Американский Научный Журнал EVALUATION OF ANTICONVULSANT ACTION OF PROPOXAZEPAM ON PENTYLENETETRAZOLE-KINDLING MODEL OF SEIZURE IN MICE

58 American Scientific Journal № ( 22 ) / 201 8
ФАРМАКОЛОГИЯ , ТОКСИКОЛ ОГИЯ И
ФАРМАЦЕВТИЧЕСКИЕ НАУКИ

EVALUATION OF ANTICONVULSANT ACTION OF PROPOXAZEPAM ON
PENTYLENETETRAZOLE -KINDLING MODEL OF SEIZURE IN MICE

Golovenko Nikolay Yakovlevich ,
Doctor of science (Biology), professor,
Leading Researcher, V. Bogatsky Physico -Chem ical Institute of NAS of Ukraine, Odessa.,
Larionov Vitalii Borisovich ,
Doctor of science (Biology), Senior Researcher,
V. Bogatsky Physico -Chemical Institute of NAS of Ukraine, Odessa.,
Reder Anatolii Semenovich
PhD (Chemistry), Senior Researcher,
V. Bo gatsky Physico -Chemical Institute of NAS of Ukraine, Odessa.,
Andronati Sergiy Andriyovich ,
Doctor of science (Chemstry), professor,
Head of department of Medicinal Chemistry, V. Bogatsky Physico -Chemical Institute of NAS of Ukraine,
Odessa.,
Valivodz Iri na Petrivna ,
Junior Research Fellow,
V. Bogatsky Physico -Chemical Institute of NAS of Ukraine, Odessa.,

Abstract
Kindling model of epilepsy on mice was induced by subcutaneous pentylenetetrazol (PTZ) injections (30 mg/kg)
each second -third day during 29 days. Experimental animals were treated with propoxazepam (0,2; 0,6; 1,0 and 2.0
mg/kg, intraperitonealy) 30 min prior to convulsive agent injection. Quantities of different seizures types (in accordance
to standard scale) were counted during 1 hour after PTZ injection. The highest quantity of minimal severity seizures (1
point) was registered at animals of control group from 3 th to 8 th and after 8 th PTZ injections. Propoxazepam administra-
tion did not lead to reliable changes of seizures manifestations, but constrained second type seizures development,
reducing their quantity. The statistically significant redistribution in seizures quantity of second and fourth severity
types with simultaneous increasing of higher severity seizures was noted. At the same ti me seizures of fourth severity
type were diminished. For animals of control and experimental groups the reducing of first and second severity seizures
types latent time was noted. With propoxazepam administration reducing of higher severity seizures types (3th and 4 nd
type) was in accordance with reducing of the latency time for seizures of 1 th and 2 nd types.
Keywords : propoxazepam, pentylenetetrazol kindling epilepsy, antiepileptic effects, components of seizure.

INTRODUCTION
Epilepsy refers to chronic p olyethyologic diseases of
the brain characterized by recurrent seizures that occur as
a result of excessive neuronal discharges and accompa-
nied by various clinical and paraclinical symptoms. Anti-
convulsant therapy remains the basis for treating patients
with epilepsy, which involves inhibition or a significant
reduction in the number of attacks. Currently, the term
“antiepileptic” is synonymous with “anticonvulsant
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use is determined predominantly by the nature of parox-
ysmal manifestations or its equivalents [1]. Depending on
the clinical manifestations of epilepsy, different anticon-
vulsants can be prescribed. Often, for the treatment of ep-
ilepsy, combined use of several medicines is rational (sim-
ulta neously or sequentially). Therefore, the success of the
treatment of epilepsy is on the way to finding new anti-
convulsants, which would have had an effect on different
pathogenesis links in the formation of all variability of sei-
zure states [2].
A novel 3-substituted 1,4 -benzodiazepine, 7 -bromo -
5-(o-chlorophenyl) -3-propoxy -1,2 -dihydro -3H -1,4 -ben-
zodiazepin -2-one (named propoxazepam ), has been found
to have a potent anticonvulsant effect [3]. In models of
chemically induced seizures we determined [4] the ave r-
age effective doses (ED 50) of propoxazepam as an antag-
onist to picrotoxin (1.67 ± 0.09 mg/kg), pentylenetetrazol
(0.9 ± 0.04 mg/kg), and strychnine (4.24 ± 0.47 mg/kg),
which reflect the high activity level of the substance.
Propoxazepam had shown high activity on the model of
GABA -deficient thiosemicarbazide -induced convulsions
[5]. Propoxazepam mean effective doses in acute (3 hours )
and remote (24 hours ) periods of supervising have no sta-
tistically significant difference.
In this study, the anticonvuls ant effect of propoxaze-
pam was determined in pentylenetetrazol -induced kin-
dling model of seizures in mice for further determination
of antiepilectic properties.

