Pharmacodynamic and pharmacokinetic analysis of CNS-active constitutional isomers of valnoctamide and sec-butylpropylacetamide - Amide derivatives of valproic acid

Article abstract of DOI:10.1016/j.yebeh.2015.02.040

Valnoctamide (VCD) and sec-butylpropylacetamide (SPD) are CNS-active closely related amide derivatives of valproic acid with unique anticonvulsant activity. This study evaluated how small chemical changes affect the pharmacodynamics (PD; anticonvulsant activity and teratogenicity) and pharmacokinetics (PK) of three constitutional isomers of SPD [sec-butylisopropylacetamide (SID) and tert-butylisopropylacetamide (TID)] and of VCD [tert-butylethylacetamide (TED)]. The anticonvulsant activity of SID, TID, and TED was comparatively evaluated in several rodent anticonvulsant models. The PK-PD relationship of SID, TID, and TED was evaluated in rats, and their teratogenicity was evaluated in a mouse strain highly susceptible to teratogen-induced neural tube defects (NTDs). sec-Butylisopropylacetamide and TID have a similar PK profile to SPD which may contribute to their similar anticonvulsant activity. tert-Butylethylacetamide had a better PK profile than VCD (and SPD); however, this did not lead to a superior anticonvulsant activity. sec-Butylisopropylacetamide and TED did not cause NTDs at doses 4-7 times higher than their anticonvulsant ED₅₀ values. In rats, SID, TID (ip), and TED exhibited a broad spectrum of anticonvulsant activity. However, combined anticonvulsant analysis in mice and rats shows SID as the most potent compound with similar activity to that of SPD, demonstrating that substitution of the isobutyl moiety in the SPD or VCD molecule by tert-butyl as well as a propyl-to-isopropyl replacement in the SPD molecule did not majorly affect the anticonvulsant activity.

Full text of DOI:10.1016/j.yebeh.2015.02.040

Epilepsy & Behavior 46 (2015) 72–78
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Epilepsy & Behavior
journal homepage: www.elsevier.com/locate/yebeh
Pharmacodynamic and pharmacokinetic analysis of
CNS-active constitutional isomers of valnoctamide and
sec-butylpropylacetamide — Amide derivatives of valproic acid
Hafiz Mawasi a,1, Tawfeeq Shekh-Ahmad a,1, Richard H. Finnell b, Bogdan J. Wlodarczyk b, Meir Bialer a,c,
⁎
a
School of Pharmacy, Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
Department of Nutritional Sciences, Dell Pediatric Research Institute, The University of Texas at Austin, Austin, TX, USA
b
c
David R. Bloom Center for Pharmacy, The Hebrew University of Jerusalem, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Valnoctamide (VCD) and sec-butylpropylacetamide (SPD) are CNS-active closely related amide derivatives of
valproic acid with unique anticonvulsant activity. This study evaluated how small chemical changes affect the
pharmacodynamics (PD; anticonvulsant activity and teratogenicity) and pharmacokinetics (PK) of three consti-
tutional isomers of SPD [sec-butylisopropylacetamide (SID) and tert-butylisopropylacetamide (TID)] and of VCD
[tert-butylethylacetamide (TED)]. The anticonvulsant activity of SID, TID, and TED was comparatively evaluated
in several rodent anticonvulsant models. The PK–PD relationship of SID, TID, and TED was evaluated in rats,
and their teratogenicity was evaluated in a mouse strain highly susceptible to teratogen-induced neural tube de-
fects (NTDs). sec-Butylisopropylacetamide and TID have a similar PK profile to SPD which may contribute to their
similar anticonvulsant activity. tert-Butylethylacetamide had a better PK profile than VCD (and SPD); however,
this did not lead to a superior anticonvulsant activity. sec-Butylisopropylacetamide and TED did not cause
NTDs at doses 4–7 times higher than their anticonvulsant ED50 values. In rats, SID, TID (ip), and TED exhibited
a broad spectrum of anticonvulsant activity. However, combined anticonvulsant analysis in mice and rats
shows SID as the most potent compound with similar activity to that of SPD, demonstrating that substitution
of the isobutyl moiety in the SPD or VCD molecule by tert-butyl as well as a propyl-to-isopropyl replacement
in the SPD molecule did not majorly affect the anticonvulsant activity.
