In vivo screening of diarylimidazoles as anticonvulsant agents

Article abstract of DOI:10.1007/s00044-011-9869-9

In this study, the anticonvulsant activity evaluation of a series of new 2,4(1H)-diarylimidazoles, characterized by the presence of different substituents on the aryl rings, was described. The anticonvulsant activity profile of those compounds was determined by Maximal Electroshock Seizure (MES), subcutaneous Metrazol Seizure (scMet) tests, and 6-Hz seizure model, whereas their neurotoxicity was examined using rotarod test. The trifluoromethoxy derivative, obtained starting from phenyμglyoxal, p-OCF3-benzaldehyde, and ammonium acetate in methanol (r.t.), exhibited a great ability to prevent the seizures induced in the 6-Hz test, becoming a new promising molecule to develop for the treatment of therapyresistant partial seizures. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9869-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3428–3434
DOI 10.1007/s00044-011-9869-9
ORIGINAL RESEARCH
In vivo screening of diarylimidazoles as anticonvulsant agents
•
Mirko Rivara Valentina Zuliani
Received: 22 July 2011 / Accepted: 31 October 2011 / Published online: 19 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, the anticonvulsant activity eval-
uation of a series of new 2,4(1H)-diarylimidazoles, char-
acterized by the presence of different substituents on the
aryl rings, was described. The anticonvulsant activity
profile of those compounds was determined by Maximal
Electroshock Seizure (MES), subcutaneous Metrazol
Seizure (scMet) tests, and 6-Hz seizure model, whereas
their neurotoxicity was examined using rotarod test. The
trifluoromethoxy derivative, obtained starting from phe-
nylglyoxal, p-OCF3-benzaldehyde, and ammonium acetate
in methanol (r.t.), exhibited a great ability to prevent the
seizures induced in the 6-Hz test, becoming a new prom-
ising molecule to develop for the treatment of therapy-
resistant partial seizures.
treatments are available that can successfully prevent sei-
zures; however, approximately one-third of all cases of
seizures cannot be controlled (Kwan and Brodie, 2000;
Dodson, 2004; Zuliani et al., 2009). Moreover, many
marketed antiepileptic drugs (AEDs) cause several side
effects and drug interactions, which limit their clinical use
(Baker et al., 1997; Carrenom et al., 2008; Worthen et al.,
2009). These evidences suggest an urgent need for more
effective and better tolerated AEDs. Previous studies
published from our group have identified analogues of the
2,4(1H)-diarylimidazole as being potent hNaV1.2 sodium
channel blockers able to provide protection against seizures
induced in the maximal electroshock seizure (MES) model
(Rivara et al., 2008; Fantini et al., 2009; Zuliani et al.,
2010). In this study, we decided to expand this class of
interesting molecules, introducing different substituents
on the aryl rings, with the aim to evaluate the ability of
these new diarylimidazoles to inhibit seizures in various
in vivo tests.
Keywords Epilepsy Á Diarylimidazoles Á
In vivo evaluations Á MES Á 6 Hz Á Toxicity
Introduction
Epilepsy is a very common neurological disease that affect
about 0.5–1.0% of worldwide population (Sander, 2003).
The major numbers of cases is detected among children, or
people over 65 years; however, it can occur at any time.
There are different types of epilepsy: in generalized sei-
zures, for example, the origin is in both hemispheres, while
focal seizures involve only a portion of the brain, in par-
ticular structures located in the temporal and frontal lobes
(McCormick and Contreras, 2001). Currently, many
Results and discussion
Chemistry
The molecules here presented were synthesized through
parallel synthesis employing a synthetic protocol previ-
ously described by us (Zuliani et al., 2007). In particular,
compounds 1–7 were prepared through parallel synthesis,
starting from the appropriate phenylglyoxal a, benzalde-
hyde b, ammonium acetate as ammonia source, and a polar
protic solvent, such as methanol, at room temperature
(Scheme 1). The structures of the compounds were proved
M. Rivara Á V. Zuliani (&)
`
Dipartimento Farmaceutico, Universita degli Studi di Parma,
1
by their H-NMR spectroscopic data and elemental anal-
V.le G.P. Usberti, 27/A, 43124 Parma, Italy
e-mail: valentina.zuliani@unipr.it
ysis data (CHN).
