Modification of NO-cGMP Pathway Differentially Affects Diazepam- and Flunitrazepam-Induced Spatial and Recognition Memory Impairments in Rodents.

This study investigated the influence of sildenafil and methylene blue (MB), two modulators of the nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway on amnesic effects of two benzodiazepines (BZs) (diazepam (DZ) and flunitrazepam (FNZ)), in rodents-mice and rats. In the modified elevated plus maze (mEPM) and novel object recognition (NOR) tests, MB given ip at a dose of 5 mg/kg 5 min prior to DZ administration (0.25 or 1 mg/kg, sc) enhanced/induced memory impairment caused by DZ. When MB (2.5, 5, and 10 mg/kg) was applied 5 min prior to FNZ administration (0.05 and 0.1 mg/kg), an effect was opposite and memory impairment induced by FNZ was reduced. When sildenafil (2.5 and 5 mg/kg, ip) was applied 5 min prior to DZ, we observed a reduction of DZ-induced memory deficiency in the mEPM test. A similar effect of sildenafil was shown in the NOR test when the drug was applied at doses of 1.25, 2.5, and 5 mg/kg prior to DZ. In the mEPM test, sildenafil at abovementioned doses had no effects on FNZ-induced memory impairment. In turns, sildenafil administered at doses of 2.5 and 5 mg/kg increased the effect of FNZ on memory impairment in the NOR test. In conclusion, the NO-cGMP pathway is involved differentially into BZs-induced spatial and recognition memory impairments assessed using the NOR and mEPM tests. Modulators of the NO-cGMP pathway affect animal behavior in these tests in a different way depending on what benzodiazepine is applied.

Synthesis and Pharmacological Evaluation of Novel 1-(1,4-Alkylaryldisubstituted-4,5-dihydro-1H-imidazo)-3-substituted Urea Derivatives.

Novel 1-(1,4-alkylaryldisubstituted-4,5-dihydro-1H-imidazo)-3-substituted urea derivatives have been synthesized and evaluated for their central nervous system activity. Compounds 3a-m were prepared in the reaction between the respective 1-alkyl-4-aryl-4,5-dihydro-1H-imidazol-2-amines 1a-c and appropriate isocyanates 2 in dichloromethane. The compounds were subjected to in silico ADMET studies in order to select best candidates for in vivo experiments. The effects of the compounds on the spontaneous locomotor activity and amphetamine-evoked hyperactivity were estimated. Analgesic activity, without or in the presence of naloxone, was assessed in the writhing test. The tendency to change the HTR, evoked by l-5-HTP and the involvement in alteration in body temperature in mice was studied. Additionally, to check possible occurrence of drug-induced changes in the muscle relaxant activity of mice, which may have contributed to their behaviour in other tests, the rota-rod and chimney tests were performed. The new urea derivatives exerted significant activities in the performed pharmacological tests, although the presented results show a preliminary estimation, and thus, need to be extended for identification and understanding the complete pharmacological profile of the examined compounds.

The adenosinergic system is involved in sensitization to morphine withdrawal signs in rats-neurochemical and molecular basis in dopaminergic system.

RATIONALE: Experimental data informs that not only do the dose and time duration of dependent drugs affect the severity of withdrawal episodes. Previous withdrawal experiences may intensify this process, which is referred as sensitization to withdrawal signs. Adenosine and dopamine (DA) receptors may be involved in this sensitization. OBJECTIVES: Rats were continuously and sporadically treated with increasing doses of morphine for 8 days. In rats, sporadically treated with morphine, morphine administration was modified by adding three morphine-free periods. Adenosine agonists were given during each of the morphine-free periods (six injections in total). On the 9th day, morphine was injected. One hour later, naloxone was administered to induce morphine withdrawal signs. Then, the animals were placed into cylinders and the number of jumpings was recorded. Next, the rats were decapitated and brain and brain structures (striatum, hippocampus, and prefrontal cortex) were dissected for neurochemical, molecular, and immunohistochemical experiments within DAergic pathways. RESULTS: We demonstrated that previous experiences of opioid withdrawal intensified subsequent withdrawal signs. Adenosine ligands attenuated the sensitization to withdrawal signs. In a neurochemical study, the release of DA and its metabolites was impaired in all structures. Significant alterations were also observed in mRNA and protein expression of DA receptors. CONCLUSIONS: Results demonstrate that intermittent treatment with morphine induces alterations in the DAergic system which may be responsible for sensitization to morphine withdrawal signs. Although adenosine ligands attenuate this type of sensitization, they are not able to fully restore the physiological brain status.

l-NAME differential effects on diazepam and flunitrazepam responses of rats in the object recognition test.

