Stereoselective pharmacodynamic and pharmacokinetic analysis of sec -butylpropylacetamide (SPD), a new CNS-Active derivative of valproic acid with unique activity against status epilepticus

Article abstract of DOI:10.1021/jm4007565

sec-Butylpropylacetamide (racemic-SPD) is a chiral CNS-active amide derivative of valproic acid (VPA). This study describes synthesis and stereospecific comparative pharmacodynamics (PD, anticonvulsant activity and teratogenicity) and pharmacokinetic (PK) analysis of four individual SPD stereoisomers. SPD stereoisomers' anticonvulsant activity was comparatively evaluated in several anticonvulsant animal models including the benzodiazepine-resistant status epilepticus (SE). SPD stereoisomers' PK-PD relationship was evaluated in rats. Teratogenicity of SPD stereoisomers was evaluated in SWV mice strain, susceptible to VPA-induced neural tube defect (NTD). SPD stereoisomers (141 or 283 mg/kg) did not cause NTD. SPD has stereoselective PK and PD. (2R,3S)-SPD and (2S,3R)-SPD higher clearance led to a 50% lower plasma exposure that may contribute to their relative lower activity in the pilocarpine-induced SE model. (2S,3S)-SPD, (2R,3R)-SPD, and racemic-SPD have similar anticonvulsant activity and a PK profile that are better than those of (2R,3S)-SPD and (2S,3R)-SPD, making them good candidates for development as new, potent antiepileptics with a potential in benzodiazepine-resistant SE.

Full text of DOI:10.1021/jm4007565

Article
pubs.acs.org/jmc
Stereoselective Pharmacodynamic and Pharmacokinetic Analysis of
sec-Butylpropylacetamide (SPD), a New CNS-Active Derivative of
Valproic Acid with Unique Activity against Status Epilepticus
Naama Hen,†,⊥ Tawfeeq Shekh-Ahmad,†,⊥ Boris Yagen,†,‡ John H. McDonough,§ Richard H. Finnell,∥
Bogdan Wlodarczyk,∥ and Meir Bialer*,†,‡
†Institute for Drug Research, Faculty of Medicine, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
‡David R. Bloom Center for Pharmacy, The Hebrew University of Jerusalem, Israel
§Pharmacology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground,
Maryland, United States
∥Department of Nutritional Sciences, Dell Pediatric Research Institute, The University of Texas at Austin, Austin, Texas, United
States
ABSTRACT: sec-Butylpropylacetamide (racemic-SPD) is a chiral CNS-active amide derivative of valproic
acid (VPA). This study describes synthesis and stereospecific comparative pharmacodynamics (PD,
anticonvulsant activity and teratogenicity) and pharmacokinetic (PK) analysis of four individual SPD
stereoisomers. SPD stereoisomers’ anticonvulsant activity was comparatively evaluated in several
anticonvulsant animal models including the benzodiazepine-resistant status epilepticus (SE). SPD
stereoisomers’ PK−PD relationship was evaluated in rats. Teratogenicity of SPD stereoisomers was evaluated in SWV mice
strain, susceptible to VPA-induced neural tube defect (NTD). SPD stereoisomers (141 or 283 mg/kg) did not cause NTD. SPD
has stereoselective PK and PD. (2R,3S)-SPD and (2S,3R)-SPD higher clearance led to a 50% lower plasma exposure that may
contribute to their relative lower activity in the pilocarpine-induced SE model. (2S,3S)-SPD, (2R,3R)-SPD, and racemic-SPD
have similar anticonvulsant activity and a PK profile that are better than those of (2R,3S)-SPD and (2S,3R)-SPD, making them
good candidates for development as new, potent antiepileptics with a potential in benzodiazepine-resistant SE.
Treatment of SE is aimed at rapid controlling of convulsive
seizures before compensatory mechanisms fail and the patient
enters into a “refractory” state. Benzodiazepines (lorazepam
and diazepam), phenobarbital, and phenytoin are generally
considered the first line drugs for the early treatment of SE.
Second drugs of choice includes iv administration of VPA,
levetiracetam, or lacosamide. SE can quickly become
pharmacologically refractory when initial attempts to control
the seizures fail despite adequate treatment. In addition to
diazepam and lorazepam, other anesthetic drugs that might be
considered for refractory SE include pentobarbital, thiopental,
propofol, and ketamine.8 A recent study showed that in
contrast to diazepam or VPA, SPD (180 mg/kg) was as
efficacious as propofol (100 mg/kg) and pentobarbital (30 mg/
kg) in suppressing pilocarpine-induced electrographic SE
(ESE) when administered 60 min after first seizure.9 There is
a clear need for more effective treatments for refractory SE that
displays rapid onset and effective seizure control without
producing dose-limiting sedation and respiratory depression.
Further, the development of an effective therapy that attenuates
refractory SE particularly when given 30−60 min after seizure
onset and offers some neuroprotective potential would
represent an important advance in the treatment of SE.
INTRODUCTION
■
All 16 new antiepileptic drugs (AEDs) developed since 1990
for the symptomatic treatment of epilepsy have evolved from
compounds receiving early evaluation in highly predictive
animal seizure and epilepsy models.1−3 sec-Butylpropylaceta-
mide (SPD, Figure 1) is a one-carbon homologue of
valnoctamide (VCD, Figure 1), a chiral constitutional isomer
of VPA’s corresponding amide valpromide (VPD) (Figure
1).4−6 Similar to VCD, SPD possesses two stereogenic carbons
in its structure.7 Therefore, it exists as a racemic mixture of four
stereoisomers in equal proportion as depicted in Figure 2.
SPD (racemate) was recently reported by us to possess a
unique and broad-spectrum antiseizure profile superior to
(lower ED50 values) valproic acid (VPA) and better than that of
VCD.8 In addition, SPD blocked behavioral and electrographic
status epilepticus (SE) induced by pilocarpine (muscarinic
agonist) and soman (organophosphate nerve gas) and afforded
in vivo neuroprotection that was associated with cognitive
sparing.8,9 SPD’s activity against SE is superior to that of
diazepam in terms of rapid onset, potency, and ability to block
SE when given 20−40 min after seizure onset. When
administered 20 and 40 min after SE onset, SPD (100−174
mg/kg) produced long-lasting efficacy (e.g., 4−8 h) against
soman-induced convulsive and electrographic SE in both rats
and guinea pigs. SPD ED50 values in rats and guinea pigs were
71 and 92 mg/kg when administered 40 min after SE onset,
respectively.8
Received: May 22, 2013
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of valproic acid (VPA), valpromide (VPD), valnoctamide (VCD), and sec-butylpropylacetamide (SPD).
pharmacokinetic profile of each SPD stereoisomer following
ip administration to rats and to explore a possible
pharmacokinetic−pharmacodynamic (PK−PD) correlation in
regard to their anticonvulsant activity.
RESULTS
■
Chemistry. The general synthesis of the four individual
SPD stereoisomers is depicted in Schemes 1 and 2 and detailed
in the Experimental Section. The synthesized products were
1
purified by crystallization. H NMR spectra of the synthesized
compounds were measured in DMSO using TMS as an internal
standard. Elemental analyses were performed for all the
synthesized compounds.