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Med Chem Res (2012) 21:3428–3434
3429
seizure model, to have a complete view of their possible
action. In fact, in this model, a low-frequency (6 Hz), long-
duration (3 s) stimulus is delivered through corneal elec-
trodes, and the ability of the molecules to prevent the
raising of the seizures so induced is tested (Chen et al.,
2007).
O
O
H
R1
R
H
+
O
b
a
The results are reported in Tables 1 and 2 and they are
expressed as the number of animals protected out of the
number of animals tested over time. Compounds 8 and 9
were added for comparison and they have been previously
reported (Zuliani et al., 2010).
CH3COONH4/CH3OH
R1
N
R
In the Phase I, preliminary anticonvulsant screening all
the compounds administered at 30, 100, and 300 mg/kg
doses showed some degree of protection in MES test,
whereas they were virtually inactive in the scMet screen. In
the neurotoxicity screening, some molecules were com-
pletely devoid of minimal motor impairment (1, 2, 6, 7),
and the others showed only low toxicity, usually at high
doses (300 mg/kg). Considering the results showed in
Table 1, it is possible to observe an interesting behavior. In
fact, the introduction of a chlorine on the phenyl ring in the
4 position on the imidazole is detrimental for the anti-
convulsant activity at medium doses (100 mg/kg) and 0.5 h
after administration of the drug, regardless of the position
of the chlorine substituent, as demonstrated comparing
compounds 6 and 7 with 8. Nevertheless, considering a
different time interval (4 h), the presence of the chlorine
did not modify the activity (cf. 7 and 9) or increased it (cf.
6 and 9). These compounds, active at 100 and 300 mg/kg,
after 4 h, have quick onset and long duration of action at
relatively higher dose. It is also very important to consider
that compounds 6 and 7 are completely devoid of toxicity,
also at high doses, unlike the non-chlorine compound 8.
Another interesting observation is that the introduction of
an electron releasing substituent similar to –OCH3, as
–OCF3 is detrimental for the anticonvulsant activity, in the
MES test, as can be evidenced by comparison of the
diarylimidazole 5 with the reference molecules 8 and 9.
The synthesized compounds were also tested in the 6-Hz
model to identify their activity at five different time points,
i.e., 0.25, 0.5, 1.0, 2.0, and 4 h. All the compounds
administered at 100 mg/kg showed some activity in this
test but, surprisingly, the best molecule is the trifluoro-
methoxy derivative 5. It displayed 100% protection against
induced convulsions at 0.25, 0.5, and 1 h at the dose of
100 mg/kg, and also had moderate activity at 2 and 4 h
after drug administration, being more active that the ref-
erence compound 8. The most informative compounds (6
and 7) were evaluated quantitatively in phase II screening
(MES and scMet) in which the ED50 and TD50 were
determined. Moreover, for compounds 1, 5, and 8, the
ED50 in the 6-Hz test were determined. The results
N
H
1-7
Scheme 1 Synthesis of the 2,4(1H)-diarylimidazoles 1–7. Reagents
and conditions 1 equiv. a in 3.8 ml CH3OH; 1 equiv. b and 4 equiv.
CH3COONH4 in 3.5 ml CH3OH. Overnight at r.t
Pharmacology
The compounds here presented were tested at the National
Institute of Neurological Disorders and Stroke (NINDS),
via the Anticonvulsant Screening Program (ASP). The in
vivo anticonvulsant activity evaluation was performed
following the procedures proposed by the NINDS, via the
ASP. In particular, various electrical seizures models were
used: MES test, subcutaneous Metrazol Seizure (scMet)
test, and 6-Hz minimal clonic seizure model. All the
compounds synthesized were tested i.p to determine their
ability to prevent seizures at different times and at different
doses. For the best molecules, the ED50 was also measured.
The in vivo evaluation involved the assessment of toxicity
using the standardized rotarod test, which determines the
minimal motor impairment of the animals, which are not
genetically modified and predisposed to seizures (Dunham
and Miya, 1957).
Maximal electroshock seizure model produces the
spread of seizure similar to grand mal epilepsy, resulting
useful for finding drugs to treat human generalized tonic-
clonic seizures. Instead, scMet model identifies those
compounds which raise seizure threshold, being useful for
finding drugs to treat human generalized myoclonic sei-
zures (White et al., 2002). Both these tests are widely used
for the first screening and early identification of new AEDs,
but sometimes they can fail and do not identify molecules
able to treat different types of seizures. For example, this
has happened with levetiracetam, a very potent anticon-
vulsant drug useful for the treatment of therapy-resistant
partial seizures. For this reason, all the molecules synthe-
sized were submitted to an additional test, named 6-Hz
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Med Chem Res (2012) 21:3428–3434
Table 1 Anticonvulsant activity and toxicity of compounds 1–9 administered intraperitoneally to mice
Comp.