Received 5 January 2015
Revised 26 February 2015
Accepted 27 February 2015
Available online 8 April 2015
Keywords:
New antiepileptic drugs (AEDs)
Valnoctamide (VCD)
sec-Butylpropylacetamide (SPD)
CNS drugs
Structure–pharmacokinetic
(PK)–pharmacodynamic (PD) relationship
Constitutional isomers
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
as recently as 2005 [1–3]. Following a successful phase IIa study in pa-
tients with mania funded by the Stanley Medical Research Institute
Valnoctamide (VCD, Fig. 1) is a CNS-active chiral constitutional iso-
mer of valpromide (VPD, Fig. 1), the corresponding amide of valproic
acid (VPA) [1–3]. Unlike VPD that in humans acts as a prodrug to VPA,
VCD acts as a drug on its own with minimal biotransformation to its cor-
responding acid, valnoctic acid (VCA) [1,4–6]. sec-Butylpropylacetamide
(SPD, Fig. 1) is a one-carbon homologue of VCD currently in the preclin-
ical stage that has unique activity against status epilepticus and organo-
phosphate neuronal damage [7–9]. Both VCD and SPD (Fig. 1) possess
two stereogenic carbons in their structure and exist as racemic mixture
of 4 stereoisomers.
(SMRI) [10], teratogenicity studies (by the SMRI) comparing racemic
to VPA (head-to-head) in mice, rats, and rabbits at Covance Labora-
tories were conducted. In these additional studies, VCD, in contrast
to VPA, failed to demonstrate teratogenic potential in mice and rabbits.
In rats, a modest teratogenic signal was observed at plasma concentra-
tions 15 times higher than VCD therapeutic plasma levels [11]. Conse-
quently, VCD recently underwent a 3-week SMRI-funded phase IIb,
randomized, double-blind, multicenter, monotherapy study in patients
with bipolar manic episodes [11]. Interim analysis of the primary study
endpoint for the modified intent-to-treat (mITT) cohort demonstrated
the superiority of the risperidone treatment compared to placebo
(p = 0.0446) while VCD did not. In contrast, the Completers Cohort
analysis demonstrated the superiority of both VCD (p = 0.0159) and
risperidone (p = 0.0188) over the placebo treatment. The difference
in the VCD effect between the mITT and Completers Cohorts is unclear
and is in contrast to the phase IIa results, but it shows that patients
with bipolar manic episodes who stayed on VCD benefitted from it
[12]. Because of the mITT interim analysis results, the SMRI decided to
early terminate the phase IIb study.
Valnoctamide (racemate) was commercially available as an anxio-
lytic drug (Nirvanil®) in several European countries from 1964 until
⁎
Corresponding author at: School of Pharmacy, Institute for Drug Research, Faculty of
Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel.
Tel.: +972 2 6758610; fax: +972 2 6757246.
E-mail address: bialer@md.huji.ac.il (M. Bialer).
These authors contributed equally.
1
http://dx.doi.org/10.1016/j.yebeh.2015.02.040
1525-5050/© 2015 Elsevier Inc. All rights reserved.

H. Mawasi et al. / Epilepsy & Behavior 46 (2015) 72–78
73
Fig. 1. Chemical structure of valproic acid (VPA), valpromide (VPD), valnoctamide (VCD), sec-butylpropylacetamide (SPD), sec-butylisopropylacetamide (SID), tert-butylisopropylacetamide
(TID), and tert-butylethylacetamide (TED).
Racemic VCD and racemic SPD have a wide spectrum of anticonvul-
sant activity, and their ED50 values are 2–16 times (depending on
the model) more potent than those of VPA [7,9,13–15]. Valnoctamide
(65 mg/kg, ip) also provided a full protection in the pilocarpine-
induced status epilepticus (SE) rat model when administered at seizure
onset. However, in contrast to its one-carbon homologue, SPD, VCD
lost its behavioral SE protection when administered (80 mg/kg)
30 min after seizure onset [14], but it did block pilocarpine-induced
electrographic SE at a higher dose (180 mg/kg) [8]. Following a single
dose (600 mg/kg, ip) at day 8.5 of gestation, VCD and SPD and their
individual stereoisomers failed to exert any significant teratogenic effect
in Swiss Vancouver (SWV)/Fnn mice, an inbred mouse strain that is
highly susceptible to VPA-induced teratogenicity [9,14].
