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0BIOLOGICAL SCIENCES
DIFFERENTIAL EFFECTS OF PROPOXAZEPAM ON PENTYLENETETRAZOLE- AND MAXIMAL ELECTROSHOCK-INDUCED CONVULSIONS IN MICE
Larionov V.B.,
Doctor of science, Biology Reder A.S., PhD, Chemistry Golovenko N. Ya.
Doctor of Science, Biology, professor A. V.Bogatsky Physico-Chemical Institute of NAS of Ukraine
Abstract
Propoxazepam ED50 in pentylenetetrazole-induced seizures test (PTZ-test) is 0.9 ± 0.04 mg/kg. At low doses administration seizures of mice are mostly presented by the tonic component, which intensity is reduced with increased dose of administered substance, proving the reducing of paroxysmal activity foci decreasing as well as inhibition process activation in the central nervous system, associated with GABAA-receptors. At maximal elec-troshock test (MES test) propoxazepam effective dose is 0,57 ± 0,23 mg/kg what indicates the non-specific protective action mechanism in this pharmacological model. The propoxazepam dose increase leads to redistribution of different seizures representation - tonic convulsions are gradually reduce with simultaneous dose-dependent increase of clonic component as well as time of seizures appearing.
Keywords: propoxazepam, pentylenetetrazol, maximal electroshock, epilepsy
Epilepsy refers to chronic polyetiological diseases of the brain characterized by recurrent seizures that occur as a result of excessive neuronal discharges and accompanied by various clinical and paraclinical symptoms. Anticonvulsant 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 agents" as they all selectively suppress seizures and their use is determined predominantly by the nature of paroxysmal manifestations or its equivalents [1]. Depending on the clinical manifestations of epilepsy, different anticonvulsants can be prescribed. Often, for the treatment of epilepsy, combined use of several medicines is rational (simultaneously or sequentially). Therefore, the success of the treatment of epilepsy is on the way to finding new anticonvulsants, which would have had an effect on different pathoge-netic links in the formation of all variability of seizure states [2].
Antiepileptic drugs act on different molecular targets, selectively changing the excitability of neurons in such a way that the neuronal activity associated with attacks is blocked without disturbing the normal activity required to transmit signals between neurons. Various mechanisms can lead to reducing the excitability of the neurons of the epileptogenic cell [3]. Basically they consist either in inhibition of activating neurons, or in activation of depressing nerve cells, i.e., they are reduced to three major pharmaco-neurophysiological effects: relief of GAbA or glycine-dependent transmission, reduction of excitatory (glutamate or aspartate) transmission and non-specific modification of ion currents (sodium, calcium and potassium channels). Despite the fact that these medicines take maximum account of the mechanisms of epileptogenesis, the issue of improving their effectiveness is still relevant.
Our studies [4, 5] gave reports that the inhibition of the development of pathological excitation by propoxazepam - 7- bromo-5-(o-chlorophenyl)-3-propoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-one occurs primarily through GABAergic mechanisms. The use of selective chemoconvulsants also suggests the involvement of mixed GABA/glycine synapses, whose contribution to the overall effect, however, does not exceed 70%. In our radio-receptor studies [6], it was found that affinity (K is the inhibition by the propoxazepam binding of [3H] flumazenil with synaptic membranes of the rat brain) is 3.5 ± 0.3 nM. Compared with other benzodiazepine drugs, this is a fairly significant value. For diazepam, chlordiazepoxide, nitrazepam and oxazepam, these figures are 6.3. 220; 6.4; 14.0, respectively. Calculations have shown that GABA-shift for propoxazepam is 1.9, which allows it to be attributed to a full agonist of GABA-RC.
It is known that pentylenetetrazole (PZT) causes the first-generalized seizures and absence attack in experimental animals. At moderate doses PZT administration leads to appearing of clonic and at high doses - tonic-clonic and generalized convulsions (status epi-lepticus) and even animals death. Biotargets of PZT action are GBA- and glycineergic systems. Maximal electroshock (MES) simulates first-generalized seizures (status epilepticus) and partial paroxysms and is the basic test for estimating substances' potential antiseizure activity, if they act on Na+-channels.