Structure
Dose (mg/kg)
MESa
0.5 hd
scMetb
Toxc
4 hd
0.5 hd
4 hd
0.5 hd
4 hd
1
30
100
300
30
0/1
2/3
1/1
0/1
0/3
1/1
0/1
2/3
1/1
1/1
3/3
1/1
0/1
0/3
1/1
0/1
1/3
1/1
0/1
0/3
1/1
1/1
3/3
1/1
0/1
3/3
1/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
1/3
1/1
0/1
3/3
1/1
0/1
2/3
1/1
0/1
0/3
1/1
0/1
2/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/5
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
4/4
0/4
0/8
4/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
1/8
4/4
0/4
0/8
4/4
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
1/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
1/2
0/2
0/4
2/2
N
CH3
N
H
2
3
N
100
300
30
N
H
N
100
300
30
O
O
N
H
4
N
100
300
30
N
H
5
CF3
O
N
100
300
30
N
H
Cl
6
100
300
30
N
OCH3
N
H
Cl
7
100
300
30
N
OCH3
N
H
8e
9e
a
N
100
300
30
N
H
N
100
300
OCH3
N
H
Maximal electroshock test (number of animals protected/number of animals tested)
b
c
d
e
Subcutaneous metrazol test (number of animals protected/number of animals tested)
Neurotoxicity (number of animals protected/number of animals tested)
Time after drug administration
Fantini et al. (2009)
obtained are reported in Tables 3 and 4, respectively, along
with the data on the standard drug phenytoin.
the 6 Hz, with a protective index higher than 8 and than the
standard drug phenytoin.
The data reported in Table 3 showed that the newly
synthesized compounds 6 and 7 exhibited similar activity
to the lead compounds 8 and 9 in the MES test, but com-
pound 7 had lower neurotoxicity and a PI value superior to
that of 8 and 9. Interestingly, the PI value of 7 in the scMet
test is much higher than that of the reference molecules.
Table 4 reports the quantitative anticonvulsant activity of
compounds 1, 5, and 8 in the 6-Hz screen. The results
obtained are very interesting, in fact the new molecule 5
(–OCF3), less active of the parent unsubstituted compound
8 in the qualitative MES test, is absolutely more active in
In conclusion, in this study, as a continuation of our
research on AEDs, the anticonvulsant activity evaluation of
a series of new 2,4(1H)-diarylimidazoles have been
described. The data obtained revealed that almost all of the
derivatives synthesized were effective in the MES screen
and some of the active compounds are devoid of toxicity.
The most interesting result concerns compound 5, charac-
terized by the presence of an electron donor trifluoro-
methoxy group introduced instead of the methoxy one of
the reference compound 9. In fact, 5 showed a lower
activity in the MES screen, but a great ability to prevent the
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Med Chem Res (2012) 21:3428–3434
3431
Table 2 Anticonvulsant activity by psychomotor seizure test of
compounds 1–9 (6-Hz, dose of 100 mg/kg)
Table 4 Quantitative anticonvulsant activity (6 Hz) and toxicity in
mice of compounds 1, 5, and 8
Comp.
0.25 ha
0.5 ha
1.0 ha
2.0 ha
4.0 ha
Comp.
Test
ED50
PIa
1
2
3
4
5
6
7
8
9
3/4
1/4
2/4
2/4
4/4
1/4
3/4
4/4
2/4
2/4
1/4
1/4
1/4
4/4
3/4
3/4
4/4
2/4
2/4
0/4
0/4
1/4
4/4
2/4
0/4
4/4
1/4
1/4
1/4
0/4
0/4
1/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
1
6-Hz
TOX
6-Hz
TOX
6-Hz
TOX
6-Hz
TOX
62.4
188.0
28.2
3.0
5
4.8
2.7
1.3
135.8
47.2
8
126.8
27.5
Pheb
35.6
Phe phenytoin
a
Protective index (PI = TD50/ED50
Number of animals active over the number of animals tested
a
Time after drug administration
)
b
Lenkowski et al. (2007)
seizures induced in the 6-Hz test compared to the unsub-
stituted 8 and to the drug phenytoin. Considering that this
test has been validated as a model of therapy-resistant
epilepsy, compound 5 can be considered as a new prom-
ising molecule to develop for the treatment of therapy-
resistant partial seizures.