All studies so far have focused on VCD and SPD and their individual
stereoisomers [7,9,13,14]. The current study comparatively evaluated
the pharmacodynamics (PD; anticonvulsant activity and teratogenicity)
and pharmacokinetics (PK) of three constitutional isomers of SPD [sec-
butylisopropylacetamide (SID) and tert-butylisopropylacetamide (TID)]
and of VCD [tert-butylethylacetamide (TED)]. tert-Butylethylacetamide
and TID are the two new constitutional isomers of VCD and SPD, respec-
tively, where the sec-butyl moiety was substituted with tert-butyl. sec-
Butylisopropylacetamide is an SPD constitutional isomer where SPD's
n-prorpyl moiety was replaced by isopropyl. The study also aimed to ex-
plore the impact of small chemical changes on the unique anticonvulsant
activity of VCD and SPD.
housed in an Association for Assessment and Accreditation of Laboratory
Animal Care International (AAALAC)-accredited temperature- and
humidity-controlled facility and maintained on a standard 12 h/12 h
light–dark (lights on at 06:00) cycle with free access to standard labora-
tory chow (Prolab RMH 3000, Charles River, Wilmington, MA, USA) and
water ad libitum. All animal experiments were performed in accor-
dance with the guidelines set by the National Institutes of Health and
the University of Utah Institutional Animal Care and Use Committee
(IACUC). All animals were allowed free access to both food and water
except when they were removed from their cages for the experimental
procedure. Except for the kindling studies, animals were used once. All
animals were euthanized in accordance with the Institute of Laboratory
Resources policies on the humane care of laboratory animals. In
0.5% methylcellulose, TID, SID, or TED was administered (ip or po) in a
volume of 0.04 mL/10 g body weight to rats.
2.2. Biological testing/anticonvulsant activity
2.2.1. Pharmacokinetic studies: analysis of TID, SID, and TED in plasma
The plasma concentrations of each compound were quantified by
a GC/MS assay. Plasma (200 μL) was added to the test tubes, followed
by 25 μL of methanol and 25 μL of internal standard solution [α-fluoro-
2,2,3,3-tetramethylcyclopropanecarboxamide (α-F-TMCD) 250 μg/mL
in methanol] [14], and the tubes were thoroughly vortexed. Chloro-
form (2 mL) was used for the extraction of the compounds. The
dry residues obtained after evaporation of 1.8 mL chloroform were
reconstituted with 50 μL methanol, of which 1 μL was injected into
the GC/MS apparatus. The temperature program was as follows:
injector temperature of 200 °C, initial temperature of 60 °C for 5 min,
gradient of 15 °C/min until 180 °C, gradient of 20 °C until 310 °C, and
hold time of 1 min. The MS parameters were set as follows: source tem-
perature of 200 °C, transfer line of 280 °C, and positive ion monitoring
using EI-MS at 70 eV. The pressure of the carrier gas, helium, was set
2. Materials and methods
2.1. Animals and test substances used for seizure testing
Male and female albino CF1 mice (18–25 g, Charles River, Portage,
MI) and male albino Sprague–Dawley rats (100–150 g, Charles River,
Wilmington, MA) were used as experimental animals. Animals were

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H. Mawasi et al. / Epilepsy & Behavior 46 (2015) 72–78
Table 1
Anticonvulsant activity (ED50) and neurotoxicity (TD50) of TED, TID, and SID in comparison to their respective constitutional isomers (VCD and SPD) following ip and oral administration
to rats.a
Anticonvulsant test
po administration
ip administration
ED50 (95% confidence interval) (mg/kg)
SPD
TID
SID
VCD
TED
Maximal electroshock seizure (MES)
Metrazol-induced seizure (scMet)
29 (18–53)
18 (13–25)
154 (124–192)
20 (15–27)
21 (15–27)
8/8 protected
at 65 mg/kgc
84 (62–103)c
49 (43–55)
N60
–
25 (20–30)
38 (27–46)
57 (45–66)
32 (24–36)
23 (19–28)
8/8 protected
at 65 mg/kg
73 (50–94)
35 (30–39)
29 (19–38)
54 (46–63)
58 (47–66)
66 (50–74)
17 (10–24)
8/8 protected
at 65 mg/kgd
0/8 at 80 mg/kgd
72 (63–80)
68 (45–104)
29 (22–38)
178 (162–192)
59 (41–77)
b
Neurotoxicity (TD50
)
N60
Maximal electroshock seizure (MES)
Metrazol-induced seizure (scMet)
Pilocarpine-induced status epilepticus (0 min)
30 (14–48)
15 (7–25)
4/8 protected
at 65 mg/kg
15 (6–27)
8/8 protected
at 200 mg/kgc
257 (192–317)c
124 (115–134)
b
Pilocarpine-induced status epilepticus (30 min)
–
Neurotoxicity (TD50
)
48 (32–36)
a
Four to 8 rats were used to determine the ED50 and TD50 values.
Not determined.
Data are taken from ref. [7].
b
c
d
Data are taken from ref. [18].
at 5 psi. For EI analysis, the ionization energy was 70 eV with a source
pressure of 10−6 Torr. Retention times of SID, TED, TID, and the internal
standard were 12.5, 12.7, 11.2, and 10.1 min, respectively. Calibration
curves were constructed for each analytical run and were linear on the
concentration range between 1 and 100 μg/mL.
