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Aims: GABAergic modulation involved in cognitive processing appears to be substan- tially changed in Alzheimer's disease (AD). In a widely used 5xFAD model of AD, we aimed to assess if negative and positive allosteric modulators of α5 GABA A receptors (NAM and PAM, respectively) would affect social interaction, social, object and spatial memory, and neuroinflammation. Methods: After 10-day treatment with PAM, NAM, or solvent, 6-month-old trans- genic and non-transgenic 5xFAD mice underwent testing in a behavioral battery. Gene expressions of IL-1β, IL-6, TNF-α, GFAP, and IBA-1 were determined in hippocampus and prefrontal cortex by qPCR. Results: PAM treatment impaired spatial learning in transgenic females compared to solvent-treated transgenic females, and social recognition in transgenic and non- transgenic males. NAM treatment declined social interaction in transgenic and non- transgenic males, while had beneficial effect on cognitive flexibility in non-transgenic males compared to solvent-treated non-transgenic males. Transgenic animals have not fully displayed cognitive symptoms, but neuroinflammation was confirmed. NAM reduced proinflammatory gene expressions in transgenic females and astrogliosis in transgenic males compared to pathological controls. Conclusion: PAM and NAM failed to exert favorable behavioral effects in transgenic animals. Suppression of neuroinflammation obtained with NAM calls for more studies with GABAergic ligands in amyloid beta- and/or tau-dependent models with promi- nent neuroinflammation.

Several pyrazoloquinolinone (PQ) ligands were recently discovered as functionally selective positive modulators at the PQ site of α6-containing GABAA receptors. PQs are also neutral modulators at the benzodiazepine site. We assessed the influence of PQ compounds from three structural groups (PZ-II-029 and related deuterated analogues DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related analogues DK-I-58-1 and DK-II-58-1; and DK-I-87-1), alone and in combination with diazepam, on the behaviour of male Sprague–Dawley rats. An excellent behavioural safety profile of all tested PQs was demonstrated in the spontaneous locomotor activity, rotarod, loss of righting reflex and pentylenetetrazol tests. In interaction studies, only PZ-II-029 and its analogues prevented the ataxic effects of the benzodiazepine, as assessed in the rotarod test and during monitoring of rat locomotor activity after awakening from the loss of righting reflex. Published electrophysiological profiles of PQ ligands imply that positive modulation elicited at α6-GABAA receptors that contain the γ2 and δ subunit, rather than their neutral modulatory action at the benzodiazepine site, may prevent the ataxic action of diazepam. Thus, PZ-II-029 and its deuterated analogues are not prone to untoward interactions with benzodiazepines and may indeed completely abolish their ataxic action, seen at therapeutic, and especially toxic concentrations.

The imidazodiazepine, (5‐(8‐ethynyl‐6‐(pyridin‐2‐yl)‐4H‐benzo [f]imidazole[1,5‐α] [1,4]diazepin‐3‐yl) oxazole or KRM‐II‐81) is a new α2/3‐selective GABAkine (gamma aminobutyric acid A receptor potentiator) with anticonvulsant, anxiolytic, and antinociceptive activity in preclinical models. Reducing metabolism was utilized as a means of potentially extending the half‐life of KRM‐II‐81. In vitro and in vivo studies were conducted to evaluate metabolic liabilities. Incubation of KRM‐II‐81 in hepatocytes revealed sites of potential metabolism on the oxazole and the diazepine rings. These sites were targeted in the design of a deuterated analog (D5‐KRM‐II‐ 81) that could be evaluated as a potentially longer‐acting analog. In contrast to computer predictions, peak plasma concentrations of D5‐KRM‐II‐81 in rats were not significantly greater than those produced by KRM‐II‐81 after oral administra- tion. Furthermore, brain disposition of KRM‐II‐81 was higher than that of D5‐KRM‐ II‐81. The half‐life of the two compounds in either plasma or brain did not statis- tically differ from one another but the tmax for D5‐KRM‐II‐81 occurred slightly earlier than for KRM‐II‐81. Non‐metabolic considerations might be relevant to the lack of increases in exposure by D5‐KRM‐II‐81. Alternative sites of metabolism on KRM‐II‐81, not targeted by the current deuteration process, are also possible. Despite its lack of augmented exposure, D5‐KRM‐II‐81, like KRM‐II‐81, significantly prevented seizures induced by pentylenetetrazol when given orally. The present findings introduce a new orally active anticonvulsant GABAkine, D5‐KRM‐II‐81.