BACKGROUND: The present study was undertaken to better understand possible interaction(s) between a non-selective nitric oxide inhibitor: N(G)-nitro-l-arginine methyl ester (l-NAME) and benzodiazepines (BZs) in recognition memory. METHODS: The study was carried out on adult male albino Wistar rats. A novel object recognition (NOR) task was used to evaluate memory process. RESULTS: Combined administration of l-NAME (50mg/kg, ip) with a threshold dose of DZ (0.25mg/kg) induced amnesic effects in rats, participating in the NOR test. On the other hand, following a combined administration of l-NAME (100mg/kg, ip) with flunitrazepam (FNZ; 0.1mg/kg), it was found out that l-NAME inhibited the amnesic effects of FNZ on rats in the NOR test. CONCLUSIONS: The obtained results suggest that suppressed NO synthesis may lead to a facilitation of DZ-induced memory impairment but surprisingly may prevent amnesic effect after FNZ in rats, submitted to NOR task.

Effects of NMDA antagonists on the development and expression of tolerance to diazepam-induced motor impairment in mice.

The goal of the study was to investigate the effects of ketamine and memantine on the development and expression of tolerance to diazepam (DZ)-induced motor impairment in mice. DZ-induced motor incoordination was assessed by the rotarod and chimney tests. It was found that (a) ketamine, at the dose of 5mg/kg (but not 2.5mg/kg), decreased the expression, but not the development, of tolerance to the motor impairing effects of DZ, (b) memantine, at the doses of 5 and 10mg/kg decreased both the development and expression of DZ tolerance in the rotarod test (also in the chimney test but at the higher dose of 10mg/kg) and (c) ketamine and memantine alone had no effect, either in the rotarod or the chimney test in mice. Those findings provided behavioral evidence that the glutamatergic system could contribute an important role in the development and/or expression of tolerance to DZ in mice.

Effects of the adenosinergic system on the expression and acquisition of sensitization to conditioned place preference in morphine-conditioned rats.

In the presented study, we attempt to investigate if the sensitization to conditioned place preference (CPP) induced by low doses of morphine was developed in rats which have been previously conditioned with morphine. The experiments were performed in the CPP test. Firstly, it has been demonstrated that administration of ineffective dose of morphine on the 9th day induces the increase in time spent of rats at a morphine-paired compartment, confirming that sensitization to CPP has been developed in these animals. Secondly, it has been shown that stimulation of A1 receptor significantly inhibits the expression of morphine-induced of sensitization, and blockade of these receptors produces the opposite effect. Finally, it has been indicated that both stimulation and blockade of A1 and/or A2A receptors inhibit the acquisition of sensitization to CPP. The obtained results have strongly supported the significance of adenosinergic system in both expression and acquisition of studied sensitization. These results seem to be important for the identification of connections in the central nervous system which can help finding new strategies to attenuate rewarding action of morphine.

Pharmacological effects of primaquine ureas and semicarbazides on the central nervous system in mice and antimalarial activity in vitro.