In the scMet seizure test, a convulsant dose of pentylenetetrazole was
injected subcutaneously (85 mg/kg in mice) at the time to peak effect
of the test substance, followed by observation of seizure occurrence.
Absence of seizures indicates that the tested compound/dose can
elevate the pentylenetetrazole seizure induction threshold. Systemic
administration of pilocarpine, a cholinergic agonist, has been used to
induce status epilepticus. This seizure state is clinically defined as
continuous seizure activity or multiple seizures without regaining
consciousness lasting more than 30 min. To determine if a test substance
can prevent acute pilocarpine-induced status epilepticus, candidate
drugs were administered to male Sprague–Dawley rats via the ip route,
followed by administration of a challenge dose of pilocarpine immedi-
ately (0 min) and 30 min after treatment with a candidate drug. The
outcome measures were “protection” or “no protection” from epileptic
seizures. In addition, morbidity was also determined 24 h after each
test was completed. Quantitative determination of the protective effect
was undertaken for compounds found to possess significant protection.
This included calculations of the peak time response as well as determi-
nation of ED50 and 95% confidence limits.
2.2.2. Calculation of pharmacokinetic (PK) parameters
The PK parameters of SID, TED, and TID were calculated by
noncompartmental analysis based on statistical moment theory using
PK software Phoenix Winnonlin Tripos L.P. version 6.3 (Pharsight Co.,
Mountain View, CA) as previously described [7,9,14].
2.2.3. Anticonvulsant tests in animals
The antiepileptic potential of the tested compounds was established
in mice and/or rats using the following seizure models: maximal
electroshock (MES), chemically induced shock with subcutaneous
pentylenetetrazole (metrazol) (scMet), bicuculline (scBIC), picrotoxin
(scPIC), the 6-Hz psychomotor test, the corneally kindled mouse,
Frings audiogenic seizure mouse, and rat pilocarpine tests. In the
MES test, 60 Hz 50 mA of alternating current was delivered through
corneal electrodes for 0.2 s. Four to 8 mice or rats were used to deter-
mine the ED50 and TD50 values. During the time of administration
of the test substance, a drop of 0.5% tetracaine in saline is applied
to the eyes of the animals. Animals were restrained by hand during
administration of the electrical stimulus and then released for observa-
tion of the seizure throughout its entire course. A test substance/dose
that is able to abolish the hind limb tonic extensor component of the
seizure, indicating prevention of the MES-induced seizure spread, is
considered “active”. Tonic extension was considered abolished if the
hind limbs were not fully extended at 180° to the plane of the body.
2.2.4. Minimal behavioral toxicity tests
Minimal toxicity was identified in rats and mice as minimal motor
impairment (MMI) as determined by overt evidence of ataxia, abnormal
gait, and stance.
2.2.5. Teratogenicity
For this study, the highly inbred SWV/Fnn mouse strain with a
known susceptibility to AED-induced neural tube defects (NTDs) was
used according to the previously published procedure [16,17]. Dams
were allowed to mate overnight with male mice and were examined
Table 2
Anticonvulsant activity and neurotoxicity of TED, TID, and SID in comparison to their respective constitutional isomers (VCD and SPD) following ip administration to mice.a
Anticonvulsant test
ED50 (95% confidence interval) (mg/kg)
SPD
TID
SID
VCD
TED
6 Hz 32 mA
6 Hz 44 mA
27 (24–30)
118 (81–162)
Negative
100 (91–109)
70 (60–78)
N140
40 (31–52)
53 (34–81)
35 (23–50)
39 (32–47)
60 (46–76)
39 (24–56)
15 (8–25)
10 (9–12)
49 (46–54)
37 (26–50)
67 (61–72)
58 (41–71)
32 (22–45)
162 (130–211)
N200
35 (29–48)
10 (7–12)
81 (72–87)
153 (125–174)
~150
163 (155–173)
58 (50–76)
N210
45 (40–49)
71 (55–90)
62 (47–71)
94 (87–103)
17 (9–28)
39 (31–45)
20 (18–22)
114 (100–134)
Maximal electroshock seizure (MES)
Metrazol-induced seizure (scMet)
Bicuculline-induced seizure (scBIC)
Picrotoxin-induced seizure (scPIC)
Corneally kindled mouse
Frings audiogenic seizures
N210
N210
–
b
–
44 (38–56)
127 (113–143)
30 (24–35)
202 (183–230)
Neurotoxicity (TD50
)
a
Four to 8 rats were used to determine the ED50 and TD50 values.