Avance spectrometer (300 MHz); chemical shifts (d scale)
are reported in parts per million (ppm) relative to the
central peak of the solvent. Selective isolation of either
non-basic or basic compounds was obtained using
ISOLUTEÒ SCX-2 columns (Particles size: 30–90 lM).
Reactions were monitored by TLC, on Kieselgel 60 F 254
(DC-Alufolien, Merck).
Experimental
General procedure for the synthesis of 2,4(1H)-
diarylimidazoles 1–7
Synthesis
A solution of phenylglyoxal monohydrate (0.70 mmol) in
methanol (3.8 ml) was added dropwise to a stirred sus-
pension of arylaldehyde (0.70 mmol) and ammonium
acetate (3.41 mmol) in methanol (3.5 ml). The reaction
mixture was stirred overnight at room temperature, then the
solvent was evaporated and the residue was partitioned
between saturated aqueous NaHCO3 solution (20 ml) and
methylene chloride (20 ml). The organic phase was dried
Melting points are not corrected and were determined using
a Gallenkamp melting point apparatus. Final compounds
were synthesized in parallel using Bu¨chi SyncoreÒ Reactor
and were analyzed on a ThermoQuest (Italia) FlashEA
1112 Elemental Analyzer, for C, H, and N. The percent-
ages recorded were within 0.4% of the theoretical values.
The 1H-NMR spectra were recorded on a Bruker 300
Table 3 Quantitative anticonvulsant activity (MES and scMet) and toxicity in mice (i.p.; tested at 0.25 h) of compounds 6–9
MESa EDb50
PIc
scMetd EDb50
PIe
b,f
TD
50
Comp.
6
68.8 (56.1–88.4)
82.1 (73.1–88.8)
61.7 (51.9–71.4)
46.8 (44.0–52.0)
9.5 (8.1–10.4)
3.5
4.7
2.1
4.3
6.9
158.1 (134.4–189.5)
102.7 (69.8–133.6)
160.9 (138.6–187.0)
142.2 (103.6–203.4)
–
1.5
3.8
0.8
1.4
–
238.0 (209.8–258.6)
390.3 (333.6–518.2)
126.8 (107.6–140.2)
200.8 (163.0–243.6)
65.5
7
8g
9g
Pheh
Phe phenytoin
a
Maximal electroshock test
b
c
d
e
f
The interval in parentheses stands for the 95% confidence interval
Protective index (PI = TD50/ED50) in the MES test
Subcutaneous metrazol test
Protective index (PI = TD50/ED50) in the scMet test
Neurotoxicity
g
h
Fantini et al. (2009)
Cui et al. (2009) and Lenkowski et al. (2007)
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Med Chem Res (2012) 21:3428–3434
over Na2SO4 and the solvent was removed in vacuo. The
isolation of the target compounds from the crude reaction
mixture was obtained using SCX-2 column (2 g,
30–90 lm, loading 0.4 meq/g). The column was pre-
washed with DCM:methanol = 1:1 (10 ml), the side
products were eluted with methanol (10 ml), and then the
desired 2,4(5)-arylimidazoles were eluted with a metha-
nolic ammonia 5% w/w solution (10 ml). All the products
were then crystallized as the hydrochloride salt from
absolute ethanol/diethyl ether.
C16H11N2OF3ÁHCl: C, 56.40; H, 3.55; N, 8.22. Found: C,
56.21; H, 3.80; N, 8.09.
2-(4-Methoxyphenyl)-4(5)-(4-chlorophenyl)imidazole
(6)
76% yield, mp. [270°C (dec.). 1H NMR (300 MHz,
DMSO-d6): d 8.25 (s, 1H), 8.19 (d, 2H, J = 8.9 Hz),
8.02 (d, 2H, J = 6.7 Hz), 7.63 (d, 2H, J = 8.7 Hz), 7.23
(d, 2H, J = 8.9 Hz), 3.88 (s, 3H). Anal. Calcd. for
C16H13N2OClÁHCl: C, 59.83; H, 4.39; N, 8.72. Found: C,
59.44; H, 4.37; N, 8.59.