Background/Aim: In addition to well-established central effects, benzodiazepines, but also some other allosteric modulators of gamma-amino-butyric acid (GABA) receptor exhibit significant vascular effects. However, there are currently no elucidated mechanisms for manifested vasodilatory properties and very little is known about GABA gamma-amino-butyric acid function and GABAA receptor expression within peripheral blood vessels. Methods: In the present study, we demonstrated the vasorelaxant properties of Diazepam, GABA and novel imidazobenzodiazepine amide ligands GL-II-73 and GLII-74, which are characterized as positive allosteric modulators of a5-containing GABAA receptor. Using isometric organ bath system, we examined the vascular responses to phenylephrine, in the presence and absence of various ligands, in the rat thoracic aorta. Results: The observed significant and strong attenuation of the maximal contractile response of phenylephrine indicates a non-competitive antagonism of Diazepam, GL-II-73 and GL-II-74 (p < 0.001), whereas GABA does not affect phenylephrine contraction. Since the strongest inhibitory effect was observed with compound GL-II-74, that, compared to other tested ligands, exhibited a higher potentiation at a5 GABAARs, it could be assumed that the a5 subunit plays a significant role in the structure of putatively present "vascular" GABAARs. Conclusion: This work emphasizes the importance of GABAARs research in the periphery and also points to the possibility of using a5 selective GABAAR modulators as potential therapeutic targets for novel vasodilators.

The current study describes the experimental design guided development of PEGylated nanoemulsions as parenteral delivery systems for curcumin, a powerful antioxidant, as well as the evaluation of their physicochemical characteristics and antioxidant activity during the two years of storage. Experimental design setup helped development of nanoemulsion templates with critical quality attributes in line with parenteral application route. Curcumin-loaded nanoemulsions showed mean droplet size about 105 nm, polydispersity index

Altered gamma-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging, and neurodegenerative disorders and reduced function of GABA interneurons is associated with both mood and cognitive symptoms. Benzodiazepines (BZ) have broad anxiolytic, but also sedative, anticonvulsant and amnesic effects, due to nonspecific GABA-A receptor (GABAA-R) targeting. Varying the profile of activity of BZs at GABAA-Rs is predicted to uncover additional therapeutic potential. We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands and tested them for positive allosteric modulation at multiple α-GABAA-R (α-positive allosteric modulators), pharmacokinetic properties, as well as anxiolytic and antidepressant activities in adult mice. Efficacy at reversing stress-induced or age-related working memory deficits was assessed using a spontaneous alternation task. Diazepam (DZP) was used as a control. Three ligands (GL-II-73, GL-II-74, and GL-II-75) demonstrated adequate brain penetration and showed predictive anxiolytic and antidepressant efficacies. GL-II-73 and GL-II-75 significantly reversed stress-induced and age-related working memory deficits. In contrast, DZP displayed anxiolytic but no antidepressant effects or effects on working memory. We demonstrate distinct profiles of anxiolytic, antidepressant, and/or pro-cognitive activities of newly designed IBZD amide ligands, suggesting novel therapeutic potential for IBZD derivatives in depression and aging.