Not determined.
b

H. Mawasi et al. / Epilepsy & Behavior 46 (2015) 72–78
75
Table 3
Solubility (mg/mL) and lipophilicity data (ClogP) of VCD, TED, SPD, SID, and TID.
2.4. Water solubility and ClogP calculations
The solubility of SID, SPD, TED, TID, and VCD in water and various
other solvents was determined by adding gradually 10–100 mg of
each compound to 1 mL of double-distilled water or any other solvent
until the compound precipitated. The solution was stirred for 2 h. At
the end of the 2-h period, the sample was centrifuged and 1-μL aliquots
were taken for the GC analysis.
Solvent
SPD
TID
SID
VCD
TED
Water
1.5
5
27
50
2.2
3
16
17
22
6
39
33
66
7
27
50
80
1.7
9
41
31
63
1.6
Saline:PG:EtOH (5:4:1)
PG:water:EtOH (4:5:1)
PG:water:EtOH (5:4:1)
ClogP
2.0
2.1
PG—propylene glycol and EtOH—ethanol.
ClogP was calculated by utilizing the ChemDraw Ultra version 8 software.
ClogP was calculated by utilizing the ChemDraw Ultra version 12
software.
3. Results
3.1. Chemistry
on the following morning for the presence of vaginal plugs. The onset
of gestation was set at 1 a.m. of the previous night. On gestational day
8.5, pregnant females received a single ip injection (10 μL per gram of
body weight) of sodium valproate at a dose of 1.1, 1.8, or 2.7 mmol/kg or
TID, SID, and TED at a dose of 1.8 or 2.7 mmol/kg. A 25% Cremophor EL
water solution was injected ip to dams constituting the control group.
On gestation day 18.5, the dams were euthanized by CO2 asphyxiation,
the abdomen opened, and the gravid uteri removed. The locations of
all viable, dead, and resorbed fetuses were recorded, and the fetuses
were grossly examined for the presence of exencephaly. For statistical
purposes, either ANOVA with Tukey's posttest multiple comparison
(fetus weight) or contingency table analysis with Fisher's exact test
(number of resorptions and NTDs) was performed. The p-value was
set at 0.05.
The syntheses of SID, TID, and TED were previously described by
Kaufmann et al. [18]. Briefly, the starting material for SID was 3-
methylvaleric acid and for TED and TID, dimethylbutyric acid. The
acids were converted to their corresponding enolates by LDA, followed
by substitution of a hydrogen atom on the α-carbon by the appropriate
alkyl using specific alkyl iodide. The carboxylic acids were treated with
thionyl chloride in order to produce the corresponding acyl chloride
followed by treatment with NH4OH. The chemical structures of the syn-
thesized amides were identified by 1H NMR GC–MS, while purity was
established using elemental analysis.
3.2. Time to peak effect (TPE) of SID, TID, and TED and determination of
their median effective (ED50) or behavioral toxic (TD50) dose
All quantitative in vivo anticonvulsant/behavioral neurotoxicity
studies were conducted at time to peak effect (TPE) values previously
determined in a qualitative analysis. The TPE of SID, TED, and TID
was 0.25 h following ip and oral administration. Groups of 4–8 mice
or rats were tested with various doses until at least two points were
established between the limits of 100% protection or minimal toxicity
and 0% protection or minimal toxicity. The dose of drug required to pro-
duce the desired endpoint in 50% of animals (ED50 or TD50) in each test,
the 95% confidence interval, the slope of the regression line, and the
SEM of the slope were then calculated by a computer program based
on the method described by Finney [19].
2.3. Methods
A gas chromatography–mass spectrometry (GC/MS) assay was per-
formed on a HP5890 series II GC equipped with a Hewlett-Packard MS
engine (HP5989A) single quadrupole mass spectrometer, HP7673
autosampler, HP MS-DOS Chemstation, and HP-5MS capillary column
(0.25 μm × 15 m × 0.25 mm). The temperature program was as follows:
injector temperature of 180 °C, initial temperature of 60 °C for 5 min,
gradient of 20 °C/min until 180 °C, gradient of 10 °C/min until 300 °C,
and hold time of 3 min. The MS parameters were set as follows: source
temperature of 180 °C, transfer line temperature of 280 °C, and positive
ion monitoring using electron ionization-mass spectrometry (EI-MS) at
70 eV. The molecular ion and the five most pronounced ions are provid-
ed. Elemental analyses were preformed on a 2400-2 Perkin-Elmer CHN
analyzer. The C, H, and N analyses of all newly synthesized compounds
were within 0.4 of theoretical values and, thus, were considered
satisfactory.