Therefore, the aim of this study was to investigate the effects of propoxazepam on PTZ - and MES -induced convulsions in mice for better understanding the propoxazepam s antiepileptic profile and its action mechanisms.
2. Methods
2.1. Experimental Animals
Experiments were performed on outbreed white mice (20-26 g) of both sexes. All experimental procedures were conducted in accordance with the rules of the "European Convention for the Protection of Vertebrate Animals, Used for Experimental and Other Scientific Purposes" in accordance with the Directive of the Council of the European Union 86/609 of the EU of November 24 1986. During experiments, the animals were kept under standard conditions (12 hour light-shade mode and with access to water and food ad libitum).
The test compound (propoxazepam) was administered intraperitoneally (in a Twin 80 emulsion) at doses ranges, determined in the previous pilot studies. Chemoconvulsant solution (PZT, 120 mg/kg, causing lethal effect in 95% of animals) was administered sub-cutaneously to animals (6-8 mice in each experimental group) 30 minutes after propoxazepam. The counting time of the experiment started from the moment of seizure agent administration. During the observation period, the number of myoclonic tremors and generalized attacks in the form of tonic extensia, as well as the time before the onset of a lethal effect was recorded. To characterize the representativeness of each seizure types, the experimental data are presented in relative form (M ± m) of the total number of recorded seizure episodes.
2.2. Drugs, Chemicals, and Solutions
Propoxazepam was synthesized according to the method described in (Andronati et al., 2010). The structure of the substance was determined by a complex of physicochemical methods (IR and mass spectroscopy, as well as X-ray diffraction analysis). Chemical purity was confirmed by elemental analysis (99%). Pentylene-tetrazole (Sigma) was used in isotonic saline (0.9 % NaCl) at dose 120 mg/kg.
2.3. Pentylenetetrazole-Induced Seizures Test
The anticonvulsant effect of propoxazepam was
evaluated in experiments on mice as the relative number of surviving animals that were recorded 2 hours after the convulsant administration. The tested compound was administered intraperitoneally (in a Tween-80 emulsion) at doses whose limits were cho-2.4. chosen after previous pilot studies and according to the requirements of statistical and calculation methods. Chemoconvulsant solution (pentylenetetrazol 120 mg/kg are dose that cause lethal effects in 95% of tested animals) was administered subcutaneously to animals (six to eight animals in each experimental group) 30 min after propoxazepam administration. The start of the experiment was the moment of the administration of the convulsive agent. During the follow-up period, the number of myoclonic tremors and generalized seizures in the form of a tonic extension, as well as the time to the onset of the lethal effect were recorded. To characterize the representativeness of each type of seizure, the data
are presented as a relative number (partial contribution, M ± m) of total convulsive episodes. The lethal effect was evaluated in an alternative form (presence or absence of effect). The protective effect of the substance (ED50) was evaluated by the number of animals (the frequency of effect manifestation) that survived in each individual group.
2.4 Maximal Electroshock (MES)-Induced Seizure Test
Mice were divided into six groups each containing eight animals and treated with vehicle and propoxaze-pam (0,2 - 20 mg/kg i.p.), respectively. Thirty minutes later, seizures were induced by a current stimulus (50 mA, 50 Hz for 0.2 seconds) delivered using ear electrodes by an electroconvulsometer. To improve electrode contact, the electrodes were moistened with normal saline before they were attached to the ears of mice [7]. The current used was predetermined before experimentation. The current that caused hind limb tonic extension and death did not occur in all mice in the control trials. Protection was defined as the complete absence of hindlimb tonic extension.
2.5. Statistical Analysis
Evaluation of the lethal effect was carried out in an alternative form (presence or absence of effect). The protective effect of the substance (the value of ED50) was estimated by the number of animals (frequency of effect) that survived in each individual group.