MATERIAL AND METHODS
Male mice (18 -26 g), obtained from Institute of Phar-
macology and toxicol ogy NAMS of Ukraine housed at the
local animal department, were used. The animals were ex-
posed to a 12 h light -dark cycle and were provided with
food and water ad libitum . All experiments were con-
ducted during the light part of the day (9.00 -14.00). The
experiments were carried out according to the recommen-
dations of the Committee for Research and Ethical Issues

American Scientific Journal № (22 ) / 201 8 59
of the IASP (1983) and were approved by the regional eth-
ical committee for animal research. All manipulations
were made to minimize animal sufferin g and to reduce the
number of animals used.
The test compounds were suspended in tween 80
(1%) emulsion, and the control animals received corre-
sponding amount of vehicle (1% tween 80).
Propoxazepam was synthesized according to the
method described in [3]. The structure of the substance
was determined and approved by a complex of physico-
chemical methods (IR and mass spectroscopy, as well as
X-ray diffraction analysis). Chemical purity was con-
firmed by elemental analysis (99%). pentylenetetrazol
(PTZ) was obt ained from Sigma, USA.
Induction of kindling by pentylenetetrazol (PTZ )
injections
All the animals except the normal control were in-
jected with a subconvulsive dose of PTZ (30 mg/kg) on
every alternate day to induce kindling. Propoxazepam was
injected int raperitoneally 30 mins prior to each PTZ injec-
tion for 30 days. Prolonged administration of PTZ in sub-
doses (25 -35 mg/kg) is a characteristic condition for kin-
dling. In this study, the chemoconvulsant administration
scheme was chosen in 2 -3 days, which com bines the re-
duction of traumatic in animals (due to parenteral admin-
istration) and the ability to achieve the necessary kindling
indicators.
As recorded indicators of propoxazepam activity, the
number of convulsive episodes of varying severity and the
time of their development from the chemoconvulsant ad-
ministration time were used [4]. Seizure attacks were
grouped according to severity depending on external signs
according to the standard 5 -point scale:
O points: No response
1 point: Ear and facial twitchi ng
2 points: myoclonic body jerks
3 points: clonic forelimb convulsions
4 points: generalized clonic convulsions, turning
onto one side position
5 points: generalized clonic -tonic convulsions (or
death within 30 minutes)
For studying on the model of the kindling -epilepsy
were taken doses of 1.0 mg/kg (close to ED 50) and doses
with an average step of 0.33 on a logarithmic scale - twice
(2.0 mg/kg) and doses within the range one (0.6 mg/kg)
and two (0.2 mg/kg) standard deviations from the average
dose, corr esponding to an effect of ~ 22% and ~ 7% of
animals according to the dose -effect curve. The data were
presented in the form of "mean ± standard deviation in the
data set".
The relative contribution of convulsive attacks of
varying degrees of severity to th e total amount of seizure
readiness in each group was determined as the normalized
ratio of the sum of seizure attacks of each group to the
total number of events in the group:
where I i - contribution (in %) of seizure attacks of
groups of severity i (1 -5), N i - number of convulsive at-
tacks of the corresponding group, N - total number of sei-
zure attacks in the group of animals.
The integral characteristic of the development of the
kindling state in each experimental animal group w as car-
ried out using the index of convulsions (kindling):
Where A, B, C, D and E are the number of convulsive
attacks of varying severity for each animal, respectively.

Statistical analysis
A preliminary assessment of the nature of the distri-
bution of each type of data for compliance with the normal
distribution law was carried out using asymmetry and ex-
cessive indices. If the indicators of these values did not
exceed the value of the corresponding errors by more than
2 times, th e Student's criteria was used as the characteris-
tic of the difference, and the data was represented as
"mean  standard deviation in the data set" . In the case of
a significant deviation of the distribution from the Gauss
one the assessment of the probabil ity of the difference be-
tween the groups performed using methods of nonpara-
metric statistics (Mann -Whitney U -test) [5].