3.3. Anticonvulsant efficacy of SID, TED, and TID in seizure and epilepsy
rodent models
The anticonvulsant activity of SID, TID, and TED in comparison to their
respective isomers (SPD and VCD) is depicted in Tables 1 (rats) and 2
Fig. 2. Plasma concentration–time plots of SID (50 mg/kg), TID (50 mg/kg), and TED (70 mg/kg) following ip administration to rats. Each time point represents a mean from three rats with
% coefficient of variation (%CV) of 20–30%.

76
H. Mawasi et al. / Epilepsy & Behavior 46 (2015) 72–78
Table 5
(mice). In rats, SID and TED had a wide spectrum of anticonvulsant ac-
tivity and were equipotent to their respective isomers (SPD and VCD)
following ip administration to rats. In mice, SID was more potent than
TED and TID and exhibited a similar wide spectrum of anticonvulsant ac-
tivity as SPD. tert-Butylisopropylacetamide was the least active among
the tested compounds.
Teratogenic effect in the SWV mouse model of the constitutional isomers of VCD and SPD.
Compound Dose,
mg/kg
No. of No. of
litters implants resorptions fetuses
No. of
No of live No. of
fetuses
with NTDs
(%)
(mmol/kg)
(%)
(%)
Control 7
NaVPA a
NaVPA a
NaVPA c
SPD
SPD
VCD
VCD
TID
TID
TED
TED
SID
25% CEL
10
13
12
12
12
11
9
10
10
10
10
10
10
10
140
160
154
156
179
160
119
132
113
142
127
142
142
141
9 (6.4)
131 (93.6)
0
452 (2.7)
301 (1.8)
181 (1.1)
283 (1.8)
141 (0.9)
389 (2.7)
257 (1.8)
424 (2.7)
283 (1.8)
386 (2.7)
257 (1.8)
283 (1.8)
141 (0.9)
19 (11.9)
21 (13.6)b
12 (7.7)
141 (88.1) 41 (29.1)b
133 (86.4) 2 (1.5)
3.4. Solubility, lipophilicity, and pharmacokinetics of SID, TED, and TID in
rats
144 (92.3)
155 (86.6)
148 (92.5)
0
0
0
24 (13.4)b
12 (7.5)
The solubility of SID, TED, and TID (in comparison to SPD and VCD)
in water and other solvents is depicted in Table 3 which also includes
their lipophilicity expressed as ClogP.
The PK of SID, TED, and TID was studied following ip administration
(50, 70, and 50 mg/kg, respectively) of each amide to rats. The doses
chosen for the PK study were the intermediate doses among the various
ED50 values. The plasma concentration–time plots of SID, TED, and TID
are presented in Fig. 2. Each time point in Fig. 2 represents a mean of
three rats with % coefficient of variation (%CV) of 20–30%.
The PK parameters, calculated by noncompartmental analysis,
are summarized in Table 4. tert-Butylisopropylacetamide and SID
had similar clearance and volume of distribution values: 2.7 L/h/kg
and 2–3.3 L/kg, respectively (Table 4). Consequently, the half-life of
TID and SID was rather short and ranged between 0.6 and 0.8 h. tert-
Butylethylacetamide had a different PK profile compared to TID and
SID, its clearance (0.13 L/h/kg) was the smallest, and consequently,
its half-life (4.9 h) was the longest. The tmax value of SID, TID, and TED
was 0.5 h, a similar value to that obtained previously with SPD and
VCD and their individual stereoisomers [7,9,14].
25 (21.0)b
5 (3.8)d
94 (79.0) 0d
127 (96.2) 1 (0.8)
19 (16.8) 6 (31.6)b
121 (85.2) 5 (4.1)b
117 (92.1) 2 (1.7)d
129 (90.9) 1 (0.8)
94 (83.2)b,d
21 (14.8)b
10 (7.9)
13 (9.1)
13 (9.1)
10 (7.1)
129 (90.9)
131 (92.9)
0
0
SID
For statistical purposes, either ANOVA with Tukey's posttest multiple comparison (fetus
weight) or contingency table analysis with Fisher's exact test (number of resorptions
and NTDs) was performed. p-Value was set at 0.05.
SID (2.7 mmol/kg) was a lethal dose (2 pregnant mice died in 2 h after injection).
a
Results from ref. [21].
Significantly different when compared to the control group.
Results from ref. [22].
Significantly different when compared to the group treated with an equimolar dose
b
c
d
of VPA.
Recently, Spampanato and Dudek showed that VCD induces a spe-
cific, rapid, dose-dependent, and reversible slowing of the decay of
miniature inhibitory postsynaptic current in CAI pyramidal cells [23].