The ED50 values were calculated by probit analysis [8] (with the Barens corrections). The significance of the differences in indices of convulsive action were evaluated using the Student's t test or by nonparametric statistics (Wilcoxon-Mann-Whitney test) [9]. The data are presented as mean ± standard deviation (M ± m), or as a median (first-third quartile), i.e., Me (Q1-Q3).
3. Results
3.1. Effect of propoxazepam PTZ-induced seizures
The main point index of the compound pharmacological activity is the dose at which the probability of effect developing in 50% of the animals will be maximum (ED50). However, not always the initial experimental data correspond to such a distribution, being a generalized characteristic of the organism response. Thus, the initial indices of anticonvulsant activity of propoxazepam for antagonism with PTZ are characterized by an achievement of 100% effect, while the antagonism with strychnine administration of even high doses of propoxazepam (up to 30 mg/kg) does not allow to achieve such an indicator [4].
The analysis of the "propoxazepam dose-anticon-vulsant effect" curves is presented in Table 1. The value ED50 of propoxazepam for antagonism with PTZ 0.9 ± 0.04 mg/kg indicates the high activity of the substance. Since the antagonism with the PTZ compounds and exhibits a higher activity, then GABAergic and partially glycinergic system are likely to be involved.
Table 1
Indicators of the components of the pharmacological action of propoxazepam (lifetime of animals and the
The average effective dose (M ± m), ED50, 2,24 ± 0,93 mg/kg (0,9 ± 0,04 mg/kg)
The slope of the curve "dose-effect" 0.821
control 6.0 (4.25 J 7,00)
propoxazepam dose (mg/kg)
2 11.0 (10.0L 12.0)
5 11.5 (10.00L 12,25)
8 7.0 (6.5 J23.0)
12.8 16.5 (15.00L 17.75)
16.2 22.0 (19.5L34.5)
25 18.0 (14.25L24,25)
32 20.0 (15.25L36.75)
3.2. Effect of propoxazepam on MES-Induced Seizures
Propoxazepam administration to mice in increasing doses (0.2-20 mg/kg) leads to an increase of the protective effect in the MES test, with a maximum protective effect (100%) at a dose of 20 mg/kg. However, attention is drawn to the fact that the dose-effect relationship in this test does not correspond to the normal distribution in the range of doses used. Thus, doses in the range of 10-90% of the protective effect cause
almost linear dependence, whereas the part of the curve within the maximum frequency of protective action has a more decreasing inclination. According to the protective effect (Table 2) in the MES test, propoxazepam exhibits a significant protective (antiepileptic) effect (ED50 0.57 ± 0.23 mg/kg). The inclination of the curve, equal to 0.51, indicates a slow increase in the protective effect under conditions of dose increase (within the value of ED50).
Table 2
Anticonvulsive action (protective effect) and effect on the frequency of individual seizure attacks of
Indicator The protective effect (number of survived animals) States of convulsive attack (ED50 (ED82-ED18))
Tonic seizures/ tonic extension clonic seizures refractory period
The median effective dose, D, mg/kg 0,57 ± 0,23 0.92 (0.021-39.9) 1.53 (0.28-8,5) 0.56 (0.001-237)
mmol/kg 1,39 ± 0,56 2.25 (0.05-97.9) 3.7 (0.7-20.9) 1.29 (0.003-581)
The slope of the curve "dose-effect», s 0.51 -21.2 -2.3 -4,2
DISCUSSION
As a chemoconvulsant, PTZ is most commonly used. In this procedure, convulsions simulate primary-generalized seizures in so-called abscess seizures. In moderate doses, the administration of PTZ leads to the development of clonic, and in high tonic-clonic and generalized seizures (status epilepticus) and even the death of an animal [10]. PTZ is a ligand of both GABA-RC and glycine-erosive system, although its binding sites are identified in the chloro-ionophore channel of GABA-receptor complex, which is why its effect is often characterized as "noncompetitive" with respect to derivatives of 1.4-benzodiazepines, which exhibit anticonvulsant effect. For conducting an effector analysis of the mechanism of anticonvulsant activity of propox-azepam on models of chemically induced convulsions, its values of mean weight effective doses (ED50) were determined.