New primaquine (PQ) urea and semicarbazide derivatives 1-4 were screened for the first time for central nervous system (CNS) and antimalarial activity. Behavioural tests were performed on mice. In vitro cytotoxicity on L-6 cells and activity against erythrocytic stages of Plasmodium falciparum was determined. Compound 4 inhibited 'head-twitch' responses and decreased body temperature of mice, which suggests some involvement of the serotonergic system. Compound 4 protected mice against clonic seizures and was superior in the antimalarial test. A hybrid of two PQ urea 2 showed a strong antimalarial activity, confirming the previous findings of the high activity of bis(8-aminoquinolines) and other bisantimalarial drugs. All the compounds decreased the locomotor activity of mice, what suggests their weak depressive effects on the CNS, while PQ derivatives 1 and 2 increased amphetamine-induced hyperactivity. None of the compounds impaired coordination, what suggests a lack of their neurotoxicity. All the tested compounds presented an antinociceptive activity in the 'writhing' test. Compounds 3 and 4 were active in nociceptive tests, and those effects were reversed by naloxone. Compound 4 could be a useful lead compound in the development of CNS active agents and antimalarials, whereas compound 3 may be considered as the most promising lead for new antinociceptive agents.

Synthesis, central nervous system activity and structure-activity relationships of novel 1-(1-Alkyl-4-aryl-4,5-dihydro-1H-imidazo)-3-substituted urea derivatives.

A series of 10 novel urea derivatives has been synthesized and evaluated for their central nervous system activity. Compounds 3a-3h were prepared in the reaction between the respective 1-alkyl-4-aryl-4,5-dihydro-1H-imidazol-2-amines 1a and 1b and appropriate benzyl-, phenethyl-isocyanate or ethyl 4-isocyanatobenzoate and ethyl isocyanatoacetate 2 in dichloromethane. Derivatives 4c and 4g resulted from the conversion of 3c and 3g into the respective amides due to action of an aqueous ammonia solution. The results obtained in this study, based on literature data suggest a possible involvement of serotonin system and/or the opioid system in the effects of tested compounds, and especially in the effect of compound 3h. The best activity of compound 3h may be primarily attributed to its favourable ADMET properties, i.e., higher lipophilicity (related to lower polar surface area and greater molecular surface, volume and mass than for other compounds) and good blood-brain permeation. This compound has also the greatest polarizability and ovality. The HOMO and LUMO energies do not seem to be directly related to activity.

Synthesis, central nervous system activity and structure-activity relationship of N-substituted derivatives of 1-arylimidazolidyn-2-ylideneurea and products of their cyclization.

A series of 20 N-substituted derivatives of 1-arylimidazolidyn-2-ylideneurea and products of their cyclization was designed as compounds having double antinociceptive and serotoninergic activity. Ethyl {[(1-arylimidazolidin-2-ylidene)carbamoyl]amino}acetates were prepared from 1-aryl-4,5-dihydro-1H-imidazol-2-amines and ethyl isocyanatoacetate, and then converted with ammonia solution to 2-{[(1-phenylimidazolidin-2-ylidene)carbamoyl]amino}acetamides. Both series of N-substituted derivatives of 1-arylimidazolidyn-2-ylideneureas were subjected to cyclization to respective imidazo[1,2-a][1,3,5]triazines. Chain and cyclic compounds bearing ester moiety affected spontaneous locomotor activity, body temperature of mice as well as showed antinociceptive and serotoninergic activity. Interestingly, their antinociceptive activity was not reversed by naloxone, thus it is not mediated through the opioid system. Chain and cyclic compounds bearing amide moiety were devoid of central nervous system (CNS) activity which may be attributed to unfavorably low lipophilicity (connected with too high polar surface area and too small molecular volume) and poor blood-brain barrier permeation properties.

Divergent effects of L-arginine-NO pathway modulators on diazepam and flunitrazepam responses in NOR task performance.

The goal of the study was an evaluation of the degree, in which nitric oxide (NO) is involved in the benzodiazepines (BZs)-induced recognition memory impairment in rats. The novel object recognition (NOR) test was used to examine recognition memory. The current research focused on the object memory impairing effects of diazepam (DZ; 0.5 and 1mg/kg, sc) and flunitrazepam (FNZ; 0.1 and 0.2mg/kg; sc) in 1-hour delay periods in rats. It was found that acute ip injection of L-arginine (L-arg; 250 and 500 mg/kg; ip), 5 min before DZ administration (0.5mg/kg, sc) prevented DZ-induced memory deficits. On the other hand, it was also proven that L-arg (125, 250 and 500 mg/kg; ip) did not change the behaviour of rats in the NOR test, following a combined administration with FNZ at a threshold dose (0.05 mg/kg; sc). It was also found that 7-nitroindazole (7-NI; 10, 20 and 40 mg/kg; ip) induced amnesic effects in DZ in rats, submitted to the NOR test, following a combined administration of 7-NI with a threshold dose of DZ (0.25mg/kg; sc). However, following a combined administration of 7-NI (10, 20 and 40 mg/kg; ip) with FNZ (0.1mg/kg; sc), it was observed that 7-NI inhibited the amnesic effects of FNZ on rats in the NOR test. Those findings led us to hypothesize that NO synthesis suppression may induce amnesic effects of DZ, while preventing FNZ memory impairment in rats, submitted to NOR tasks.