This effect was similar to that of benzodiazepines (BZDs), but the effect
of VCD persisted in the presence of flumazenil (BZD-binding site
antagonist) and was additive to the effect of diazepam. These results
indicate that VCD acts through a different binding site than BZDs, a
fact which may contribute the effect of VCD in BZD-refractory SE [23].
tert-Butylethylacetamide (a VCD isomer) and its one-carbon homo-
logue, SID, are active in the BZD-resistant pilocarpine-induced SE model
when given 30 min after seizure onset and may thus share a similar
mechanism of action as VCD or SPD. In the rat (ip) MES and scMet
models, TED was found to be equipotent to VCD and less neurotoxic.
However, following oral administration, TED was found to be less
potent than VCD in the MES test but was more potent at the scMet
model as well as less neurotoxic. TED's best PK profile compared to
all other closely related investigated compounds did not translate
into a better anticonvulsant activity. TED's constitutional isomer in
which the tert-butyl moiety was substituted by n-butyl as well as its
nonbranched isomer, octanamide, were previously found to be less
potent than TED [18,24,25]. In contrast, another constitutional isomer
of TED and VCD, propylispopropyl acetamide, in which the isobutyl
(VCD) or tert-butyl (TED) moiety was replaced by isopropyl and one
additional methyl was added to the ethyl side chain (shared by TED
and VCD), was previously found to be equipotent to VCD [26].
3.5. Teratogenicity
The teratogenic potential of SID, TED, and TID was assessed for their
ability to induce gross morphological defects in the SWV/Fnn mice that
are highly susceptible to VPA-induced exencephaly. Valproic acid, at a
dose of 2.7 mmol/kg, was embryotoxic and teratogenic, causing almost
a twofold increase in the resorption rate compared to the control group
(11.9% vs 6.3%, respectively) and NTDs in 29.1% of live fetuses. At a lower
dose of 1.8 mmol/kg, VPA was still embryotoxic (13.6% of resorptions),
but the number of fetuses with exencephaly (2) was not statistically
significant. In contrast to VPA, SID and TED did not cause a statistically
significant increase of NTDs at doses of 141 and 283 mg/kg (Table 4).
These doses are 4–7 times higher than their anticonvulsant ED50 values.
tert-Butylisopropylacetamide (283 mg/kg) was embryotoxic and in-
duced resorptions in 14.8% of conceptions (Table 5).
4. Discussion
Antiepileptic drug therapy, particularly with VPA, is associated with
severe side effects (e.g., teratogenicity). In addition, currently, approxi-
mately 30% of patients with epilepsy are not seizure-free despite thera-
py with the existing medications [6].
In mice and rats (ip), SID exhibited a similar broad spectrum of anti-
convulsant activity as SPD but was less potent in the rat (po) scMet
and mouse 6-Hz (32 mA) tests and was more neurotoxic. In contrast,
SID was two times more potent than SPD in the corneal kindled and
Frings audiogenic seizure mouse models. tert-Butylisopropylacetamide
was less active than SPD in the mouse MES and 6-Hz tests but was
equipotent to SPD in the rat MES and mouse and rat scMet tests. tert-
Butylisopropylacetamide was less potent than SPD in the pilocarpine-
induced SE model when given at seizure onset (0 time) but was not
tested at 30 min. tert-Butylisopropylacetamide was less potent in the
rat MES model following oral dosing than after ip administration. This
might be caused by low water solubility (3 mg/mL) coupled with a
first-pass effect. As the rat liver blood flow (Q) is 4 L/h/kg and assuming
a blood-to-plasma ratio of about 1, TID's liver extraction ratio (E) is 68%.
Table 4
PK parameters of SPD and VCD and their constitutional isomers TID, SID, and TED as
obtained after ip administration to rats.
PK parameter
SPDa
TID
0.8
2.7
3.3
18
11
0.5
1.5
SID
0.6
2.7
2.0
22
16
0.5
1.2
VCDb
TED
4.9
0.13
0.96
515
71
0.5
6.8
t1/2 (h)
1.1
1.8
3.0
31
14
0.75
1.8
1.6
0.18
0.41
413
94
CL/F (L/h/kg)
V/F (L/kg)
AUC (mg/L/h-1
)
C
max (mg/L)
t
max (h)
–
MRT (h)
3.6
a
Data taken from ref. [7].