The technique of maximum maximal electroshock seizure (MES) simulates primary-generalized seizures (status epilepticus) and partial paroxysms and is a basic test for the evaluation of the action of substances with anticonvulsant activity. It is believed that the MES test
is a model for antiepileptic drugs that act on Na+ channels (eg carbamazepine, phenytoin). At the same time, some anticonvulsants are effective in the MES model, but they interact with other targets: GABA-RC (phenobarbital) and glutamate receptors (topiramate) [11].
Despite the individual differences of experimental animals with regard to convulsive sensitivity, the MES test makes it possible to determine the effect of the compound on the threshold of the process in a particular animal or group of animals. This makes the mentioned method more sensitive and extends its susceptibility to anticonvulsant testing of anticonvulsants. The given model allows not only to determine the efficiency of the compound, but also to characterize some mechanisms of its action.
Despite the complex nature of the influence of the electric current on the brain activity, certain phases of functioning impairment that lead to the development of an epileptic attack, can be defined and above all, it concerns the emergence of generalized depolarization of neurons with the simultaneous de-functionalization of brain structures.
This condition corresponds to the first phase, in which tonic contractions of limbs are observed. The phase of the clonic contractions of the limbs corresponds to the formation of short-term oscillating excitation cells against the background of the extinction of the previous electrostimulation on the one hand and the blocking of the central nervous system's brains on the other. In the absence of brain control for excitatory processes by braking systems (to a greater extent, GA-BAergic), or its hyperactivation in the pathogenesis of development by the seizures for the most part at the expense of glutamate receptors (NMDA-, and AMPA-type) there is synchronization of several paroxysmal cells with the formation of a powerful epileptogenic complex, as well as generalization of excitation and contraction of skeletal muscles, which is externally registered as a phase of tonic extension. In the presence of a depressant compounds in the brain, the inhibitory systems are in a hyperpolarized state, the constancy of which depends on factors such as the affinity of the compound to these receptors, compound's internal activity and the concentration of the compound in the brain tissue. The compounds possessing a powerful an-tiepileptic action, more effectively return the inhibitory system to the physiological (native) state, which blocks the development of paroxysmal activity in the subsequent phases of convulsive attack caused by MES.
Taking into account the described nature of the pathogenesis in MES, it is also expedient to fix the time of each phase, since this indicator is a general characteristic of the change in processes during this period. In addition, after the restoration of the native state of the central nervous system, the animals are in a state of a certain stupor - a refractory period during which behavioral reflexes are restored. The duration of this period is a characteristic of the rate of return of the central nervous system to the physiological.
Our studies indicate that in animals of control group electrostimulation did not cause tonic-clonic seizures, and the structure of the epileptic attack was represented only by a tonic component associated with irreversible processes of excitation propagation, which caused a high level of lethality in this group (100%, n = 6). The average duration of the tonic component in animals was 14,9 ± 2,6 s, and this indicator did not change due to the effects of various doses of propoxa-zepam.
The results obtained regarding the protective effect of propoxazepam (ED50 0,57 ± 0,23 mg/kg) in the MES model are somewhat unusual, not only for 1,4-benzodiazepine derivatives, but also for most antiepi-leptic drugs. The calculated ED50 value for propoxaze-pam protective effect in the PTZ-induced convulsions test was 0,92 1 0,38 mg/kg. Taking into account the errors of experiments, it is possible to recognize the indices obtained for MES and PTZ as similar. At the same time, from literary sources [12] is aware of the lack of similarity between the noted indicators. So diazepam has the following ratio MES/PTZ (mg/kg, mouse), -3,5/0,52; clonazepam (7,5/0,06); nitrazepam (4,5/0,27); phenazepam (10,2/0,037); gidazepam (41,2/0,36). Barbituric acid derivatives (phenobarbital, primidon) have respectively the following indices:
21,8/13,2; 11,4/58,6, and diphenylhydantoin (12,8/149,8). Some anticonvulsants (phenytoin, car-bamazepine) do not affect seizures caused by PTZ, but are active in relation to MES (9,5 and 8,8, respectively). Only for metsuximide (76,3/68,3) and valproic acid (271,7/148,6) there are approximately similar indicators. Obviously, the protective properties of the drugs in these models depend, at least, on two components: the effect of seizure agents on various anatomical and morphological structures of the central nervous system [12] and molecular mechanisms of seizures.