2-(4-Methylphenyl)-4(5)-phenylimidazole (1)
79% yield, mp. [250°C (dec.). 1H NMR (300 MHz,
DMSO-d6): d 8.23 (s, 1H), 8.14 (d, 2H, J = 8.3 Hz), 7.99
(d, 2H, J = 8.5 Hz), 7.45–7.56 (m, 5H), 2.42 (s, 3H). Anal.
Calcd. for C16H14N2ÁHClÁH2O: C, 66.55; H, 5.93; N, 9.70.
Found: C, 66.68; H, 5.96; N, 9.69.
2-(4-Methoxyphenyl)-4(5)-(3-chlorophenyl)imidazole
(7)
1
70% yield, mp. 238–240°C. H NMR (300 MHz, DMSO-
d6): d 8.32 (s, 1H), 8.23 (d, 2H, J = 8.9 Hz), 8.16 (t, 1H,
J = 1.7 Hz), 7.99 (d, 1H, J = 7.7 Hz), 7.53 (t, 1H,
J = 7.8 Hz), 7.48 (d, 1H, J = 8.1 Hz), 7.22 (d, 2H,
J = 9.0 Hz), 3.88 (s, 3H). Anal. Calcd. for
C16H13N2OClÁHCl: C, 59.83; H, 4.39; N, 8.72. Found: C,
60.13; H, 4.41; N, 8.75.
2-(4-Isopropylphenyl)-4(5)-phenylimidazole (2)
1
72% yield, mp. 229–232°C. H NMR (300 MHz, DMSO-
d6): d 8.25 (d, 3H, J = 8.4 Hz), 8.00 (d, 2H, J = 7.5 Hz),
7.42–7.55 (m, 5H), 2.98–3.02 (m, 1H), 1.25 (d, 6H,
J = 7.2 Hz). Anal. Calcd. for C18H18N2ÁHClÁ1/2C2H5OH:
C, 70.91; H, 6.89; N, 8.70. Found: C, 71.15; H, 6.75; N,
8.60.
Description of animal testing methods
for MES, scMet, 6-Hz and toxicity evaluations
Animals utilized in these experiments were male albino
CF1 mice (18–25 g, Charles River, Portage, MI) experi-
mental animals. All animals were allowed free access to
both food (Prolab RMH 3000) and water except when they
were removed from their cages for testing. Animals were
used only once. All mice were housed, fed, and handled in
a manner consistent with the recommendations in the
National Research Council Publication, ‘‘Guide for the
Care and Use of Laboratory Animals.’’ No insecticides
capable of altering hepatic drug metabolism enzymes were
used in the animal facilities. All animals were euthanized
in accordance with the Institute of Laboratory Resources
policies on the humane care of laboratory animals. Com-
pounds were administered in 0.5% methylcellulose intra-
peritoneally in a volume of 0.01 ml/g body weight. In vivo
anticonvulsant activity was established using both an
electrical and chemoconvulsant seizure test which have
been described previously. The electrical tests used were
the MES test and the 6-Hz test, whereas the scMet was
used as the chemical test.
2-(4-Ethoxyphenyl)-4(5)-phenylimidazole (3)
1
82% yield, mp. 228–234°C. H NMR (300 MHz, DMSO-
d6): d 8.18 (d, 3H. J = 9.9 Hz), 7.97 (d, 2H, J = 7.5 Hz),
7.45–7.56 (m, 3H), 7.21 (d, 2H, J = 8.7 Hz), 4.16 (q, 2H),
1.37 (t, 3H, J = 6.9 Hz). Anal. Calcd. for C16H17N2OÁHCl:
C, 67.88; H, 5.70; N, 9.31. Found: C, 67.88; H, 5.65; N,
9.10.
2-(4-Isopropoxyphenyl)-4(5)-phenylimidazole (4)
1
81% yield, mp. 222–225°C. H NMR (300 MHz, DMSO-
d6): d 8.23 (d, 3H, J = 8.8 Hz), 8.00 (d, 2H, J = 8.5),
7.55–7.43 (m, 3H), 7.19 (d, 2H, J = 8.9 Hz), 4.76–4.84
(m, 1H), 1.31 (d, 6H, J = 6.0 Hz). Anal. Calcd. for
C18H18N2OÁHCl: C, 68.67; H, 6.08; N, 8.90. Found: C,
68.96; H, 6.34; N, 8.86.