RESULTS
Propoxazepam has previously been reported to have
anticonvulsant effects for acute seizures in mice [6 -8]. In
the present work we report anticonvulsant effects of
propoxazepam in a chronic model (PTZ -induced kin-
dling,). PTZ -kindling is a well -studied animal model
which simulates clinical epilepsy. It is a commonly pre-
ferred behavioral approach used for chemical kindling to
study brain excitability [9]. Absence, myoclonic and gen-
eralized tonic -clonic seizures are induced by PTZ admin-
istration.
The analysis of anticonvulsant activity of propoxaze-
pam on the model of PTZ induced kindling in mice
(equivalent to absence form of epilepsy ) was performed,
and the dynamics of convulsions redistribution of various
severity degrees was characterized. PTZ kindling is a
common pre -clinical model of temporal frontal epilepsy
in mice, which allows to investigate the effects of sub-
stances on the ep ileptogenesis of convulsions in the course
of kindling development (administration of substances
from the prophylactic scheme), as well as on the genera-
tion and generalization of convulsions in animals with
fully developed kindling (administration of subst ances in
the therapeutic scheme). The model of the PTZ kindling
reproduces the GABA -receptor (GABA -R) dependent ep-
ileptogenesis [10].
The kindling state is modeled by repeated PTZ ad-
ministration to animals of a dose that does not cause con-
vulsive effect (s ubdose). Such an effect increases the grad-
ual convulsive "readiness" of the brain to the effect of con-
vulsants. This is manifested in the fact that previously
inactive subconvulsive doses of PTZ caused seizures, the
severity of which increases over time an d ends with gen-
eralized clonic -tonic seizure. The kindling model in the
development of chronic epileptogenesis is also considered
as a pharmacologically resistant form of the epileptic syn-
drome [11]. Possible mechanism of drug resistance in-
clude the neglig ible to lack of effect of antiepileptic drugs
(narrow therapeutic index, low level of effective concen-
tration of the drug in the brain, reduced response to the 100%i i
N I N  2 3 4 5 A B C D E I A B C D E
        

60 American Scientific Journal № ( 22 ) / 201 8
drug in repeated or prolonged use as a result of adaptive
body reaction). These indicators can b e modeled in exper-
iments on animals. This approach only to some extent re-
flects the natural situation that occurs in the clinic as the
resistance of the therapy of epilepsy depends on the course
of the disease itself, and the pharmacodynamic mecha-
nisms (pr imary resistance) and the pharmacodynamics of
drugs (secondary resistance), although the search for ef-
fective pharmacological agents in such conditions is defi-
nitely the basis for their successful application in medical
practice.
The analysis of initial ex perimental data showed that
the obtained data can not be described by the normal
(Gaussian) distribution law, since their predominant part
has different indicators of asymmetry and excess (in 1,5 -
2,8 times the values of the corresponding standard devia-
tion s). In this regard, for the corresponding data of the
control and experimental groups of animals, the values of
the difference were estimated on the basis of the non -par-
ametric Wilcoxon -Mann -Whitney criteria.
It should be noted that seizures of the fifth ( highest)
severity degree were not observed in any experimental an-
imal group, and seizures of the fourth severity were ob-
served only in single animals and only on the terminal
days of the experiment. For seizures of the first to third
severity degree the ca lculated values of the difference sig-
nificant level do not show an adequate level ( р0,2 -0,6 ),
which does not allow us to conclude that the effects of the
administered doses of propoxazepam on the development
of convulsions during the chronic PTZ administr ation
have not been demonstrated. For indicators of the number
of second -degree severity seizures in the group of animals
receiving propoxazepam at a dose of 2.0 mg/kg the calcu-
lated significance levels were less than 0.05 (eighth and
ninth administration) .
For animals of control (Fig. 1A) and experimental
groups (Fig. 1B -D), there was a decrease in the latent time
of seizures manifestation of the first severity degree. In the
control group of animals and in the group receiving
propoxazepam at a dose of 0.2 mg/kg, the minimum value
of the latent time of the first degree of severity convulsion
appeared on the 8 th-11 th day of PTZ administration, what
can be regarded as the absence of significant influence of
the compound on development prolonged neurodegenera-
tive changes.
A B
C D 0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Number of PTZ injection
Impact, %
Point 1 Point 2 Point 3 Point 4 0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Number of PTZ injection
Impact, %
Point 1 Point 2 Point 3 Point 4 0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Nu mb er o f PT Z i n j ecti o n
I mp act, %
Point 1 Point 2 Point 3 Point 4 0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Number of PTZ injection
Impact, %
Point 1 Point 2 Point 3 Point 4

American Scientific Journal № (22 ) / 201 8 61
E
Fig. 1. Change in the partial contribution of convulsions of various severity deg rees to the total number of mice
attacks convulsions in the control group of animals (A) and in experimental groups (B -E) treated with propoxazepam
(0.2 -0.6 -1.0 -2,0 mg/kg, respectively).