Synthesis, central nervous system activity, and structure-activity relationship of 1-aryl-6-benzyl-7-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidine-5(1H)-ones.

A series of 24 1-aryl-6-benzyl-7-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidine-5(1H)-ones was designed as antinociceptive compounds acting through opioid receptors with additional serotoninergic activity. The compounds, similarly as previously published series, lack the protonable nitrogen atom which is a part of classical opioid receptor pharmacophore and is necessary to interact with the conserved Asp(3.32) in the opioid receptor binding pocket. The compounds were obtained in one-step cyclocondensation of 1-aryl-4,5-dihydro-1H-imidazol-2-amines diethyl 2-benzylmalonate or diethyl 2-(2-chlorobenzyl)malonate under basic conditions. Almost all the tested compounds exerted strong antinociceptive activity, but surprisingly, it was not reversed by naloxone; thus, it is not mediated through opioid receptors. It makes it possible to conclude that addition of one more aromatic moiety to the non-classical opioid receptor pharmacophore results in the compounds which are not opioid receptor ligands. The lack of activity of one of the tested compounds may be attributed to low blood-brain barrier permeation or unfavorable distribution of electrostatic potential and HOMO and LUMO orbitals.

Pharmacological and structure-activity relationship evaluation of 4-aryl-1-diphenylacetyl(thio)semicarbazides.

This article describes the synthesis of six 4-aryl-(thio)semicarbazides (series a and b) linked with diphenylacetyl moiety along with their pharmacological evaluation on the central nervous system in mice and computational studies, including conformational analysis and electrostatic properties. All thiosemicarbazides (series b) were found to exhibit strong antinociceptive activity in the behavioural model. Among them, compound 1-diphenylacetyl-4-(4-methylphenyl)thiosemicarbazide 1b was found to be the most potent analgesic agent, whose activity is connected with the opioid system. For compounds from series a significant anti-serotonergic effect, especially for compound 1-diphenylacetyl-4-(4-methoxyphenyl)semicarbazide 2b was observed. The computational studies strongly support the obtained results.

The antinociceptive effect of 4-substituted derivatives of 5-(4-chlorophenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione in mice.

The aim of the present experiments was to examine the antinociceptive activity of 4-substituted derivatives of 5-(4-chlorophenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione in mice. The compounds were synthesized using the so-called Mannich reaction and their structures were confirmed using IR and 1H-NMR spectra. The antinociceptive activity was investigated in two behavioral tests: the hot plate test and the writhing test. For preliminary estimation of other behavioral effects, the locomotor activity of mice, the motor coordination in the rota-rod test, and the myorelaxation in the chimney test were also studied. The changes in body temperature of animals were also recorded. We demonstrated that all examined compounds produced antinociceptive effect, both in the hot plate test and in the writhing test, without impact on the motor coordination and myorelaxation of animals. The pharmacological effect of all drugs has been developed within 60 min after administration of drugs; and in two cases (T-103 and T-104), it has been a short-lasting effect (up to 90 min). Two compounds (T-100 and T-102) also inhibited the locomotor activity of animals. T-104 induced the changes in body temperature of mice. Generally, we demonstrated that combination of two different heterocyclic systems (morpholine and 1,2,4-triazole) might be beneficial for reduction of nociception.

Synthesis, pharmacological activity and molecular modeling of 1-aryl-7- hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidine-5(1H)-ones and their 6-substituted derivatives.