Pharmacokinetics of SPD Stereoisomers in Rats. The
pharmacokinetics of SPD stereoisomers was studied following
ip administration (60 mg/kg) to naive rats. The dose was
chosen for the PK study as the intermediate dose among the
various ED50 values of SPD stereoisomers. SPD has a low water
solubility. Consequently, SPD was administered to rats in
multisol, namely, a pure 5:1:4 solution of propylene glycol,
alcohol, and water for injection, where SPD solubility was
increased from 1.5 to 27 mg/mL. The plasma concentration−
time plots of SPD individual stereoisomers in comparison to
racemic-SPD are presented in Figure 3. The PK parameters of
SPD and its four individual stereoisomers, calculated by
noncompartmental analysis, are summarized in Table 1. The
clearance (CL/F) of SPD stereoisomers ranged between 1.8
and 4.6 L h−1 kg−1 and their volume of distribution (Vz/F)
ranged between 3.0 and 7.2 L/kg. (2R,3S)-SPD and (2S,3R)-
SPD had 2-fold higher CL and V values compared to their
other diastereoisomers. Consequently, the half-life (t1/2) of
SPD individual stereoisomers was similar and ranged between 1
and 1.5 h.
Figure 2. Chemical structures of the four SPD stereoisomers.
The current study was designed to evaluate stereospecifically
the anticonvulsant activity, neurotoxicity, teratogenicity, and
pharmacokinetics of SPD’s four individual stereoisomers. The
objectives of the current study were: (a) to synthesize SPD four
individual stereoisomers, (2S,3S)-SPD, (2S,3R)-SPD, (2R,3R)-
SPD, and (2R,3S)-SPD; (b) to comparatively evaluate their
anticonvulsant activity, including two (pilocarpine- and soman-
induced) benzodiazepine-resistant SE models, and teratogenic-
ity in animal models in comparison to one another and to
racemic-SPD and in the case of the teratogenicity studies, in
comparison to VPA; (c) to comparatively analyze the
Time to Peak Effects (TPE) of SPD Stereoisomers and
Determination of Their Median Effective (ED50) or
Behavioral Toxic Dose (TD50). All quantitative in vivo
a
Scheme 1. Total Stereoselective Synthesis of (2R,3R)-SPD (4) and (2S,3R)-SPD
a
Reagents and conditions: (a) pivaloyl chloride, DCM, (2R,3R)-pseudoephedrine (chiral auxiliary), Et3N; (b) LDA, LiCl, 1-propyliodide, THF; (c)
H2SO4, 1,4-dioxane; (d) SOCl2, CH2Cl2, 28−30% NH4OH, ACN. For the synthesis of (2S,3R)-SPD, (2S,3S)-pseudoephedrine is the chiral auxiliary.
B
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a
Scheme 2. Total Stereoselective Synthesis of (2S,3S)-SPD (8) and (2R,3S)-SPD
a
Reagents and conditions: (a) pivaloyl chloride, DCM, (2S,3S)-pseudoephedrine (chiral auxiliary), Et3N; (b) LDA, LiCl, 1-propyliodide, THF; (c)
H2SO4, 1,4-dioxane; (d) SOCl2, CH2Cl2, 28−30% NH4OH, ACN. For the synthesis of (2R,3S)-SPD, (2R,3R)-pseudoephedrine is the chiral
auxiliary.
Figure 3. Plasma concentration−time plots of SPD (racemate), (2R,3R)-SPD, (2S,3S)-SPD, (2R,3S)-SPD, and (2S,3R)-SPD as obtained following
ip administration of 60 mg/kg (of each compound) to rats.
Table 1. PK Parameters of SPD (Racemate), (2S,3S)-SPD, (2R,3R)-SPD, (2R,3S)-SPD, and (2S,3R)-SPD As Obtained after ip
Administration of 60 mg/kg (of Each Compound) to Rats
PK parameter
t1/2 (h)
racemic-SPD
(2S,3S)-SPD
(2R,3R)-SPD
(2R,3S)-SPD
(2S,3R)-SPD
1.1
1.5
1.1
1.3
3.9
7.2
15.2
5.3
1.0
2.2
0.95
4.6
CL/F (L h−1 kg−1
)
1.8
1.8
2.2
Vz/F (L/kg)
3.0
3.9
3.6
6.3
AUC (mg L−1 h−1
Cmax (mg/L
tmax (h)
)
31.3
14.1
0.75
1.8
32.9
21.7
0.5
26.4
18.0
0.5
12.6
8.0
0.5
MRT (h)
1.8
1.5
1.5
Table 2. Anticonvulsant Activity and Neurotoxicity of SPD (Racemate) and Its Four Individual Stereoisomers after ip
Administration to Mice
ED50 (95% coinfidence interval) (mg/kg)
anticonvulsant test
SPD (racemate)
(2R,3S)-SPD
(2S,3S)-SPD
(2R,3R)-SPD
(2S,3R)-SPD
maximal electroshock seizure (MES)
metrazol-induced seizure (scMet)
neurotoxicity (TD50)
71 (55−90)
62 (47−71)
88 (81−95)
27 (24−30)
45 (40−49)
114 (100−134)
95 (80−118)
93 (76−114)
109 (97−121)
29 (26−34)
51 (29−73)
67 (56−79)
75 (58−91)
69 (64−75)
70 (57−81)
53 (45−64)
94 (88−102)
38 (27−48)
52 (43−68)
111 (100−130)
6 Hz, 32 mA
27 (24−29)
59 (48−74)
126 (103−160)
27 (19−33)
<100
6 Hz, 44 mA
neurotoxicity (TD50)
>75
anticonvulsant/toxicity studies were conducted at time to peak
effect (TPE) previously determined in a qualitative analysis.
The TPE of SPD strereoisomers was 0.25−0.5 and 0.5−1 h
following ip and oral administration, respectively. Groups of
four to eight mice or rats were tested with various doses of the
candidate drug 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
produce the desired end point 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
C
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by a computer program based on the method described by
Finney.10
Anticonvulsant Efficacy of SPD Stereoisomers in
Seizure and Epilepsy Rodents Models. The anticonvulsant
activity in various rat seizure and epilepsy models of SPD
stereoisomers (in comparison to racemic-SPD) is depicted in
Tables 2 and 3. Three SPD stereoisomers (2R,3R)-SPD,
(2S,3S)-SPD, and (2R,3S)-SPD exhibited anticonvulsant
activity similar to racemic-SPD, while (2S,3R)-SPD exhibited
less potent anticonvulsant activity at the rat (po) MES model.