In the control group of animals before the 8 th PTZ
injection, ther e is also the occurring of convulsions of the
third and fourth degree of severity, however, these indica-
tors begin to stabilize only after the 11 th administration
(Fig. 1, A). In the experimental group of animals (0.2
mg/kg of propoxazepam), seizures of th e third and fourth
degree of severity occur later (13 th administration). In ad-
dition, convulsions of the third type of severity (Fig. 1D,
E) are observed even when high doses of propoxazepam
were administrated (1.0 and 2.0 mg/kg). Obviously, the
effect of propoxazepam even in high doses on GABA -R
completely does not compensate the functional changes in
other mediator systems (NMDA -, aspartate and gluta-
mate), which are involved in the processes of kindling de-
velopment.
In terms of severity of kindling -epilep sy pathological
process development the partial contribution of each of
convulsions types to general seizure spectra is indicative.
To relatively assess the changes in qualitative composition
of seizures attack, the participation of each type of con-
vulsion was determined in relation to the total amount of
seizures (Fig. 1A -E). This method of data transformation
not only reflects the individual contribution of each type
of convulsion, but also gives an idea of the depth of influ-
ence of the long -term administ ration of PTZ on the bal-
ance of inhibitory and excitation systems.
In animals of the control group (Fig. 1A) during the
development of the kindling -state a gradual decrease in
the number of first -degree convulsions with a simultane-
ous increase in the repre sentation of the higher -severity
seizure arising from functional changes in the central
nervous system is observed. The fourth administration
leads not only to increase of third group convulsions con-
tribution increase, but also the appearance (to 3 -6%) of
more severe components. After the sixth administration,
their presence in the seizure spectrum becomes constant,
although the total contribution does not exceed 6% (Fig.
1A).
In general, the assessment of the partial contribution
is more indicative for the effect of the propoxazepam ad-
ministered doses on the spectrum of seizure attack
(Fig. 1B -E). In the spectrum of convulsive activity of an-
imals that received propoxazepam in a dose of 2.0 mg/kg,
practically there is no manifestation of attacks of even a
second degree of severity, indicating a direct effect on
GABA -R.
One of the classical parameters that characterizes the
development of the kindling -state is the index of convul-
sions ( I), which is both normalized and the average indi-
cator of the contribution of each convulsion type to the
general state of the kindling, which is registered.
Calculated values of the seizure index do not undergo
significant changes and do not exceed the value of 2.0
(Fig. 2). Both in the control group and in the experimental
grou ps of animals receiving low doses of propoxazepam
(0.2 and 0.6 mg/kg), this indicator does not show statisti-
cally significant differences, and only in the group of ani-
mals receiving propoxazepam at dose of 2.0 mg/kg, its
tendency to a significant increase is not observed.
In general, the analysis of primary data (latent time
of convulsive attacks development, their number and de-
gree of severity) does not allow to make conclusion about
the amount of contribution of propoxazepam to inhibit the
development of the kindling state in experimental ani-
mals. However, the variance analysis of these data from
control and experimental groups characterizes the effect
of the total factor of the propoxazepam administered doses
(0.2 -2.0 mg/kg) at 23 % and leaves about 77 % of other
factors (Table 1). At first glance, this is a low value (de-
spite a statistically significant result with p = 0.002) for a
compound that has shown a high anticonvulsant effect in
acute PTZ experiments, especially given the statistically
significant differences in severity of convulsions. How-
ever, this may be due to the lack of exposure to low doses
of propoxazepam, the excessive variability of control val-
ues, or a combination of mutually exclusive factors (for
example, the lack of efficacy of propox azepam in the ter-
minal stages of the kindling state). 0,60
0,80
1,00
1,20
1,40
1,60
1,80
0 2 4 6 8 10 12 14
Number of PTZ injection
Index of convulsions
Control 0,2 mg/kg 0,6 mg/kg 1 mg/kg 2 mg/kg

62 American Scientific Journal № ( 22 ) / 201 8
Fig. 2. Change in the value of the index of convulsions (I) of the control and experimental (propoxazepam in doses of
0.2 -2.0 mg/kg) animal groups during the kindling state development.