It is unclear whether GABAA receptors (GABAARs) that contain the α3-subunit are substantially involved in the anxiolytic effects of benzodiazepines (BDZs). In the present study, we tested YT-III-31, a newer BDZ ligand with functional preference for α3βγ2 GABAARs, in two paradigms of unconditioned anxiety, the open field and elevated plus maze in rats. The effective dose of YT-III-31 (2 mg/kg) displayed a clear anxiolytic-like profile, unhampered by sedative action, in both tests. At a higher dose (10 mg/kg), YT-III-31 induced ataxia in the rotarod and sedation in spontaneous locomotor activity test. The latter effect was preventable by flumazenil and βCCt, the non-selective and α1βγ2 GABAAR affinity-selective antagonist, respectively, demonstrating that sedative properties of YT-III-31, when attained, are mediated by the α1γ2 site. To elucidate the receptor substrate of subtle behavioral differences between YT-III-31 and diazepam, we approximated in vivo receptor potentiation for both ligands, based on estimated unbound concentrations in rat brains. Far different from diazepam, YT-III-31 has significantly lower affinity for the α1γ2 over other BDZ-sensitive sites, and at lower doses (1–2 mg/kg) was devoid of potentiation at α1βγ2 GABAARs. The approximation approach revealed a modest selectivity of YT-III-31 for α3γ2- in comparison to α2γ2 and α5γ2 binding sites, suggesting that its anxiolytic-like activity may not necessarily or predominantly reflect potentiation at α3βγ2 GABAARs. Nonetheless, as the anxiolytic effects are achievable at a dose devoid of any sedative potential, and having favorable safety (cytotoxicity) and metabolic stability profile, YT-III-31 represents a valuable candidate for further translational research.

This work aimed to deepen the lately acquired knowledge about parenteral nanoemulsions as carriers for brain delivery of risperidone, a poorly water-soluble antipsychotic drug, through establishing the prospective relationship between their physicochemical, pharmacokinetic, biodistribution, and behavioral performances. For this purpose, two optimized risperidone-loaded nanoemulsions, stabilized by lecithin or lecithin/polysorbate 80 mixture, and costabilized by sodium oleate, were produced by high-pressure homogenization. The characterization revealed the favorable droplet size, narrow size distribution, high surface charge, with proven stability to autoclaving and long-term stability for at least one year at 25 +/- 2 degrees C. Pharmacokinetic and tissue distribution results demonstrated improved plasma, liver, and brain pharmacokinetic parameters, resulting in 1.2-1.5-fold increased relative bioavailability, 1.1-1.8-fold decreased liver distribution, and about 1.3-fold improved brain uptake of risperidone active moiety following intraperitoneal administration of nanoemulsions relative to solution in rats. In behavioral study, investigated nanoemulsions showed pronounced reduction in basal and, more pertinently, amphetamine-induced locomotor activity in rats, with an early onset of antipsychotic action, and this effect lasted at least 90 min after drug injection. Together, these findings corroborate the applicability of parenteral nanoemulsions as carriers for enhanced brain delivery of risperidone, further suggesting their promise in acute psychosis treatment or other emergency situations.

gamma-Aminobutyric acid type A (GABA(A)) receptors containing the alpha 6 subunit are located in trigeminal ganglia, and their reduction by small interfering RNA increases inflammatory temporomandibular and myofascial pain in rats. We thus hypothesized that enhancing their activity may help in neuropathic syndromes originating from the trigeminal system. Here, we performed a detailed electrophysiological and pharmacokinetic analysis of two recently developed deuterated structurally similar pyrazoloquinolinone compounds. DK-I-56-1 at concentrations below 1 mu M enhanced gamma-aminobutyric acid (GABA) currents at recombinant rat alpha 6 beta 3 gamma 2, alpha 6 beta 3 delta and alpha 6 beta 3 receptors, whereas it was inactive at most GABA(A) receptor subtypes containing other alpha subunits. DK-I-87-1 at concentrations below 1 mu M was inactive at alpha 6-containing receptors and only weakly modulated other GABA(A) receptors investigated. Both plasma and brain tissue kinetics of DK-I-56-1 were relatively slow, with half-lives of 6 and 13 hr, respectively, enabling the persistence of estimated free brain concentrations in the range 10-300 nM throughout a 24-hr period. Results obtained in two protocols of chronic constriction injury of the infraorbital nerve in rats dosed intraperitoneally with DK-I-56-1 during 14 days after surgery or with DK-I-56-1 or DK-I-87-1 during 14 days after trigeminal neuropathy were already established, demonstrated that DK-I-56-1 but not DK-I-87-1 significantly reduced the hypersensitivity response to von Frey filaments. Significance Neuropathic pain induced by trigeminal nerve damage is poorly controlled by current treatments. DK-I-56-1 that positively modulates alpha 6 GABA(A) receptors is appropriate for repeated administration and thus may represent a novel treatment option against the development and maintenance of trigeminal neuropathic pain.