Efficacy of SPD Stereoisomers in Slice Electrophysi-
ology Studies. This model examined the ability of SPD
(racemate) and its four inidividual stereoisomers to block
spontaneous electrographic seizure-like activity in combined
medial entorhinal cortex (mEC) hippocampal brain slices
obtained from rats that have been treated with kainic acid
(KA). Extracellular field potential recordings made on layer II
of the mEC showed spontaneous bursting activity upon
introduction of 6 mM KCl, and both the frequency (rate)
and duration of these bursts are variously affected by
AEDs.11−13 As depicted in Table 4, the activity of racemic-
SPD and its four individual stereoisomers was different in this
in vitro model. Racemic-SPD significantly increased burst
duration, while (2R,3S)-SPD and (2S,2S)-SPD significantly
decreased burst duration. (2R,3R)-SPD did not affect burst
duration but deceased the burst rate, while (2S,3R)-SPD did
not change the burst duration or its rate.
SPD Stereoisomers Block Convulsive Seizures In-
duced by Cholinergic Agonist Pilocarpine. Administration
of lithium pilocarpine induces SE characterized by convulsive
and nonconvulsive seizures that can last for several hours. From
a behavioral perspective, the number and severity of the
observed convulsive seizures following pilocarpine adminis-
tration were similar in the two treatment groups (pilocarpine
alone and pilocarpine + SPD stereoisomer). The first
convulsive stage 3 or greater seizure was observed 12 min
after pilocarpine administration and lasted for approximately 60
s. Within the succeeding 30 min, rats were observed to have 4.9
0.2 seizures with an interseizure interval of 3−5 min.8 SPD
stereoisomers, administered 30 min after the first observed
stage 3 motor seizure (marking the SE onset), prevented the
expression of further convulsive seizures in a dose-dependent
fashion with ED50 values ranging between 95 and 135 mg/kg.
(2R,3R)-SPD was the least potent SPD stereoisomer (ED50
>
130 mg/kg) (Table 3).
SPD Stereoisomers Block Electrographic and Con-
vulsive Seizures Induced by Soman in Rats. In the rat
nerve agent seizure model, SPD stereoisomers dissolved in
multisol were administered at various doses along with the
standard medical countermeasures at treatment delays of 20
min after the onset of soman-induced seizures to determine
effective dose for termination of soman-induced electrographic
seizures as described previously for racemic-SPD.8 SPD
stereoisomers were capable of stopping soman-induced
seizures. The ED50 for seizure control was calculated by probit
analysis and ranged between 40 and 70 mg/kg (Figure 4, Table
3). Following administration of SPD stereoisomers the average
latency (s) for electrographic seizure termination at the 20 min
treatment delay time (mean SEM) was the following: 550
149, n = 16 [(2R,3R)-SPD]; 994 280, n = 8 [(2R,3S)-SPD];
719
216, n = 15 [(2S,3R)-SPD]; 1589
684, n = 13
[(2S,3S)-SPD] (Figure 5). All four individual SPD stereo-
isomers had a steep dose−response curve with Hill (shape)
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Table 4. Effect of SPD (Racemate) and Its Four Individual Stereoisomers (100 μM) on Spontaneous Burst in in Vitro Slice
Electrophysiology Studies (Rats)
% control burst rate SEM
% control burst duration SEM
SPD (racemate)
(2R,3R)-SPD
(2S,3S)-SPD
(2R,3S)-SPD
(2S,3R)-SPD
76 11
145 11 (p < 0.05)
94 10
77 5 (p < 0.05)
109
97
9
45 7 (p < 0.05)
78 8 (p < 0.05)
9
4
91
90
5
(2R,3R)-SPD was also teratogenic at the 1.8 mmol/kg dose,
causing exencephaly in six fetuses. In contrast three of the SPD
stereoisomers (2S,3S)-SPD, (2R,3S)-SPD, and (2S,3R)-SPD
were neither embryotoxic nor teratogenic in our study of SWV
mice (Table 5).
DISCUSSION
■
PK Analysis of SPD Stereoisomers. (2R,3S)-SPD and its
enantiomer (2S,3R)-SPD have clearance values twice higher
than racemic-SPD, (2S,3S)-SPD, and (2R,3R)-SPD (Table 1).
This relatively high clearance led to lower plasma exposure
(AUC < 50%) that may contribute to their lower
anticonvulsant activity in the pilocarpine-induced benzodiaze-
pine-resistant SE model, although the relative higher clearance
of (2R,3S)-SPD did not affect its anticonvulsant activity
[compared to racemic-SPD, (2S,3S)-SPD, or (2R,3R)-SPD]
in the rat-MES and scMet models. (2S,3S)-SPD and (2R,3R)-
SPD have similar plasma exposure and similar anticonvulsant
activity.
Figure 4. Anticonvulsant dose−response curve of SPD (racemate) and
its four individual stereoisomers administered 20 min after onset of
soman-induced (electrographic) status epilepticus (SE) in rats.
Similar to its one-carbon homologue VCD, SPD is mainly
eliminated by metabolism that presumably occurs primarily in
the liver. Rat liver blood flow (Q) is 60−70 mL/min.14
Assuming that SPD metabolism is mainly hepatic, then the liver
extraction ratio (E = CL/Q = CLm/Q) of the various SPD
stereoisomers ranges between 0.12 [(2R,3R)-SPD and (2S,3S)-
SPD] and 0.31 [(2R,3S)-SPD and (2S,3R)-SPD]. If these rat
data can be extrapolated in humans, it may indicate that the less
active SPD stereoisomers (in contrast to the active ones) might
be slightly susceptible to hepatic first pass effect after oral
dosing.
SPD Stereoisomers Activity in Anticonvulsant Animal
Models. In the present study, we describe the anticonvulsant
activity of SPD stereoisomers in a battery of traditional seizure
and epilepsy models often employed in the search for novel
AEDs. The results obtained from the study demonstrate that
three SPD stereoisomers, (2S,3S)-SPD, (2R,3R)-SPD, and
(2R,3S)-SPD, possess a broad-spectrum anticonvulsant profile
in models of focal and generalized seizures and have similar
activity like racemic-SPD. These three SPD stereoisomers
demonstrated similar anticonvulsant activity (ED50) as racemic-
SPD at the MES, scMet, and 6 Hz tests and exhibited similar
safety margin as expressed by their protective index (PI =
TD50/ED50). In contrast (2S,3R)-SPD was found to be the least
potent stereoisomer in rats. Racemic-SPD and three of its
individual stereoisomers exhibited similar anticonvulsant
activity in in vivo anticonvulsant rodent models (Tables 2
and 3). In contrast to in vivo (rodents) models, different
activity was observed in in vitro slice electrophysiology studies
that are resistant to phenytoin and VPA (Table 4). This in vitro
model showed different effects of racemic-SPD and its four
individual stereoisomers on burst duration and rate (Table 4).
Nevertheless SPD anticonvulsant data should be mainly
interpreted in light of the extensive in vivo studies, since in
Figure 5. Latency (mean and SEM) for seizure control. The time from
when SPD stereoisomers were administered to rats 20 min after
seizure onset until the last epileptiform event could be detected on the
EEG record.
coefficient of 4.5−8.9 (Figure 4) that led to a tight 95%
confidence interval around their ED50 values. (2R,3R)-SPD was
more potent than racemic-SPD as well as the three other
individual SPD stereoisomers (p < 0.05) and had the shortest
mean latency for seizure control (although it was not
statistically significantly different because of the high inter-rat
variability).