Pain management is an important medical problem with social and economic consequences. Opioid receptors are among the most important molecular targets involved in antinociception. We have previously reported several series of antinociceptive compounds with the affinity to opioid receptors. In search for novel compounds acting on central nervous system with antinociceptive activity we synthesized a series of 1-aryl-7-hydroxy-2,3-dihydroimidazo[1,2- a]pyrimidine-5(1H)-ones and their 6-phenyl derivatives. The novel compounds were subjected to extensive pharmacological studies to assess their effect on motor coordination, body temperature, clonic seizures and tonic convulsions and their antinociceptive activity. In the writhing test the antinociceptive activity of some derivatives was reversed by naloxone, thus we can assume that their activity may be associated with opioid system. We also used molecular modeling to describe active conformations of the studied compounds and to build a pharmacophore model. As in the previously reported series of the compounds, the studied substances exerted antinociceptive activity probably associated with the opioid system without possessing a protonable nitrogen atom. Furthermore, we calculated structural, electronic and ADMET parameters (volume, surface area, polar surface area, ovality, dipole moment, HOMO and LUMO energies, polarizaibility, molar refractivity, lipophilicity, the charges on the heteroatoms, aqueous solubility, and blood-brain barrier permeation parameter) for novel compounds in order to address the observed structure-activity relationship.

The effect of perinatal lead exposure on dopamine receptor D2 expression in morphine dependent rats.

The aim of this study was to investigate the behavioral and molecular effects of pre- and postnatal lead (Pb) exposure on the expression of morphine withdrawal and tolerance in adult rats. Rats were orally treated with 0.1% (1000ppm) lead acetate from conception, through gestation, up to postnatal day (PND) 28. Subsequently, behavioral experiments were conducted on adult (PND 60) male rats. To assess behavioral effects of morphine dependence in Pb-exposed rats two experimental models were used: naloxone-precipitated withdrawal signs and the assessment of morphine tolerance to antinociceptive effect in the tail-immersion test. Morphine withdrawal and tolerance were more expressed in Pb-exposed morphine administered rats than in morphine administered rats. In the case of morphine withdrawal signs the analysis of protein (Western blotting) and mRNA (RT PCR) expression revealed significantly higher dopamine D2 receptor (D2R) expression in prefrontal cortex, but not in striatum and hippocampus, in Pb-exposed morphine administered rats than in morphine administered rats. Differently, in the case of morphine tolerance the significant upregulation of D2R protein and mRNA expression in hippocampus, but not in prefrontal cortex or striatum, was demonstrated in Pb-exposed and morphine administered rats in comparison with morphine administered. These findings suggest that in morphine withdrawal and tolerant rats the perinatal Pb-exposure can affect D2R expression in brain region-specific manner. Immunohistochemical assessment of D2R expression in hippocampus showed translocation of D2R from membrane-cytoplasm in control rats to nucleus in morphine administered rats. Perinatal Pb-exposure did not induce the changes in the localization of D2R irrespective of morphine effect.

Effects of chronic flunitrazepam treatment schedule on therapy-induced sedation and motor impairment in mice.

BACKGROUND: The aim of the present study was to examine whether different treatment schedules could be associated with tolerance development to the ataxic and sedative effects of flunitrazepam in mice. METHODS: Effects of repeated flunitrazepam administration were studied in the rotarod and the chimney test for motor coordination and in a photocell apparatus for locomotor activity in mice. Flunitrazepam doses varied in particular types of injections or in different experiment duration periods. RESULTS: Repeated flunitrazepam administration (1 mg/kg, sc and 2 mg/kg, ip) for 8 consecutive days induced tolerance to the motor impairing effects of flunitrazepam in mice, both in the rotarod and the chimney test. In turn, no tolerance developed to sedative flunitrazepam effects, regarding either dose level, injection type or treatment duration. CONCLUSIONS: Those findings confirmed the previous observations that tolerance to benzodiazepines was not simultaneous for each pharmacological property of the drugs. Interestingly enough, an acute dose of flunitrazepam (1 mg/kg, sc) in our study enhanced locomotor activity of mice.

Effects of NOS inhibitors on the benzodiazepines-induced memory impairment of mice in the modified elevated plus-maze task.

