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We studied the effects of a new dipeptide neuroleptic Dilept (N-caproyl-L-prolyl-L-tyrosine methyl ester) on activity of neurotransmitter systems in the brain. Dilept possessed antidopamine, glutamate modulatory, and cholinomimetic properties. These data indicate that Dilept is of potential efficacy in relieving positive and negative symptoms of schizophrenia.
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Bulletin of Experimental Biology and Medicine, No. 5, 2003 PHARMACOLOGY AND TOXICOLOGY467Neuromodulatory Mechanism Underlying the Effectof the Atypical Dipeptide Neuroleptic DileptM. V. Retyunskaya, L. S. Guzevatykh, N. A. Bondarenko,T. A. Gudasheva, R. U. Ostrovskaya, and T. A. VoroninaTranslated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 136, No. 11, pp. 527-531, November, 2003
Original article submitted June 30, 2003We studied the effects of a new dipeptide neuroleptic Dilept (N-caproyl-L-prolyl-L-tyrosine
methyl ester) on activity of neurotransmitter systems in the brain. Dilept possessed antidopamine, glutamate modulatory, and cholinomimetic properties. These data indicate that Dilept
is of potential efficacy in relieving positive and negative symptoms of schizophrenia.Key Words: Dilept; dipeptide; neuroleptics; neurotransmitters; schizophreniaThe therapy of mental disorders accompanying schizophrenia is an urgent problem of psychiatry. Classic
neuroleptics used in clinical practice are efficient in
eliminating productive symptoms of the disease. Repeated treatment with these preparations is followed
by the appearance of extrapyramidal symptoms and
aggravates negative symptoms of the disease. Atypical
neuroleptics cause less pronounced side effects. However, their antipsychotic activity is often in sufficient.
Schizophrenia is accompanied by hyperreactivity of
the dopaminergic (DAergic) system in limbic structures of the brain [4] and dysfunction of central neurotransmitter systems. For examples, glutamatergic dysfunction develops during schizophrenia [8]. In some
patients with schizophrenia hyperactivation of the glutamatergic system in various structures of the hippocampus and cortex is accompanied by progressive
death of neurons [6]. An imbalance in glutamatergic
neurotransmission is a major cause of cognitive disorders during schizophrenia. The appearance of psychotic symptoms is followed by activation of serotoninergic transmission. The antipsychotic effect of
most atypical neuroleptics is determined by antidopamine and antiserotonin properties [11]. Recent studies showed that deficiency in cholinergic neurotransmission in the limbicofrontal brain system [5] and,
particularly, in nicotinic cholinoceptors [10] plays an
important role in the development of negative symptoms of schizophrenia.The peptidergic concept describing the action of
neurotropic preparations and developed by T. A. Gudasheva allowed synthesizing peptide analogues of -turn structure of the active site of neurotensin (neuropeptide with antipsychotic activity) and atypical neuroleptic sulpiride [9]. Further experiments were
performed with N-caproyl-L-prolyl-L-tyrosine methyl
ester (Dilept), which possesses pronounced activity.
Dilept does not cause side effects typical of most antipsychotic drugs (e.g., catalepsy, sedation, and muscle
relaxation). Dilept is a promising atypical neuroleptic
not producing extrapyramidal disorders [1].The neuromodulatory mechanism underlying influence of Dilept was studied using analytic compounds for major neurotransmitter systems (DA-, glutamate-, serotonin-, and cholinergic) involved in the
development of psychopathological disturbances during schizophrenia.MATERIALS AND METHODSExperiments were performed on male C57BL/6 mice
and male outbred albino rats weighing 20-22 and 220-
250 g, respectively. The animals were kept in a vivarium under standard conditions and had free access to
water and food. Previous studied showed that DileptInstitute of Pharmacology, Russian Academy of Medical Sciences. Ad
dress for correspondence: [email protected]. Ostrovskaya R. U.0007-4888/03/13650467$25.00 2003 Plenum Publishing Corporation468Bulletin of Experimental Biology and Medicine, No. 5, 2003 PHARMACOLOGY AND TOXICOLOGYin a dose of 0.8 mg/kg displays maximum activity [1].
A freshly prepared suspension of Dilept and Tween 80
was injected intraperitoneally.Sensitivity of the DAergic system to Dilept was
studied in tests of apomorphine-induced verticalization and haloperidol-induced catalepsy. To evaluate
individual emotionality the mice were tested for the
reaction to novelty. We recorded the duration of freezing behavior in a metal-grid chamber (3535 cm).
