Jolanta Orzelska-Górka

Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats.

Nanoscale particles have large surface to volume ratio that significantly enhances their chemical and biological reactivity. Although general toxicity of nano silver (nanoAg) has been intensively studied in both in vitro and in vivo models, its neurotoxic effects are poorly known, especially those of low-dose exposure. In the present study we assess whether oral administration of nanoAg influences behavior of exposed rats and induces changes in cerebral myelin. We examine the effect of prolonged exposure of adult rats to small (10nm) citrate-stabilized nanoAg particles at a low dose of 0.2mg/kg b.w. (as opposed to the ionic silver) in a comprehensive behavioral analysis. Myelin ultrastructure and the expression of myelin-specific proteins are also investigated. The present study reveals slight differences with respect to behavioral effects of Ag(+)- but not nanoAg-treated rats. A weak depressive effect and hyperalgesia were observed after Ag(+) exposure whereas administration of nanoAg was found to specifically increase body weight and body temperature of animals. Both nanoAg and Ag(+) induce morphological disturbances in myelin sheaths and alter the expression of myelin-specific proteins CNP, MAG and MOG. These results suggest that the CNS may be a target of low-level toxicity of nanoAg.

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.

In Vivo Characterization of the Ultrapotent Monoacylglycerol Lipase Inhibitor {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048)

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

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.

New 5-HT1A, 5HT2A and 5HT2C receptor ligands containing a picolinic nucleus: Synthesis, in vitro and in vivo pharmacological evaluation

Picolinamide derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to 5-HT1A, 5-HT2A and 5-HT2C receptors was evaluated. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine), known to play critical roles in affinity for serotoninergic receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed high affinity in nanomolar and subnanomolar range at 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1, D2, α1 and α2). N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethyl)picolinamide (3o) with Ki=0.046nM, was the most affine and selective derivative for the 5-HT1A receptor compared to other serotoninergic dopaminergic and adrenergic receptors. N-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)picolinamide (3b), instead, showed a subnanomolar affinity towards 5-HT2A with Ki=0.0224nM, whereas N-(2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)ethyl)picolinamide (3s) presented an attractive 5-HT2C affinity with Ki=0.8nM. Moreover, the compounds having better affinity and selectivity binding profiles towards 5-HT2A were selected and tested on rat ileum, to determine their effect on 5HT induced contractions. Those more selective towards 5-HT1A receptors were studied in vivo on several behavioral tests.

Synthesis, in vitro and in vivo pharmacological evaluation of serotoninergic ligands containing an isonicotinic nucleus.

Isonicotinamide derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to 5-HT1A, 5-HT2A and 5-HT2C receptors were evaluated. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine) known to play critical roles in affinity for serotoninergic receptors and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed high affinity in nanomolar and subnanomolar range at 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1, D2, α1 and α2). N-(3-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)propyl)isonicotinamide (4s) with Ki = 0.130 nM, was the most active and selective derivative for the 5-HT1A receptor compared to other serotoninergic, dopaminergic and adrenergic receptors. Compound 4o, instead, showed 5-HT2A affinity values in subnamolar range. Moreover, the compounds having better affinity and selectivity binding profile towards 5-HT1A and 5-HT2A receptors were selected in order to be tested by in vitro and in vivo assays to determine their functional activity.

Drugs modulating the L-arginine:NO:cGMP pathway – current use in therapy

Abstract Nitric oxide (NO) is a relatively novel messenger that plays a significant role in a wide range of physiological processes. Currently, it is known that, both, lack and excess of NO can cause diseases, thus a lot of substances have been discovered and utilized which can change the concentration of this molecule within the organism. The aim of the present work is to provide an overview of currently used agents modulating the L-arginine:NO:cGMP pathway, as well as to summarize current understanding of their pharmacological profiles. Nowadays, most of these agents are employed particularly in the treatment of cardiovascular diseases. Further studies can hold promise for enhancing the therapeutic equipment for a variety of other impairments, such as osteoporosis, and also in treatments of the central nervous system.

Ultrastructural and biochemical features of cerebral microvessels of adult rat subjected to a low dose of silver nanoparticles.

The widespread use of silver nanoparticles (AgNPs) in medicine and in multiple commercial products has motivated researchers to investigate their potentially hazardous effects in organisms. Since AgNPs may easily enter the brain through the blood-brain barrier (BBB), characterization of their interactions with cellular components of the neurovascular unit (NVU) is of particular importance. Therefore, in an animal model of prolonged low-dose exposure, we investigate the extent and mechanisms of influence of AgNPs on cerebral microvessels. Adult rats were treated orally with small (10 nm) AgNPs in a dose of 0.2 mg/kg b.w. over a 2-week period. A silver citrate-exposed group was established as a positive control of ionic silver effects. Alterations in the expression of tight junction proteins claudin-5, ZO-1, and occludin, were observed. These effects are accompanied by ultrastructural features indicating enhanced permeability of microvessels such as focal edema of perivascular astrocytic processes and surrounding neuropil. We did not identify changes in the expression of PDGFβR which is a marker of pericytes. Ultrastructural alterations in these cells were not identified. The results show that altered integrity of cerebral vessels under a low-dose of AgNP-exposure may be the consequence of dysfunction of endothelial cells caused by disruption of tight junction proteins.

New Drugs - From Necessity to Delivery

Abstract How to get a new drug to market? How much time does it take to go from the idea to implementation? In this study we followed the path drugs take from synthesis to introduction to the market. In doing so, articles in the PubMed and the Google Scholar database have been analyzed using the keywords: drug development, drug design, lead compound, preclinical trials, clinical trials. The available literature was subjectively selected due to its usefulness in the topic. Based on the obtained articles, we presented the stages that a would-be drug takes on the way from the idea to marketing. Herein, it is underlined that the process of creating new drugs is long, extremely labor-intensive, and involves many restrictions in the context of the use of animals, as well as humans

The Importance of l-Arginine:NO:cGMP Pathway in Tolerance to Flunitrazepam in Mice

The goal of the study was to investigate the effects of drugs modifying l-arginine:NO:cGMP pathway on the development of tolerance to flunitrazepam (FNZ)-induced motor impairment in mice. FNZ-induced motor incoordination was assessed on the 1st and 8th days of experiment, using the rotarod and chimney tests. It was found that (a) both a non-selective nitric oxide synthase (NOS) inhibitor: NG-nitro-l-arginine methyl ester (l-NAME) and an unselective neuronal NOS inhibitor: 7-nitroindazole (7-NI) inhibited the development of tolerance to the motor-impairing effects of FNZ in the rotarod and the chimney tests and (b) both a NO precursor: l-arginine and a selective inhibitor of phosphodiesterase 5 (PDE5): sildenafil did not affect the development of tolerance to FNZ-induced motor impairment in mice. Those findings provided behavioural evidence that NO could contribute an important role in the development of tolerance to FNZ in mice.