The aim of the present study was to examine the effects of nitric oxide synthase (NOS) inhibitors on responses, elicited by benzodiazepines (BZs) in a modified elevated plus-maze task in mice. It was shown that acute doses of diazepam (DZ; 1 and 2 mg/kg) and flunitrazepam (FNZ; 0.05, 0.1 and 0.2 mg/kg) significantly increased the time of transfer latency (TL2) in a retention trial, thus confirming memory impairing effects of BZs. l-NAME (N(G)-nitro-l-arginine methyl ester; 200 mg/kg), a non-selective inhibitor of NOS, and 7-NI (7-nitroindazole; 40 mg/kg), a selective inhibitor of NOS, further intensified DZ-induced memory impairment. On the other hand, L-NAME (50, 100 and 200 mg/kg) and 7-NI (10, 20 and 40 mg/kg) prevented FNZ-induced memory compromising process. The results of this study indicated that suppressed NO synthesis enhanced DZ-induced but prevented FNZ-induced memory impairment. Taken together, these findings could suggest NO involvement in BZs-induced impairment of memory processes. The precise mechanism of these controversial effects, however, remains elusive.

Disubstituted thiourea derivatives and their activity on CNS: synthesis and biological evaluation.

A series of new thiourea derivatives of 1,2,4-triazole have been synthesized. The difference in structures of obtained compounds are directly connected with the kind of isothiocyanate (aryl/alkyl). The (1)H NMR, (13)C NMR, MS methods were used to confirm structures of obtained thiourea derivatives. The molecular structure of (1, 17) was determined by an X-ray analysis. Two of the new compounds (8 and 14) were tested for their pharmacological activity on animal central nervous system (CNS) in behavioural animal tests. The results presented in this work indicate the possible involvement of the serotonergic system in the activity of 8 and 14. In the case of 14 is also a possible link between its activity and the endogenous opioid system. All obtained compounds were tested for antibacterial activity against gram-positive cocci, gram-negative rods and antifungal activity. Compounds (1, 2, 5, 7, 9) showed significant inhibition against gram-positive cocci. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Selected compounds (1-13) were examined for cytotoxicity, antitumor, and anti-HIV activity.

Synthesis of new 1-aryl-6-benzylimidazo[1,2-a][1,3,5]triazines with potential pharmacological activity.

In the reaction of ethyl N-(1-arylimidazolidine-2-ylidene) carbamic acid ester with 4-chlorobenzylamine new derivatives of 1-(1-arylimidazolidine-2-ylidene)-3-arylurea (I-VI) were obtained. Cyclic derivatives of dihydroimidazo[l ,2-a][1,3,5]trizines were synthesized by condensation of 1-(1-arylimidazolidine-2-ylidene)-3-(4-chlorobenzyl)urea with carbonyldiimidazole (CDI) (VII-XII). The effect of compounds X, XI on the central nervous system of mice in some behavioral tests was investigated.

Effect of nitric oxide synthase inhibitors on benzodiazepine withdrawal in mice and rats.

This study was undertaken to evaluate the effect of nitric oxide (NO) synthase inhibitors on benzodiazepine withdrawal syndrome in mice and rats. Diazepam withdrawal in mice was read out as intensification of the seizures induced by a subthreshold dose of pentetrazole. In rats, the withdrawal syndrome resulting from chronic administration of diazepam, chlordiazepoxide, clonazepam and temazepam was characterized by audiogenic seizures, hypermotility and weight loss. Administration of the non-selective NO synthase inhibitors N(G)-nitro-L-arginine (L-NOARG) and N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) significantly attenuated the withdrawal syndrome (i.e., pentetrazole-induced seizures) in diazepam-dependent mice. L-NOARG significantly suppressed hypermotility in clonazepam-dependent rats and inhibited the decrease in body weight observed after 12 h of withdrawal in chlordiazepoxide- and clonazepam-dependent rats. Moreover, a clear propensity of L-NOARG to protect benzodiazepine-dependent rats against audiogenic seizures was observed. These findings suggest that the cGMP/NO system may participate in causing the signs of benzodiazepine withdrawal.