1998;353:191–206.
20. L€
oscher W, Klitgaard H, Twyman RE, et al. New avenues for anti-epileptic drug discovery and development. Nat Rev Drug Discov
2013;12:757–776.
21. Klitgaard H. Levetiracetam: the preclinical profile of a new class of
antiepileptic drugs? Epilepsia 2001;42(Suppl. 4):13–18.
22. Noyer M, Gillard M, Matagne A, et al. The novel antiepileptic drug
levetiracetam (ucb L059) appears to act via a specific binding site in
CNS membranes. Eur J Pharmacol 1995;286:137–146.
23. Kaminski RM, Matagne A, Leclercq K, et al. SV2A protein is a broadspectrum anticonvulsant target: functional correlation between protein
binding and seizure protection in models of both partial and generalized epilepsy. Neuropharmacology 2008;54:715–720.
24. Lynch BA, Lambeng N, Nocka K, et al. The synaptic vesicle protein
SV2A is the binding site for the antiepileptic drug levetiracetam. Proc
Natl Acad Sci U S A 2004;101:9861–9866.
25. Kaminski RM, Gillard M, Leclercq K, et al. Pro-epileptic phenotype
of SV2A deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam. Epilepsia 2009;50:1729–1740.
26. Kaminski RM, Gillard M, Klitgaard H. Targeting SV2A for discovery
of antiepileptic drugs. In Noebels JL, Avoli M, Rogawski MA, et al.
(Eds) Jasper’s Basic Mechanisms of the Epilepsies. 4th Ed. Bethesda:
National Center for Biotechnology Information; 2012:1–12.
27. Klitgaard H, Matagne A. Mechanisms of action of levetiracetam and
newer SV2A ligands. In Shorvon S, Pedley TA (Eds) The Epilepsies 3.
Philadelphia: Butterworth Heineman Elsevier; 2008:28–38.
28. Margineanu D, Klitgaard H. The novel SV2A ligands brivaracetam
and seletractam manifest different effects against the epileptiform
markers of field potentials in a “high K + -low CA2 + ” rat
hippocampal slice model. Epilepsia 2006;47(Suppl. S3):p281
Abstract.
29. Crowder KM, Gunther JM, Jones TA, et al. Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A). Proc Natl
Acad Sci U S A 1999;96:15268–15273.
30. Xu T, Bajjalieh SM. SV2 modulates the size of the readily releasable
pool of secretory vesicles. Nature Cell Biol 2001;3:691–698.
31. Gillard M, Fuks B, Leclercq K, et al. Binding characteristics of brivaracetam, a selective, high affinity SV2A ligand in rat, mouse and
human brain: relationship to anti-convulsant properties. Eur J Pharmacol 2011;664:36–44.
32. Gillard M, Chatelain P, Fuks B. Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in
CHO cells expressing the human recombinant protein. Eur J Pharmacol 2006;536:102–108.
33. Yang XF, Rothman SM. Levetiracetam has a time- and stimulationdependent effect on synaptic transmission. Seizure 2009;18:615–619.
34. Yang X, Bognar J Jr, He T, et al. Brivaracetam augments shortterm depression and slows vesicle recycling. Epilepsia 2015;56:1899–
1909.
35. Garcia-Perez E, Mahfooz K, Covita J, et al. Levetiracetam accelerates
the onset of supply rate depression in synaptic vesicle trafficking.
Epilepsia 2015;56:535–545.
36. Daniels V, Wood M, Leclercq K, et al. Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays. Br J Pharmacol 2013;169:1091–
1101.
37. Wood M, Urbain D, Gillard M. Evidence for a differential interaction
of brivaracetam and levetiracetam with the SV2A protein. Epilepsia
2015;56(Suppl. 1):215 Abstract.
38. Zona C, Pieri M, Carunchio I, et al. Brivaracetam (ucb 34714) inhibits
Na+ current in rat cortical neurons in culture. Epilepsy Res
2010;88:46–54.
39. Niespodziany I, Andre VM, Leclere N, et al. Brivaracetam differentially affects voltage-gated sodium currents without impairing sustained repetitive firing in neurons. CNS Neurosci Ther 2015;21:241–
251.
40. Niespodziany I, Andr
e V, Leclere N, et al. Anticonvulsant properties
of brivaracetam are not mediated by its effects on voltage-gated
sodium channels. Epilepsy Curr 2014;14:1.204 Abstract.
41. Niespodziany I, Lukyanetz EA, Matagne A, et al. Brivaracetam does
not modulate the major ionic conductances in neurons. Epilepsia
2015;56(Suppl. 1):192–193 Abstract.