By freezing behavior to novelty, the animals were
divided into 2 groups. Group 1 included the animals
with a first-movement latency of not more than 1 sec
(highly reactive mice, HRM). The animals whose freezing behavior lasted more than 1 sec comprised
group 2 (low reactive mice, LRM).Antidopamine activity of Dilept in HRM and
LRM was studied in the test of apomorphine-induced
verticalization. The animals received Dilept or solvent
in an equivalent volume (physiological saline and
Tween 80) 15 min before apomorphine administration.
Apomorphine in a dose of 2 mg/kg was injected subcutaneously immediately before placing the animal
into cylindrical wire chambers (diameter 13 cm, height
16 cm). Recording of vertical activity started 10 min
after apomorphine administration and continued for
1 h at 2-min intervals. The degree of verticalization
was determined by a 4-point scale (number of limbs
put on the vertical wall) [2]. The total score of verticalization was estimated for each animal over the period of observations.Haloperidol (Gedeon Richter) in a dose of 1 mg/kg
was injected intraperitoneally to produce catalepsy.
The degree of catalepsy (ability to maintain posture)
was determined in the staircase test by a 4-point scale
30, 60, 90, and 120 min after haloperidol administration.
We evaluated the ability of mice to hold 1 or 2 forelimbs
on steps (2.5 and/or 5.0 cm in height) for 10 sec.For evaluation of the possible effects of Dilept on
the glutamatergic system we used two types of pharmacological compounds: noncompetitive NMDA receptor blocker ketamine and glutamate decarboxylase
inhibitor thiosemicarbazide inducing accumulation of
neuronal glutamate. The ability of Dilept to improve
learning impaired by ketamine was determined in the
test of conditioned passive avoidance response
(CPAR). Experiments were performed in a 2-compart-
ment device (Lafayette Instrument Co.) consisting of
a dark chamber with an electrode floor and a platform
fixed at a height of 1 m and illuminated with a 60-W
lamp. The rat was placed on a platform and the latency of the first entry into the compartment with
electrode floor was recorded. Unavoidable electroshock (5 electrical stimuli, 0.45 mA, 1 sec) was delivered after 3-min exploration of the chamber. CPAR
performance was evaluated after 24 h. The latency of
transition into the dark compartment was recorded
(testing). Passive avoidance of performance was assessed by the difference between latencies of the first
entry into the dark compartment during testing and
learning (DLP). Ketamine in a dose of 30 mg/kg was
injected intraperitoneally 5 min before learning. The
test compound or solvent in an equivalent volume was
administered 15 min before ketamine injection. The
solvent was given 2 times to animals of the passive
control group.In mice receiving Dilept or solvent the severity
(4-point scale) and latency of seizures produced by
thiosemicarbazide (7.5 mg/kg subcutaneously) and
animal death were evaluated. Thiosemicarbazide not
only increases glutamate content, but also reduced -aminobutyric acid (GABA) concentration in the brain.
We studied the effect of Dilept on the latency of seizure and death produced by the GABAA receptor blocker bicuculline (3 mg/kg). The animals received Dilept
15 min before bicuculline administration.The effect of Dilept on the serotoninergic system
was evaluated by modulation of the head-twitch response produced by serotonin precursor 5-hydroxy-
tryptophan (5-HTP). Dilept was injected intraperitoneally 15 min before 5-HTP (150 mg/kg).The effect of Dilept on the central muscarinic
cholinergic system was determined in mice with tremor produced by arecoline (25 mg/kg subcutaneously). The mean duration of tremor was estimated (sec).
The effect of Dilept on the central nicotinic cholinergic system was determined by modulation of seizures
produced by nicotinic receptor agonist nicotine hydrotartrate (25 mg/kg subcutaneously). The severity of
tremor and clonic seizures was evaluated using a
5-point scale. We summarized points that were recorded over 20-min observations. Dilept or solvent in
an equivalent volume was administered 15 min before
treatment with arecoline or nicotine.The results were analyzed by MannWhitney U
test (Statgraf software). The differences were significant at p<0.05.RESULTSPreliminary study showed that the degree of apomorphine-induced verticalization in C57BL/6 mice varies
from 26 to 70 points. We hypothesized that the sensitivity to this dopaminomimetic depends on the initial
emotional reactivity of animals. The mice were divided into groups depending on the reaction to novelty.
The degree of apomorphine-induced verticalization in
LRM was higher than in HRM. In these animals the
mean degree of verticalization was 69.714.86 and34.676.45 points, respectively. Study of sensitivity of
the DAergic system to the DA receptor antagonistM. V. Retyunskaya, L. S. Guzevatykh, et al.469Fig. 1. Effect of Dilept on apomorphine-induced verticalization in low
reactive (1) and highly reactive mice (2). Light bars: apomorphine
hydrochloride (control). Dark bars: Dilept and apomorphine hydrochloride. *p<0.05 compared to the control.haloperidol showed that the severity of catalepsy in
LRM is higher than in HRM. The mean degree of
catalepsy in HRM and LRM was 2.500.25 and3.690.16 points, respectively, 90 min after treatment.