Teratogenicity. The teratogenic potential of racemic-SPD
and its four individual stereoisomers was assessed for their
ability to induce gross morphological defects in the SWV/Fnn
mice that are highly susceptible to VPA-induced exencephaly.
VPA at a dose of 1.8 mmol/kg was embryotoxic and caused a
greater than 2-fold increase in the resorption rate compared to
the control groups (13.6% vs 6.3%, respectively). Two fetuses
with exencephaly were observed in this VPA-treated group.
(2R,3R)-SPD and racemic-SPD were embryotoxic and
induced resorptions in 22.9% and 13.4% of conceptions,
respectively, when tested at the higher 1.8 mmol/kg dose.
E
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Table 5. Teratogenicity Data Effect of SPD and Its Four Individual Stereoisomers in Comparison to VPA after a Single ip
Injection of the Tested Compounds to SWV Mice Dams on Day 8.5 of Gestation
a
dose mg/kg
(mmol/kg)
no. of
litters
no. of
implants
no. of resorptions
(%)
no. of live fetuses no. of normal fetuses no. of fetuses with NTD
compd
(%)
(%)
(%)
control
25% CEL
301 (1.8)
181 (1.1)
283 (1.8)
141 (0.9)
283 (1.8)
141 (0.9)
283 (1.8)
141 (0.9)
283 (1.8)
141 (0.9)
283 (1.8)
14
12
12
12
11
11
10
11
10
10
10
11
11
207
154
156
179
160
147
138
153
154
149
121
144
147
13 (6.3)
194 (93.7)
133 (86.4)
144 (92.3)
155 (86.6)
148 (92.5)
136 (92.5)
130 (94.2)
143 (93.5)
141 (91.6)
135 (90.6)
112 (92.6)
111 (77.1)
136 (92.5)
194 (100)
131 (99)
0
d
e
b
Na-VPA
Na-VPA
SPD
21 (13.6)
2 (1.5)
12 (7.7)
144 (100)
155 (87)
0
b
24 (13.4)
0
SPD
12 (7.5)
11 (7.5)
8 (5.8)
148 (93)
0
(2S,3S)-SPD
(2S,3S)-SPD
(2R,3S)-SPD
(2R,3S)-SPD
(2S,3R)-SPD
(2S,3R)-SPD
(2R,3R)-SPD
(2R,3R)-SPD
136 (100)
130 (100)
143 (100)
141 (100)
135 (100)
112 (100)
105 (94.6)
136 (100)
0
0
10 (6.5)
13 (8.4)
14 (9.4)
9 (7.4)
0
0
0
0
bc
,
b
33 (22.9)
11 (7.5)
6 (5.4)
0
141 (0.9)
a
b
c
Neural tube defects. Significantly different when compared to the control group. Significantly different when compared to group treated with
d
e
similar dose of VPA. Results from ref 28. Results from ref 42.
vitro differences are not always manifested in intact animal
models .
Two SPD stereoisomers, (2S,3S)-SPD and (2R,3R)-SPD,
were found to be a highly effective antiseizure drugs in the
lithium pilocarpine induced SE model. The results obtained
demonstrate that (2S,3S)-SPD and (2R,3R)-SPD as well as
racemic-SPD have a unique capability not shared by other
AEDs to arrest ongoing behavioral seizure activity when
administered 30 min after seizure onset, although it is possible
that the tested rats were exhibiting nonconvulsive SE.
In addition, all SPD strereoisomers exhibited activity in the
soman-induced SE models when given 20 min after onset of
electrographic seizures in rats (Figures 4 and 5). This unique
activity in ceasing electrographic seizures is not shared by
benzodiazepines or other AEDs. (2R,3R)-SPD was more potent
in this SE model than racemic-SPD as well as three other
individual SPD stereoisomers and had the shortest latency for
seizure control.
Although at present little can be said about the molecular
mechanism through which SPD stereoisomers exert their acute
antiseizure effects, the results from the anticonvulsant testing
conducted thus far would support the conclusion that it exerts
its effects through an ability to prevent seizure spread and
elevate seizure threshold. This conclusion is based on the
marked effect exerted by (2R,3R)-SPD, (2S,3S)-SPD, and
(2R,3S)-SPD in the rat MES test (seizure spread) and its ability
to elevate seizure threshold in the scMet seizure model. VPA is
a major AED with multiple mechanisms of action (MOA),
including histone deacetylase inhibition and others yet
undiscovered.15,16 As an amide derivative of VPA, it is likely
that SPD will also have multiple MOA.
Lack of Teratogenicity of SPD and Its Individual
Stereoisomers. Since most if not all commercially available
AEDs are teratogenic, it is essential to develop new AEDs that
are nonteratogenic.17 SPD (racemate) and three of its
individual stereoisomers were found to be nonembryotoxic
and nonteratogenic, and they all failed to induce neural tube
defects at doses 2−9 times [(2R,3R)-SPD] and 3−14 times
[(2S,3S)-SPD] higher than their anticonvulsant ED50 values
(Tables 2 and 3). SPD’s one-carbon homologue valnoctamide
(VCD, depicted in Figure 1) and two of its stereoisomers as
well as its corresponding acid valnoctic acid (VCA) were
nonteratogenic in SWV mice at similar dose as SPD (1.8
mmol/kg) and higher (2.7 mmol/kg). In contrast to VPA its
constitutional isomer VCA was nonteratogenic and had a
similar profile as VCD.18 A similar nonteratogenic profile was
observed with VCD’s constitutional isomer propylisopropyla-
cetamide (PID) and its two enantiomers.19 Thus, VPA amide
derivatives are superior to VPA not only by their more potent
anticonvulsant activity but also by their lack of teratogenicity.
Anticonvulsant Effects of SPD in Animal Models of SE.
SE is initially treated with a benzodiazepine such as diazepam
or lorazepam. Both are extremely effective when given early in
SE; however, the benzodiazepines lose their efficacy when given
after 20 min of spontaneous self-sustaining seizures; e.g.,
animals that experience prolonged SE quickly develop
pharmacoresistant SE if treatment is not initiated within a
short period from seizure onset.20
Stereoselective Pharmacokinetics (PK) and Pharma-
codyanmics (PD). Interaction of a racemic drug, with a
receptor or an enzyme, may result in a distinguished
pharmacological response of the individual stereoisomers that
may also display distinguished PK behavior that could lead to
PD stereoselectivity.21−24 Consequently, consideration of
chirality should be implemented into PK and PD studies.
Therefore, the FDA’s 1992 policy “Statement for Develop-
ment of the New Stereoisomeric Drugs” triggered the
development of single individual stereoisomers and not the
use of racemic mixtures in the development of drugs containing
stereogenic carbon atoms in their structures.25 This policy
coupled with marketing incentives of further profitability as a
“line extension” has encouraged companies to look for chiral
switches of established chiral drugs that were first introduced to
the market as racemic mixtures.21,26
Racemic-VCD was equipotent to SPD when given at SE
onset but in contrast to SPD, VCD (80 mg/kg) lost its activity
in behavioral SE when administered 30 min after the
pilocarpine-induced SE onset, while SPD had an ED50 value
of 84 mg/kg.8 At electrographic SE (ESE) VCD was found to
be less potent than SPD.9 VCD (180 mg/kg) stopped ECE
when given 30 min after seizure onset, while an identical dose
of SPD stopped ESE at 60 min. At 30 min SPD stopped ESE at
a lower dose of 130 mg/kg.9 In that regard SPD and its more
potent stereoisomers (2R,3R)-SPD and (2S,3S)-SPD top not
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line, 280 °C; positive ion monitoring; EI-MS (70 eV). The molecular
ion and the five most-pronounced ions are provided.
only VCD and VPA but also diazepam and other
benzodiazepines.