With regard to the significant difference in the ratios of MES/PTZ ratios of related compounds (1,4-ben-zodiazepines) having similar mechanisms of action, their explanation may be data on the structure of GBAergic system, for which the effect of PTZ is often characterized as "noncompetitive" in relation to anticonvulsant derivatives of 1,4-benzodiazepine [13]. It should be noted that a part of GABA-RC is represented by a number of subtypes, among which are responsible for the seizure effect of chemoconvulsants. Moreover, each benzodiazepine prefers a specific receptor subtype [14, 15]. So diazepam, clonazepam and nitrazepam are high-affinity compounds of a2 and a3 receptors.
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ОПТИМИЗАЦИЯ ПАРАМЕТРОВ КУЛЬТИВИРОВАНИЯ РЕКОМБИНАНТНОГО ШТАММА E. COLI, ПРОДУЦИРУЮЩЕГО L-ФЕНИЛАЛАНИН-АММОНИЙ-ЛИАЗУ (PAL)
Бабич О.О.,
доктор технических наук Дышлюк Л.С., кандидат биологических наук Сухих С.А.
доцент
Кемеровский государственный университет
OPTIMIZATION OF THE CULTIVATION PARAMETERS OF THE RECOMBINANT STRAIN OF E. COLI PRODUCE L-PHENYLALANINE AMMONIUM-LIAZE (PAL)
Babich O.O.,
Doctor of Technical Sciences Dyshlyuk L.S., Candidate of Biological Sciences
Sukhikh S.A.
assistant professor Kemerovo State University
Аннотация
С целью получения оптимального количества L-фенилаланин-аммоний-лиазу (PAL), используемый в качестве основного компонента для противоопухолевого препарата, произведена оптимизация параметров необходимых для культивирования рекомбинантного штамма E. coli
Abstract
In order to obtain the optimal amount of L-phenylalanine ammonia-lyase (PAL), which is used as the main component for the antitumor drug, the parameters necessary for the cultivation of the recombinant E. coli strain were optimized
Ключевые слова: L-фенилаланин-аммоний-лиаза, противоопухолевый препарат, параметры культивирования
Keywords: L-phenylalanine-ammonium-lyase, anticancer drug, cultivation parameters
В России на учете по онкозаболеваниям состоит около 3 млн человек. Каждый год специалисты в области медицины выявляют свыше 500 тыс. злокачественных новообразований [2. C. 3].
Проблема борьбы с раковыми заболеваниями на сегодняшний день имеет общенациональное и общегосударственное значение. К сожаление фармацевтический рынок России практические не имеет представителей отечественного производства, которые занимаются разработкой и выпуском противораковых препаратов [4. C. 6225].
Большая часть противоопухолевых препаратов представляют собой L-аспарагиназы, которая выделяется из различных бактериальных источников.
Наиболее перспективным является препарат L-фенилаланин-аммоний-лиаза (PAL), который предназначен для борьбы с опухолевыми новообразованиями. Эффективное действие такого препарата связано со
снижением уровня L-фенилаланина в лейкемических опухолевых клетках [3 C. 67].
Все вышеизложенное определило цель настоящего исследования, а именно оптимизация параметров культивирования рекомбинантного штамма E. coli, продуцирующего L-фенилаланин-аммоний-лиазу (PAL).
В автоинжукционной среде, содержащая в своём составе канамицин с концентрацией 100 мкг/мл, осуществляли ферментирование в на протяжении 18 и 32 ч, температурный режим процесса ферментирования составлял 37 °С и 25 °С. Средняя оптическая плотность клеток штамм E. coli BL21PAL была постоянной и находилась в пределах 7 оп.ед.
С помощью метода денситометрического анализа полученной электрофореграммы индуцирован-