These data suggest that in LRM the sensitivity of the
DAergic system to DA receptor agonists and antagonists is higher than in HRM.The effect of Dilept on the DAergic system was
studied in the test of apomorphine-induced verticalization. Dilept decreased the degree of verticalization
more pronounced in LRM, but had no effect on HRM
with low initial verticalization (Fig. 1). In some HRM
the degree of verticalization slightly increased after
Dilept administration.In the CPAR test ketamine produced an amnestic
effect, which manifested in shortening of DLP compared to control animals (28.77.9 and 11713 sec,
respectively). Dilept decreased the severity of amnesia
and increased DLP to 62.712.7 sec (Fig. 2).Dilept markedly relieved thiosemicarbazide-induced seizures, decreased mortality rate (56.2 vs. 93.7%
in the control), and delayed the time of the animal
death (164.919.6 vs. 114.67.8 min in the control).
The severity of bicuculline-induced seizures remained
practically unchanged after treatment with Dilept.The study of the effect of Dilept on head-twitch
response induced by 5-HTP showed that this preparation has no effect on hyperkinesia. It can be hypothesized that Dilept does not modulate activity of the
serotoninergic system.The mean duration of tremor produced by the
muscarinic receptor agonist arecoline increased by48.8% in animals receiving Dilept (p<0.05). Dilept
increased the degree of nicotine-induced tremor by
37% compared to the control (p<0.05).Our results indicate that a new promising dipeptide neuroleptic Dilept modulates the state of various
neurotransmitter systems. D2 receptor blockade causes
side extrapyramidal effects [12]. We found that Dilept
possesses moderate antidopamine activity. Moreover,
this substance produce no cataleptic and myorelaxing
effects [1]. These data suggest that Dilept would produce a mild antipsychotic effect not accompanied by
the development of extrapyramidal disorders. Our previous experiments showed that atypical neuroleptic
sulpiride displays similar activity in the test of apomorphine-induced verticalization. However, this effect
was observed after treatment with sulpiride in doses
20-fold exceeding those of Dilept. Moreover, administration of sulpiride in doses slightly surpassing the
effective concentration caused catalepsy.Glutamate agonistic properties of Dilept manifesting in antagonism to the effect of noncompetitive
NMDA receptor blocker ketamine suggest its potential
efficacy in relieving negative symptoms of schizophrenia. Dilept had antiglutamate activity in the test
of thiosemicarbazide-induced seizures, which is consistent with the ability of this preparation to reduce the
severity of neurocytotoxic damage to cultured cerebellar cells produced by glutamate in high concentrations. Glutamate modulatory properties of Dilept are
of considerable importance, since disturbances in glutamatergic transmission play a role in the pathogenesis
of schizophrenia. Hypoactivity of this system contributes to the development of cognitive deficit [6],
while hyperactivity is accompanied by neurotoxic
changes [8].The cholinergic system of the hippocampus and
cortex plays an important role in memory. The number
of muscarinic [5] and nicotinic receptors [10] in these
brain structures decreases during schizophrenia. TheFig. 2. Effect of Dilept on the impairment of learning in the test of
conditioned passive avoidance response produced by ketamine:
control (1), ketamine (2), and Dilept and ketamine (3). p<0.05:
*compared to the control group; +compared to active control animals
receiving ketamine.470Bulletin of Experimental Biology and Medicine, No. 5, 2003 PHARMACOLOGY AND TOXICOLOGYREFERENCESpositive effect of nicotine on cognitive function is
related to modulation of DAergic systems in the cortex
and hippocampus [7,10]. Neuroleptics possess cholinolytic activity [3], which aggravates the symptoms
of cognitive deficit. Dilept acts as the agonist of muscarinic and nicotinic cholinoceptors and, therefore, is
of potential efficacy in relieving negative symptoms
of schizophrenia.Thus, we analyzed the effect of Dilept on various
neurotransmitter systems and found that this substance
possesses antidopamine, glutamate modulatory, and
cholinomimetic properties. Modern notions about the
multifactor nature of neuromodulatory disturbances
during schizophrenia suggest that Dilept is a promising atypical neuroleptic. The efficiency of Dilept in
relieving positive symptoms of schizophrenia is probably associated with its antidopamine activity. Cholinomimetic and dopamine agonistic properties of Dilept determine its efficiency in eliminating negative
symptoms of schizophrenia. The decrease in neurotoxic activity of excess glutamate contributes to the
neuroprotective effect of Dilept.This work was supported by the Russian Foundation for Basic Research (grant No. 03-04-49049).1. L. S. Guzevatykh, R. U. Ostrovskaya, T. A. Gudasheva, et al.,
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