2-(4-Trifluoromethoxyphenyl)-4(5)-phenylimidazole
(5)
MES test
75% yield, mp. 223°C (dec.). 1H NMR (300 MHz, DMSO-
d6): d 8.40 (d, 2H, J = 6.6 Hz), 8.27 (s, 1H), 8.01 (d, 2H,
J = 5.7 Hz), 7.68 (d, 2H, J = 6.5 Hz), 7.53 (t, 2H,
J = 5.7 Hz), 7.44 (t, 1H, J = 5.5 Hz). Anal. Calcd. for
The MES is a model for generalized tonic-clonic seizures
and provides an indication of a compound’s ability to
prevent seizure spread when all neuronal circuits in the
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Med Chem Res (2012) 21:3428–3434
3433
brain are maximally active. These seizures are highly
reproducible and are electrophysiologically consistent with
human seizures. For the MES, a drop of anesthetic/elec-
trolyte 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, delivered for
0.2 s by an apparatus similar to that originally described by
Woodbury and Davenport (Woodbury and Davenport,
1952; Swinyard et al., 1989; White et al., 1995). Abolition
of the hindleg tonic extensor component of the seizure was
used as the endpoint. Mice are initially tested at various
intervals following doses of 30, 100, and 300 mg/kg of test
compound given by i.p. injection are initially screened at a
fixed dose of 30 mg/kg.
within 10 s from the stimulation (Barton et al., 2001;
Kaminski et al., 2004).
Acute toxicity—minimal motor impairment
To assess a compound’s undesirable side effects (toxicity),
animals are monitored for overt signs of impaired neuro-
logical or muscular function. In mice, the rotarod proce-
dure is used to disclose minimal muscular (MMI) or
neurological impairment (Dunham and Miya, 1957). When
a mouse is placed on a rod that rotates at a speed of 6 rpm,
the animal can maintain its equilibrium for long periods of
time. The animal is considered toxic if it falls off this
rotating rod three times during a 1 min period. In addition
to MMI, animals may exhibit a circular or zigzag gait,
abnormal body posture and spread of the legs, tremors,
hyperactivity, lack of exploratory behavior, somnolence,
stupor, catalepsy, loss of placing response, and changes in
muscle tone.
Subcutaneous Metrazol seizure threshold test (scMET)
Subcutaneous injection of the convulsant Metrazol pro-
duces clonic seizures in laboratory animals. The scMET
test detects the ability of a test compound to raise the
seizure threshold of an animal and thus protect it from
exhibiting a clonic seizure. Animals are pretreated with
various doses of the test compound given by i.p. injection.
At the previously determined TPE of the test compound,
the dose of Metrazol which will induce convulsions in 97%
of animals (CD97: 85 mg/kg mice) is injected into a loose
fold of skin in the midline of the neck. The animals are
placed in isolation cages to minimize stress and observed
for the next 30 min for the presence or absence of a seizure
(Swinyard et al., 1961). An episode of clonic spasms,
approximately 3–5 s, of the fore and/or hindlimbs, jaws, or
vibrissae is taken as the endpoint. Animals which do not
meet this criterion are considered protected.
Determination of median effective (ED50) or behavioral
toxic dose (TD50)
All quantitative in vivo anticonvulsant efficacy/toxicity
studies were conducted at the previously determined TPE.
Groups of at least eight mice 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 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 (Finney,
1971).
The 6-Hz seizure test
Acknowledgments We are grateful to the Anticonvulsant Screen-
ing Program (ASP) of the National Institute of Neurological Disorders
and Stroke (NINDS) for anticonvulsant evaluation. We are grateful to
the Centro Interpartimentale Misure of the University of Parma for
providing the NMR instrumentation.
It is an alternative electroshock paradigm that uses low-
frequency (6 Hz), long-duration (3 s) electrical stimula-
tion. Corneal stimulation (0.2 ms-duration monopolar
rectangular pulses at 6 Hz for 3 s) was delivered by a
constant-current device. During the stimulation, mice were
manually restrained and released into the observation cage
immediately after the current application. The seizures
manifest in ‘stunned’ posture associated with rearing,
forelimb, automatic movements, and clonus, twitching of
the vibrissae and Straub-tail. The duration of the seizure
activity ranges from 60 to 120 s in untreated animals. At
the end of the seizure, animals resume their normal
exploratory behavior. The experimental end point is pro-
tection against the seizure. The animal is considered to be
protected if it resumes its normal exploratory behavior
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