1H NMR spectra, in DMSO using TMS as internal standard, were
recorded on a Varian Mercury series NMR 300 spectrometer.
Chemical shifts (δ scale) are reported in parts per million (ppm)
relative to residual TMS. Multiplicity is indicated as follows: s
(singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of
doublet), br m (broad multiplet). Coupling constants (J) are given in
(Hz).
Similar to the current SPD study, stereoselectivity was
demonstrated in a recent study with two of VCD stereo-
isomers: (2R,3S)-VCD and (2S,3S)-VCD. In the MES test
(2R,3S)-VCD had a more potent ED50 value (34 mg/kg) than
its diastereoisomer (2S,3S)-VCD (64 mg/kg).18
Chemical structures of the newly synthesized compounds were
assessed by 1H NMR and elemental analyses. Melting point was
determined on a Buchi 530 capillary melting point apparatus.
Elemental analyses were performed on a 2400-2 Perkin-Elmer C, H,
N analyzer. C, H, N analyses of all newly synthesized compounds were
within 0.4 of theoretical values and thus were considered satisfactory.
SPD (racemate) was synthesized according to previously described
procedures.27 SPD stereoisomers [(2S,3R)-SPD, (2R,3R)-SPD,
(2S,3S)-SPD, and (2R,3S)-SPD] were synthesized according to the
methods outlined hereunder.
(2R,3R)-sec-Butylpropylacetamide [(2R,3R)-SPD (4), Scheme
1] and (2S,3R)-sec-Butylpropylacetamide [(2S,3R)-SPD]. (3R)-
Methylpentanoic Acid, ((1′R,2′R)-2′-Hydroxy-1′-methyl-2′-
phenylethyl)methylamide (1). Anhydrous DCM (60 mL) was
added to a round-bottomed flask cooled to −5 °C, containing (3R)-
methylvaleric acid (10 g, 0.08 mol) under nitrogen (N2), followed by
addition of Et3N (8.5 g, 0.084 mol, 1.05equiv) and pivaloyl chloride
(9.64 g, 0.08 mol, 1.00 equiv), while maintaining an internal
temperature of <0 °C. The reaction mixture was stirred for 1 h, and
then Et3N (8.5 g, 0.084 mol, 1.05 equiv) was added. (1R,2R)-
Pseudoephedrine (13.21 g, 0.08 mol, 1.00 equiv), for the synthesis of
(2R,3R)-SPD, or (1S,2S)-pseudoephedrine (13.21 g, 0.08 mol, 1.00
equiv) for the synthesis of (2R,3R)-SPD was added as a solid in
portions and allowed to stir for an additional 1 h below 5 °C, and then
water (60 mL) was added. The organic layer was isolated, washed with
1 M HCl (60 mL), 1 M NaOH (60 mL), and water (60 mL), dried
over MgSO4, filtered, and evaporated to yield 97% oily product. The
residue (oily product) was used in the next step without any further
purification or treatment.
(2R)-Propyl-(3R)-methylpentanoic Acid, ((1′R,2′R)-2′-Hy-
droxy-1′-methyl-2′-phenylethyl)methylamide (2). To a solution
of anhydrous LiCl (6.8 g, 0.16 mol, 2.00 equiv) in dry THF (80 mL)
and diisopropylamine (18.6 g, 0.184 mol, 2.3 equiv)) under N2
atmosphere at −20 °C, n- BuLi (1.6 M in cyclohexane, 0.184 mol,
2.3 equiv) was added slowly while maintaining an internal temperature
of less than −10 °C. After 30 min, a solution of amide 1 (0.08 mol) in
THF (40 mL) was slowly added while maintaining an internal
temperature of less than −10 °C. After 30 min, 1-iodopropane (27.2gr,
0.16 mol, 2.00 equiv) was slowly added while maintaining an internal
temperature of less than −10 °C. After 2 h, the reaction mixture was
warmed to 20 °C and stirred overnight. Saturated NH4Cl (82 mL) was
added to quench the reaction. The organic layer was separated and
washed with water (82 mL). The combined aqueous layers were
extracted with MTBE (82 mL). The layers were separated, and the
MTBE layer was washed with water (82 mL). The organic layer was
dried over MgSO4, filtered, and evaporated to yield 96% oily product.
The residue (oily product) was used in the next step without any
further purification or treatment.
(2R,3R)-sec-Butylpropylcarboxylic Acid (SPA) (3). 1,4-Dioxane
(50 mL) was added to a round-bottomed flask containing amide 2
(0.08 mol) followed by the addition of 3 M H2SO4 (40 mL, 0.12 mol,
1.5 equiv). The reaction mixture was refluxed (∼90 °C) for 6 h and
then cooled to ∼30 °C. Concentrated H2SO4 (27 mL, 0.48 mol, 6.00
equiv) was added, and the mixture was refluxed for 16 h followed by
cooling to 20 °C. The black mixture was extracted with n-heptane (60
mL × 3). The combined organic layers were washed with water (40
mL). The organic layer was extracted with 4 M NaOH (40 mL × 3).
The combined basic aqueous extracts were washed with n-heptane (40
mL). Concentrated HCl (30 mL) was carefully added to the combined
basic aqueous extracts to obtain an acidic solution (pH < 2) and then
was extracted with n-heptane (30 mL × 3). The combined organic
CONCLUSIONS
■
SPD exhibits strereoselective PK and PD in rats with a similar
time to peak plasma concentration (tmax) and time to peak
effect (TPE). The higher clearance (2R,3S)-SPD and (2S,3R)-
SPD led to a 50% lower plasma exposure (AUC) and
contributed to their lower activity (compared to racemic-SPD
and its other two stereoisomers) in the benzodiazepine-
resistant SE model. SPD and its individual stereoisomers
(141 or 283 mg/kg) did not cause NTD. These doses are 2−9
times higher than their anticonvulsant ED50 values. (2S,3S)-
SPD was found to be significantly less embryotoxic than
racemic-SPD and its enantiomer (2R,3R)-SPD. (2R,3R)-SPD
was the most active compound in the soman-induced SE
model.