Data following iv administration (74 mg/kg) taken from ref. [20].
b

H. Mawasi et al. / Epilepsy & Behavior 46 (2015) 72–78
77
This high E value makes TID susceptible to hepatic first-pass effect after
Abbreviations
oral administration. Although SID had a similar clearance value as TID
(Table 4), its solubility was 2–3 times higher than that of TID. The better
solubility of SID may contribute to its similar rat MES and scMet
ED50 values following oral and ip administration (Table 1). In the
pilocarpine-induced SE model, SID was equipotent to SPD at 0 and
30 min after seizure onset but was more potent than TID, TED, and
VCD. Although SID, SPD, TED, TID, and VCD have similar lipophilicity,
SPD had the highest ClogP value and the lowest water solubility
(Table 3), a fact that may contribute to its wide and potent spectrum
of anticonvulsant activity.
CNS
AED
MES
scMet
SE
central nervous system
antiepileptic drug
maximal electroshock seizure
subcutaneous metrazol
status epilepticus
protective index
valproic acid
PI
VPA
Acknowledgment
Since numerous commercially available AEDs are teratogenic, it is
essential to develop new AEDs that are nonteratogenic [27,28]. This
is particularly crucial for VPA as on November 21st, 2014, the EMA's Co-
ordination Group for Mutual Recognition and Decentralized Procedures
(CMDh) decided to strengthen the warning on the use of VPA in women
and girls because of the risk of malformation and developmental prob-
lems in children exposed to VPA in the womb that might be associated
with autistic spectrum disorder and childhood autism [29]. In contrast
to VPA, SID and TED did not cause a statistically significant increase of
NTDs at doses of 141 and 283 mg/kg (Table 5). These doses are 4–7
times higher than their anticonvulsant ED50 values. Hereon, we demon-
strate that SID and TED failed to induce NTDs in SWV mice at the dose of
283 mg/kg (1.8 mmol/kg). Thus, SID and TED are superior to VPA not
only by exhibiting a more potent anticonvulsant activity but also by
their reproductive safety.
This study was supported by unrestricted research grants (#039-
4489 and #039-4576) from the Israel Ministry of Defense, Medical
Branch, Nuclear Biological Chemical (NBC) Protection Division.
This work is abstracted from the PhD thesis of Mr. Hafiz Mawasi in
partial fulfillment for the requirements of a PhD degree at The Hebrew
University of Jerusalem.
The authors wish to thank Drs. John H. Kehne, H. Steve White, and
Tracy Chen of the NIH-NINDS Anticonvulsant Screening Program
(ASP) for testing the compounds and their cooperation. The authors
also wish to acknowledge the technical assistance of the faculty and
staff at the University of Utah Anticonvulsant Drug Development
(ADD) Program who conducted the in vivo anticonvulsant testing and
pilocarpine SE studies described in this manuscript. The anticonvulsant
studies were supported by NINDS, NIH Contract No. NO1-NS-4-2359.
Preclinical strategies in new drug (AEDs) discovery used to identify
potential drug candidates include target-based screening, phenotypic
screening utilizing animal models, and modifications of existing drugs
or natural substances [30,31]. Screening in rodent models has been
the engine in AED discovery since phenytoin (1938). Anticonvulsant
animal models are effective in identifying new AEDs, are nonselective
with respect to the mechanism of action, and provide insight into
AEDs' PK–PD relations including the ability to penetrate the brain and
exert a CNS effect [6,32]. Animal models with a similarly high predictive
value do not exist in other nonepileptic CNS disorders. In light of the
highly heterogeneous nature of seizure disorders in humans and the
complexity of seizure phenotypes, it is unlikely that any single animal
model will predict the full therapeutic potential of an investigational
AED [33]. Consequently, promising investigational new AEDs have to
demonstrate activity in a battery of anticonvulsant models that may
indicate a wide antiepileptic spectrum in humans. Bialer et al. demon-
strated a correlation between AEDs' ED50 values in the mouse and rat
MES models and AEDs' therapeutic dose and steady-state plasma
concentrations in patients with epilepsy [34]. This analysis showed
VPA to be the least potent AED in anticonvulsant rodent models. The
constitutional isomers of SPD or VCD designed and evaluated in the
current study by utilizing the phenotypic approach preserve VPA's
wide anticonvulsant spectrum but are significantly more potent than
VPA and, thus, may have a potential to be efficacious in patients who
have seizures resistant to VPA.
Disclosure/conflict of interest
Dr. Meir Bialer has received in the last three years speakers' or con-
sultancy fees from Bial, CTS Chemicals, Desitin, Janssen-Cilag, Johnson &
Johnson, Medgenics, Rekah, Sepracor, Teva, UCB Pharma, and Upsher-
Smith. Dr. Bialer has been involved in the design and development of
new antiepileptic and CNS drugs as well as new formulations of existing
drugs.
None of the other authors has any conflict of interest to disclose.
We, the authors, 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.
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