Separation of a complex factor (a combination of
propoxazepam doses) into separate subgroups and an as-
sessment of the effect of each dose administered (relative
to the control group) can reveal an increase in the dose
effect of the propoxaz epam (from 11.8% to 51.9% for
doses 0.2 -2.0 mg/kg). On the basis of the calculated val-
ues of the contribution of these factors, it is possible not
only statistically significantly (for each individual group,
the probability level was <0.001) to detect the existence
of dose -dependent intermittent effects of propoxazepam,
but also to explain the impossibility of detecting this effect
by other methods of data analysis.
Table 1
Dispersion analysis of the effects of doses of propoxazepam on the process of kind ling formation in mice.
Group
Factor dis-
persion
(factor
vari ence )
Non -factor
dispersion
(group vari-
ence)
The total
dispersion
The
contribution
of factors
taken into
account,%
Contribu-
tion of un-
accounted
factors,%
The calculated
level of
statistucal sig-
nifi cance , p
Estimation of the overall effect of propoxazepam
(control, doses 0.2
mg/kg, 0.6 mg/kg, 1.0
mg/kg, 2.0 mg/kg)
1.114 0.336 1,450 23 77 0,002
Estimation of the influence of dose -dependent factors on the propoxazepam administration during the devel opment
of kindling process
0.2 mg /kg 0.077 0.574 0.651 11.8 88.2 <0.001
0.6 mg /kg 0,032 0.463 0,495 6.5 93.5 <0.001
1.0 mg /kg 0.086 0.457 0,544 15.9 84.1 <0.001
2.0 mg /kg 0.318 0,295 0.613 51.9 48.1 <0.001

It has been found that administration of lo w doses
(0.2 -0.6 mg/kg) of propoxazepam during the period of for-
mation of the kindling does not have a significant anticon-
vulsant effect, although it inhibits the manifestation of the
third and fourth severity degree convulsions. Propoxaze-
pam high doses (1 .0-2.0 mg/kg) inhibit the development
of high severity degree seizures; at the administration of
high doses (2.0 mg/kg), with practically no manifestation
of attacks even a second degree of severity. According to
the results of the dispersion analysis, the contribution to
the factor of the propoxazepam dose on the model of kin-
dling -epilepsy ranges from 11.8% to 51.9% for doses 0.2 -
2.0 mg/kg).
The presented data also indicate that there is no re-
duction in the response to propoxazepam at doses of 0.2 -
2.0 mg/k g under repeated and continuous administration
schemes, which indicates that there is no experimental
pharmacodynamic resistance to the antiepileptic action of
the substance. According to the literature [12], valproate -
one of the mostly used antiepilepti c drug - in high doses
(100 -200 mg/kg) in chronic oral prophylactic administra-
tion maximally suppresses the development of general-
ized clonical -tonic convulsions of a 4 th severity degree and
only in a small number of mice prevents the development
of local clonic convulsions with severity of 1 -2th degree.
The deficiency of valproate can also be attributed to the
weakening of its anticonvulsant effect by 8 -11% to 21
days of kindling formation, which may indicate the devel-
opment of tolerance to valproate in hi gh doses with pro-
longed use. 0,60
0,80
1,00
1,20
1,40
1,60
1,80
0 2 4 6 8 10 12 14
Number of PTZ injection
Index of convulsions
Control 0,2 mg/kg 0,6 mg/kg 1 mg/kg 2 mg/kg

American Scientific Journal № (22 ) / 201 8 63
PTZ -induced kindling may be related to permanent
attenuation of inhibitory function of GABAergic system
in the brain. [10] Repetitive single dose application ends
up with decreased GABAergic activity [13]. According to
this sug gestion the functioning of the GABA A-receptor in
the CNS can be examined in vivo by estimation of the
competitive effects of benzodiazepines and seizure -induc-
ing agents (pentylenetetrazol, strychnine, and picrotoxin ).
On the basis of dose –effect curves, us ing comparative
quantile analysis for chemoconvulsants with different
mechanisms of action, we showed different stages of
propoxazepam interaction with GABA and glycine recep-
tors under in vivo conditions [4].
CONCLUSIONS
It was found that propoxazepam did not lead to reli-
able changes of seizures manifestations, but limited sec-
ond type seizures development, reducing their quantity.
The reliable redistribution in seizures quantity with two
and four points with simultaneous increasing of higher se-
verity degre e seizures was noted. At the same time sei-
zures of fourth severity points were diminished. For ani-
mals of control and experimental groups the reducing of
first and second seizures type latent time. With propoxa-
zepam administration reducing of higher severi ty seizures
(3 and 4 points) is in accordance with reducing of the la-
tency time for seizures of 1 th and 2 nd types. In the late pe-
riod of kindling development the increase of seizure index
in the experimental group of animals was registered, what
can be due to the glutaminergic system activation in the
terminal stage of the epileptic activity formation .

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