(2S,3S)-SPD and (2R,3R)-SPD and racemic-SPD have
similar anticonvulsant activity and a PK profile that is better
than that of (2R,3S)-SPD and (2S,3R)-SPD. If SPD would exert
its broad-spectrum anticonvulsant activity due to a single MOA
it is likely that it would exhibit stereoselective PD. The fact that
there was no significant difference between (2R,3S)-SPD and
(2S,3R)-SPD and racemic-SPD in the various anticonvulsant
rodent models (except the soman-induced SE) may indicate
that SPD anticonvulsant activity is due to multiple MOA. The
choice for further drug development between racemic-SPD,
(2S,3S)-SPD, or (2R,3R)-SPD will be based on comparative
toxicological analysis and additional anticonvulsant testing that
will discriminate between these three CNS-active compounds.
EXPERIMENTAL SECTION
■
Chemicals. All the solvents were of analytical grade or HPLC
grade and were purchased from J.T. Baker (The Netherlands). Pivaloyl
chloride, (1S,2S)-pseudoephedrine, (1R,2R)-pseudoephedrine, lithium
chloride, anhydrous triethylamine, n-butyllithium 1.6 M in hexane,
anhydrous diisopropylamine, 1-iodopropane, methyl tert-butyl ether
(MTBE), anhydrous 1,4-dioxane, n-heptane, sulfuric acid, thionyl
chloride, ammonium hydroxide 28−30% in water, and 4 Å molecular
sieves were purchased from Sigma-Aldrich Chemical Company Inc.
(Rehovot, Israel). (3S)-Methylvaleric acid was purchased from Asta
Tech Product Inc. (U.S.). (3R)-Methylvaleric acid was purchased from
Avonyx Laboratories LLC (U.S.). Tetrahydrofuran (THF), dichloro-
methane (DCM), petroleum ether, acetonitrile (ACN), and ethyl
acetate were purchased from Frutarom (Israel). Dry tetrahydrofuran,
dichloromethane, and acetonitrile were obtained by drying over
calcium hydride and distillation.
Materials and Methods. Product formation follow-up was
performed by means of GC−MS and TLC. TLC analyses were
performed on precoated silica gel on aluminum sheets (Kieselgal 60
F254, Merck). A gas chromatography−mass spectrometry assay was
performed on a HP5890 series II gas chromatograph 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, 180 °C;
initial temperature, 40 °C for 6 min; gradient of 20 °C/min until 140
°C; gradient of 10 °C/min until 200 °C; hold time, 3 min. The MS
parameters were set as follows: source temperature, 180 °C; transfer
G
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layer was washed with water (30 mL). The organic layer was dried
over MgSO4, filtered, and evaporated to yield 50% oily product. The
residue (oily product) was used in the next step without any further
purification or treatment.
MgSO4, filtered, and evaporated to yield 58% oily product. The
residue (oily product) was used in the next step without any further
purification or treatment.
(2S,3S)-sec-Butylpropylacetamide (SPD) (8). To acid 7 solution
(6 g, 0.038 mol) in dry DCM (50 mL) under N2 atmosphere at 0 °C, a
1:1 solution of thionyl chloride (3.32 mL, 0.045 mol, 1.2 equiv)
dissolved in dry DCM (7 mL) was added dropwise, and the mixture
was allowed to stir overnight followed by distillation of the solvents
under normal pressure. The oily product was dissolved in dry ACN
(20 mL) and was added dropwise to a 50 mL ammonium hydroxide
solution (28−30%) at 0 °C and left to stir for 2 h. Reaction mixture
was extracted with ethyl acetate (3 × 30 mL). The organic phase was
washed with 2 N NaOH, dried over MgSO4, and evaporated, and the
oily product was recrystallized with ethyl acetate petroleum ether (3:1)
to obtain the product as a white powder. Purity was assessed using
(2R,3R)-sec-Butylpropylacetamide [(2R,3R)-SPD] (4). To acid
3 solution (6 g, 0.038 mol) in dry DCM (50 mL) under N2
atmosphere at 0 °C, a 1:1 solution of thionyl chloride (3.32 mL,
0.045 mol, 1.2 equiv) dissolved in dry DCM (7 mL) was added
dropwise, and the mixture was allowed to stir overnight followed by
distillation of the solvents under normal pressure. The oily product
was dissolved in dry ACN (20 mL) and was added dropwise to a 50
mL ammonium hydroxide solution (28−30%) at 0 °C and left to stir
for 2 h. Reaction mixture was extracted with ethyl acetate (3 × 30
mL). The organic phase was washed with 2 N NaOH, dried over
MgSO4, and evaporated, and the oily product was recrystallized with
ethyl acetate petroleum ether (3:1) to obtain the product as a white
1
melting point, GC−MS, H NMR, and elemental analysis.
1
(2R,3R)-sec-Butylpropylacetamide [(2R,3R)-8]. White powder,
powder. Purity was assessed using melting point, GC−MS, H NMR,
60% yield, mp 129−132 °C. MS-EI, m/z (%): 142 (M+ − 15, 0.3), 101
and elemental analysis.
1
(20), 86 (25), 72 (100), 55 (34). H NMR (300 MHz, DMSO, δ
(2S,3S)-sec-Butylpropylacetamide [(2S,3S)-SPD (8), Scheme
2] and (2R,3S)-sec-Butylpropylacetamide [(2R,3S)-SPD]. (3S)-
Methylpentanoic Acid, ((1′S,2′S)-2′-Hydroxy-1′-methyl-2′-
phenylethyl)methylamide (5). Anhydrous DCM (60 mL) was
added to a round-bottomed flask cooled to −5 °C, containing (3S)-
methylvaleric acid (10 g, 0.08 mol) under nitrogen (N2), followed by
addition of Et3N (8.5 g, 0.084 mol, 1.05equiv) and pivaloyl chloride
(9.64 g, 0.08 mol, 1.00 equiv), while maintaining an internal
temperature of <0 °C. The reaction mixture was stirred for 1 h, and
then Et3N (8.5 g, 0.084 mol, 1.05 equiv) was added. (1S, 2S)-
Pseudoephedrine (13.21 g, 0.08 mol, 1.00 equiv) for the synthesis of
(2S,3S)-SPD or (1R, 2R)-pseudoephedrine (13.21 g, 0.08 mol, 1.00
equiv) for the synthesis of (2R,3S)-SPD was added as a solid in
portions and allowed to stir for an additional 1 h below 5 °C, and then
water (60 mL) was added. The organic layer was isolated, washed with
1 M HCl (60 mL), 1 M NaOH (60 mL), and water (60 mL), dried
over MgSO4, filtered, and evaporated to yield 97% oily product. The
residue (oily product) was used in the next step without any further
purification or treatment.
TMS): 0.78− 0.88 (m, 9H), 0.98−1.22 (br m, 7H), 1.88− 1.98 (m,
1H), 6.6− 6.7 (s, 1H), 6.15−6.25 (s, 1H). Anal. (C9H19NO) C, H, N.
[α]D +14.8 0.4 (c 1, MeOH).
(2S,3R)-sec-Butylpropylacetamide [(2S,3R)-8]. White powder,
62% yield, mp 151−154 °C. MS-EI, m/z (%): 142 (M+ − 15, 0.3), 101
1
(33), 86 (31), 72 (100), 55 (18). H NMR (300 MHz, DMSO, δ
TMS): 0.78− 0.88 (m, 9H), 0.98−1.22 (br m, 7H), 1.88−1.98 (m,
1H), 6.6−6.7 (s, 1H), 6.15−6.25 (s, 1H). Anal. (C9H19NO) C, H, N.