42. Margineanu D, Schlobohm I, Klitgaard H. UCB 34714, a new pyrrolidone anticonvulsant, had no effect on voltage-gated potassium in cultured mouse hippocampal neurons. 6th European Congress on
Epileptology (ECE), Vienna, Austria, May 30-June 3, 2004. Abstract.
43. Kostyuk PG, Lukyanetz EA, Klitgaard H, et al. UCB34714, a new
pyrrolidone derivative without impact on high and low voltage activated calcium currents in rat isolated neurons. 58th Annual Meeting of
the American Epilepsy Society (AES), New Orleans, USA, 3-8
December 2004. Abstract.
44. Niespodziany I, Klitgaard H, Margineanu DG. Levetiracetam inhibits
the high-voltage-activated Ca(2 + ) current in pyramidal neurones of
rat hippocampal slices. Neurosci Lett 2001;306:5–8.
45. Vogl C, Mochida S, Wolff C, et al. The synaptic vesicle glycoprotein
2A ligand levetiracetam inhibits presynaptic Ca2 + channels through
an intracellular pathway. Mol Pharmacol 2012;82:199–208.
46. Lukyanetz EA, Shkryl VM, Kostyuk PG. Selective blockade of N-type
calcium channels by levetiracetam. Epilepsia 2002;43:9–18.
47. Rigo JM, Nguyen L, Hans G, et al. UCB 34714: effect on inhibitory
excitatory neurotransmission. Epilepsia 2004; 45(Suppl. 3):011
Abstract.
48. Carunchio I, Pieri M, Ciotti MT, et al. Modulation of AMPA receptors
in cultured cortical neurons induced by the antiepileptic drug levetiracetam. Epilepsia 2007;48:654–662.
49. Yates SL, Fakhoury T, Liang W, et al. An open-label, prospective,
exploratory study of patients with epilepsy switching from levetiracetam to brivaracetam. Epilepsy Behav 2015;52:165–168.
50. Chanteux H, Kervyn S, Gerin B, et al. In vitro pharmacokinetic profile
of brivaracetam reveals low risk of drug-drug interaction unrestricted
brain permeability. Epilepsy Curr 2015;15(Suppl. 1):2.310 Abstract.
51. Nicolas JM, Hannestad J, Holden D, et al. Brivaracetam, a selective
high-affinity synaptic vesicle protein 2A (SV2A) ligand with preclinical evidence of high brain permeability and fast onset of action. Epilepsia 2016;57:201–209.
52. van de Waterbeemd H, Camenisch G, Folkers G, et al. Estimation of
blood-brain barrier crossing of drugs using molecular size and shape,
and H-bonding descriptors. J Drug Target 1998;6:151–165.
53. Sargentini-Maier ML, Espie P, Coquette A, et al. Pharmacokinetics
and metabolism of 14C-brivaracetam, a novel SV2A ligand, in healthy
subjects. Drug Metab Dispos 2008;36:36–45.
54. Rolan P, Sargentini-Maier ML, Pigeolet E, et al. The pharmacokinetics, CNS pharmacodynamics and adverse event profile of brivaracetam
after multiple increasing oral doses in healthy males. Br J Clin Pharmacol 2008;66:71–75.
55. Nicolas JM, Chanteux H, Rosa M, et al. Effect of gemfibrozil on the
metabolism of brivaracetam in vitro and in human subjects. Drug
Metab Dispos 2012;40:1466–1472.
56. Stockis A, Watanabe S, Rouits E, et al. Brivaracetam single and multiple rising oral dose study in healthy japanese participants: influence of
CYP2C19 genotype. Drug Metab Pharmacokinet 2014;29:394–399.
57. Stockis A, Chanteux H, Rosa M, et al. Brivaracetam and carbamazepine interaction in healthy subjects and in vitro. Epilepsy Res
2015;113:19–27.
58. Stockis A, Sargentini-Maier ML, Brodie M. Brivaracetam and carbamazepine interaction study in adult patients with epilepsy. Epilepsia
2015;56(Suppl. 1):2 Abstract.
59. Matagne A, Margineanu DG, Kenda B, et al. Anti-convulsive and antiepileptic properties of brivaracetam (ucb 34714), a high-affinity ligand
for the synaptic vesicle protein, SV2A. Br J Pharmacol
2008;154:1662–1671.
60. Detrait E, Leclercq K, Matagne A, et al. Protective activity of brivaracetam in the 6 Hz model of partial epilepsy: comparison with levetiracetam older an…