[α]D +12.0 0.5 (c 1, MeOH).
(2S,3S)-sec-Butylpropylacetamide [(2S,3S)-8]. White powder,
72% yield, mp 131−134 °C. MS-EI, m/z (%): 142 (M+ − 15, 0.3), 101
1
(33), 86 (32), 72 (100), 55 (20). H NMR (300 MHz, DMSO, δ
TMS): 0.78− 0.88 (m, 9H), 0.98−1.22 (br m, 7H), 1.88−1.98 (m,
1H), 6.6−6.7 (s, 1H), 6.15−6.25 (s, 1H). Anal. (C9H19NO) C, H, N.
[α]D −16.5 0.6 (c 1, MeOH).
(2R,3S)-sec-Butylpropylacetamide [(2R,3S)-8]. White needles,
79% yield, mp 153−156 °C. MS-EI, m/z (%): 142 (M+ − 15, 0.4), 101
1
(34), 86 (35), 72 (100), 55 (19). H NMR (300 MHz, DMSO, δ
(2S)-Propyl-(3S)-methylpentanoic Acid, ((1′S,2′S)-2′-Hy-
droxy-1′-methyl-2′-phenylethyl)methylamide (6). To a solution
of anhydrous LiCl (6.8 g, 0.16 mol, 2.00 equiv) in dry THF (80 mL)
and diisopropylamine (18.6 g, 0.184 mol, 2.3 equiv) under N2
atmosphere at −20 °C was added n- BuLi (1.6 M in cyclohexane,
0.184 mol, 2.3 equiv) slowly while maintaining an internal temperature
of less than −10 °C. After 30 min, a solution of amide 5 (0.08 mol) in
THF (40 mL) was slowly added while maintaining an internal
temperature of less than −10 °C. After 30 min, 1-iodopropane (27.2 g,
0.16 mol, 2.00 equiv) was slowly added while maintaining an internal
temperature of less than −10 °C. After 2 h, the reaction mixture was
warmed to 20 °C and stirred overnight. Saturated NH4Cl (82 mL) was
added to quench the reaction. The organic layer was separated and
washed with water (82 mL). The combined aqueous layers were
extracted with MTBE (82 mL). The layers were separated, and the
MTBE layer was washed with water (82 mL). The organic layer dried
over MgSO4, filtered, and evaporated to yield 96% oily product. The
residue (oily product) used in the next step without any further
purification or treatment.
(2S,3S)-sec-Butylpropylcarboxylic Acid (SPA) (7). 1,4-Dioxane
(50 mL) was added to a round-bottomed flask containing amide 6
(0.08 mol) followed by addition of 3 M H2SO4 (40 mL, 0.12 mol, 1.5
equiv). The reaction mixture was refluxed (∼90 °C) for 6 h and then
cooled to ∼30 °C. Concentrated H2SO4 (27 mL, 0.48 mol, 6.00 equiv)
was added, and the mixture was refluxed for 16 h followed by cooling
to 20 °C. The black mixture was extracted with n-heptane (60 mL ×
3). The combined organic layers were washed with water (40 mL).
The organic layer was extracted with 4 M NaOH (40 mL × 3). The
combined basic aqueous extracts were washed with n-heptane (40
mL). Concentrated HCl (30 mL) was carefully added to combined
basic aqueous to provide an acidic solution (pH < 2) and then was
extracted with n-heptane (30 mL × 3). The combined organic layer
was washed with water (30 mL). The organic layer was dried over
TMS): 0.78− 0.88 (m, 9H), 0.98−1.22 (br m, 7H), 1.88−1.98 (m,
1H), 6.6−6.7 (s, 1H), 6.15−6.25 (s, 1H). Anal. (C9H19NO) C, H, N.
[α]D −10.8 0.4 (c 1, MeOH).
Biological Testing/Anticonvulsant Activity. Pharmacokinetic
Studies. Analysis of SPD Stereoisomers in Plasma. Plasma
concentrations of each SPD stereoisomer were quantified by a gas
chromatography−mass spectrometry (GC−MS) assay.8 The GC−MS
analysis was performed on a Hewlett-Packard (HP) 5890 series II GC
apparatus (Hewlett-Packard, Palo Alto, CA, U.S.) equipped with an
HP5989A single quadruple mass spectrometer operating in electron
impact (EI) mode, an HP7673 autosampler, an HP MS-DOS
Chemstation, and an HP-5MS capillary column (0.25 μm × 15 m ×
0.25 mm).
Plasma (200 μL) was added to the test tubes followed by 25 μL of
methanol and 25 μL of internal standard solution (α-F-TMCD 250
μg/mL in methanol),28 and the tubes were vortexed thoroughly.
Chloroform (2 mL) was used for the extraction of the compounds.
The dry residues obtained after evaporation of 1.8 mL of chloroform
were reconstituted with 60 μL of methanol, of which 1 μL was injected
into the GC−MS apparatus. The temperature program was as follows:
injector temperature, 200 °C; initial temperature, 50 °C for 6 min;
gradient of 20 °C/min until 140 °C; gradient of 10 °C until 200 °C;
hold time, 3 min. The MS parameters were set as follows: source
temperature, 200 °C; transfer line, 280 °C; positive ion monitoring,
EI-MS (70 eV). The pressure of the carrier gas, helium, was set at 5
psi. For EI analysis, the ionization energy was 70 eV with a source
pressure of 10−6 Torr. Retention times of SPD and internal standard
were 9.9 and 7.9 min, respectively. Calibration curves were constructed
for each analytical run and were linear in the concentration range
between 0.5 and 50 μg/mL.
Calculation of Pharmacokinetic (PK) Parameters. The PK
parameters of each SPD stereoisomer was calculated by non-
compartmental analysis based on statistical moment theory using PK
H
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software Phoenix Winnonlin Tripos L.P. (Pharsight Co., Mountain
View, CA).29 The terminal half-life was calculated as 0.693/λz where λz
is the linear terminal slope of the log of the drug plasma concentration
(C) vs time (t) curve. The plasma exposure or area under the C vs t
curve (AUC) from zero to infinity was calculated by using the
trapezoidal rule with extrapolation to infinity. The mean residence
time (MRT) was calculated from the quotient AUMC/AUC, where
AUMC is the area under the C × t product vs t (first moment-time)
curve from zero to infinity. The apparent (total) clearance (CL/F) was
calculated from the quotient of Dose/AUC, with F being the absolute
bioavailability of the drug after ip administration. The apparent volume
of distribution (V/F) was calculated from the quotient of CL and λz.
The peak plasma concentration (Cmax) and the time to reach Cmax
(tmax) were determined by visual inspection.
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 rats30 (100−150 g, Charles River,
Wilmington, MA) were used as experimental animals. Animals were
housed in an Association for Assessment and Accreditation of
Laboratory Animal Care International (AAALAC) accredited temper-
ature and humidity controlled facility and maintained on a standard 12
h/12h light−dark (lights on at 06:00) cycle with free access to
standard laboratory chow Prolab RMH 3000) and water ad libitum. All
animal experiments were performed in accordance with the guidelines
set by 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.
Each of the SPD stereoisomers was administered (ip or po) in 0.5%
methylcellulose in a volume of 0.04 mL/10 g body weight in rats.
Anticonvulsant Tests. In vivo anticonvulsant activity was
established by both electrical and chemoconvulsant seizure tests
which have been described previously.30−32 The electrical tests used
were the maximal electroshock (MES) seizure test, and the 6 Hz
psychomotor seizure test. The chemical test was the sc metrazol.
MES Test and 6 Hz Test. For the MES and 6 Hz tests, a drop of
anesthetic/electrolyte solution (0.5% tetracaine hydrochloride in 0.9%
saline) was applied to the eyes of each animal prior to placement of the
corneal electrodes. The electrical stimulus in the MES test was 50 mA,
60 Hz for mice and 150 mA, 60 Hz for rats delivered for 0.2 s by an
apparatus similar to that originally described by Woodbury and
Davenport.33 Abolition of the hind leg tonic extensor component of
the seizure was used as the end point.
The ability of the test substance to prevent seizures induced by 6 Hz
corneal stimulation (32 and 44 mA, 3 s duration) in mice was
evaluated at a convulsive current that evokes a seizure in 97% of the
population tested (CC97). The 6 Hz seizures are characterized by a
minimal clonic phase that is followed by stereotyped, automatistic
behaviors described originally as being similar to the aura of human
patients with partial seizures.34,35 Animals not displaying this behavior
were considered protected.
returned to their home cages. All animals were given 1 mL of 0.9%
saline (oral) to compensate for the fluid loss induced by excessive
cholinergic activation. The median effective dose (ED50) for
anticonvulsant activity for the various SPD stereoisomers was
determined by probit analysis.36
Effect of SPD Stereoisomers on in Vitro Electrophysiology
Testing on Rat Hippoacamapal Brain Sliced from Kainic Acid
Treated Rats. Male Sprague Dawley rats were injected with either
saline or kainic acid (KA) per hour to the onset of stage 4/5 seizure on
the Racine scale.12 Extracellular recording from combined medial
entorhinal cortex (mEC) hippocamppal (HC) horizontal brain slices
(400 μm) was performed 1 week later. Baseline electrographic
responses from layer II medial EC were recorded in normal
oxygenated Ringer solution (3 mM KCl). The extraceleulr solution
was then switched to one containing 6 mM KCl and 50 μM picrotoxin
to compare differences in spontaneous burst (SB) rate and SB
duration in slices from control and KA-treated rats. Further, the ability
of SPD and its individual stereoisomers (100 μM) to block SBs was
compared in slices from KA- and saline-treated rats. All experiments
were recorded at 31 1 °C.12
Effect of SPD Stereoisomers on Soman-Induced SE. An
established rodent model of nerve agent-induced SE was used: a rat
HI-6 pretreatment model.8,37−39 The model utilizes a pretreatment
and adjunctive drug that counters the acute immediate lethal effects of
the nerve agent but that does not inhibit the development of SE. In
this model the challenge dose of soman is sufficient to elicit SE in all
animals 5−8 min following soman challenge. The animals were
typically tested in squads of eight and were randomized among
treatment groups each test day. The animals were weighed, placed in
individual recording chambers, and connected to the recording
apparatus. EEG signals were recorded using CDE 1902 amplifiers
and displayed on a computer running Spike2 software (Cambridge
Electronic Design, Ltd., Cambridge, U.K.). Baseline EEG was recorded
for at least 20 min. The animals were then pretreated with 125 mg/kg,
ip, of the oxime HI-6 to prevent the rapid lethal effects of the soman
challenge. At 30 min after pretreatment the rats were challenged with
180 μg/kg, sc, soman (1.6 × LD50) and 1 min later treated with 2.0
mg/kg, im, atropine methyl nitrate to inhibit peripheral secretions.
The rats were then closely monitored both visually and on the EEG for
seizure onset. Seizure onset was operationally defined as the
appearance of >10 s of continuous rhythmic high amplitude spikes
or sharp waves that were at least twice the baseline amplitude
accompanied by a rhythmic bilateral flicking of the ears, facial clonus,
and possibly forepaw clonus. The rats received standard medical
countermeasures [0.1 mg/kg atropine sulfate + 25 mg/kg 2-PAM Cl
admixed to deliver 0.5 mL/kg, im, and 0.4 mg/kg, im, diazepam] at 20
min after seizure onset and then were immediately given a dose (10−
165 mg/kg), ip, of SPD (racemate or an individual stereoisomer)
dissolved in mutisol (a 5:1:4 solution of propylene glycol, alcohol, and
water for injection ). These standard medical countermeasures
(atropine, 2-PAM, and in the rat model, diazepam), at the doses
and times used, are insufficient by themselves to terminate soman-
induced seizures in either model. The rats were monitored for at least
5 h after exposure and then returned to the animal housing room. At
24 h after the exposure, surviving animals were weighed and the EEG
was again recorded for at least 30 min.
Minimal Behavioral Toxicity Tests. Minimal toxicity was
identified in rats as minimal motor impairment (MMI) as determined
by overt evidence of ataxia, abnormal gait, and stance.
Teratogenicity. For this study, the highly inbred SWV/Fnn mouse
strain with a known susceptibility to AED-induced NTDs40 was used
according to the previously published procedure.41 Dams were allowed
to mate overnight with male mice and were examined 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 body weight) of
sodium valproate at doses 1.8 or 1.1 mmol/kg or SPD (racemate or its
individual stereoisomers) at doses 1.8 or 0.9 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,
Effect of SPD Stereoisomers on Lithium Pilocarpine Induced
SE. Seizures were induced by systemic administration of pilocarpine
hydrochloride (50 mg/kg, ip), a muscarinic cholinergic agonist.
Administration of LiCl (20 mg/kg, ip) 24 h prior to pilocarpine
reduces the dose of pilocarpine needed to induce status epilepticus
(SE). Pilocarpine induces behavioral seizures within a few minutes,
and those animals showing no seizures after 45 min of pilocarpine
were removed from the study. At the time of the first stage 3 (Racine
scale) seizure or higher, rats were randomized into two groups,
pilocarpine alone and pilocarpine + an individual SPD stereoisomer.
The latter group received the test compound (130 mg/kg, ip, SPD
stereoisomer) 30 min after the first stage 3 seizure. Animals were
observed and scored for seizure severity for 1.5 h before being
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and the fetuses were grossly examined for the presence of gross
malformation.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 972-2-6758610. Fax: 972-2-6757246. E-mail: bialer@
md.huji.ac.il.
Author Contributions
⊥N.H. and T.S.-A. contributed equally.
Notes
Dr. Meir Bialer has received in the past 3 years speakers or
consultancy 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 antiepileptics and CNS
drugs as well as new formulations of existing drugs. None of the
other authors have 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. The authors declare no
competing financial interest.
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ABBREVIATIONS USED
■
CNS, central nervous system; AED, antiepileptic drug; MES,
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