31st ECNP Congress, 6 - 9 October 2018, Barcelona, Spain

Altered γ-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging and neurodegenerative disorders, and reduced function of somatostatin - expressing GABA interneurons is associated with both mood and cognitive symptoms. Somatostatin-neurons signal in part through α5-subunit containing GABAA receptors (α5-GABAA-Rs) which are localized in brain regions implicated in emotion and cognition. We hypothesize that enhancing α5-GABAA-R activity has therapeutic potential for both mood and cognitive symptoms in stress-based and aging rodent models.We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands, tested them for positive allosteric modulation at α5-GABAA-R (α5-PAM), pharmacokinetic properties, and for anxiolytic and antidepressant activities in adult mice. Pro-cognitive activity was tested in adult mice submitted to chronic stress and in old mice. Diazepam (DZP), with broad PAM activity at GABAA-Rs, was used as a control.Three novel IBZD amide ligands (GL-II-73, GL-II-74 and GL-II-75) demonstrated adequate brain penetration, affinity and α5-PAM activity, and metabolic stability for in vivo studies. GL-II-73/74/75 showed significant anxiolytic and antidepressant efficacies in adult mice. GL-II-73 and GL-II-75 significantly reversed cognitive deficits induced by stress or occurring throughout normal aging. This activity was maintained after sub-chronic administration for GL-II-73. In contrast DZP displayed anxiolytic but no antidepressant or pro-cognitive activities.We demonstrate for the first time the potential for combined anxiolytic, antidepressant and pro-cognitive therapeutic, mediated by newly designed IBDZ amide ligands with efficacy at α5-GABAA-Rs. These results suggest a novel therapeutic approach targeting both mood and cognitive symptoms in depression and/or aging.

Reportedly, negative modulation of α5 GABAA receptors may improve cognition in normal and pharmacologically-impaired animals, and such modulation has been proposed as an avenue for treatment of cognitive symptoms in schizophrenia. This study assessed the actions of PWZ-029, administered at doses (2, 5, and 10 mg/kg) at which it reached micromolar concentrations in brain tissue with estimated free concentrations adequate for selective modulation of α5 GABAA receptors, in three cognitive tasks in male Wistar rats acutely treated with the noncompetitive N-methyl-d-aspartate receptor antagonist, MK-801 (0.1 mg/kg), as well in tests of locomotor activity potentiated by MK-801 (0.2 mg/kg) or amphetamine (0.5 mg/kg). In a hormetic-like manner, only 5 mg/kg PWZ-029 reversed MK-801-induced deficits in novel object recognition test (visual recognition memory), whereas in the Morris water maze, the 2 mg/kg dose of PWZ-029 exerted partial beneficial effects on spatial learning impairment. PWZ-029 did not affect recognition memory deficits in social novelty discrimination procedure. Motor hyperactivity induced with MK-801 or amphetamine was not preventable by PWZ-029. Our results show that certain MK-801-induced memory deficits can be ameliorated by negative modulation of α5 GABAA receptors, and point to the need for further elucidation of their translational relevance to cognitive deterioration in schizophrenia.

The chapter provides a review of diverse pharmacokinetic, pharmacodynamic and biodistribution studies conducted by eminent researchers in their work with various types of nanostructured drug delivery systems. Although main accent is on the available in vivo studies, certain critical physicochemical attributes will be discussed along the way for the sake of better understanding of the relationship between the drug, the nanocarrier and the resulting efficacy in vivo. The importance of supporting bioanalytical methodology is also addressed. Special focus is placed on two groups of nanocarriers: those targeting the brain, that always prove to be an additional challenge, and those intended for topical application, in which case in vivo studies need to discern dermal from transdermal drug delivery. Hence, more detailed description of the variety of in vivo methodologies conducted in the case of risperidone and aceclofenac as model drugs will be given.

Multiple sclerosis (MS) treatment options include several biologic and numerous non- biologic drugs. Although three recombinant preparations of interferon-beta (IFN-β) represent the cornerstone of the disease-modifying therapy, the introduction of pegylated IFN-β-1a should ensure a less frequent drug administration. At the moment, natalizumab is the sole monoclonal antibody registered for relapsing-remitting MS in our country. Even though the biologics are primarily limited to parenteral routes of administration, symptomatic therapy, which aims to alleviate or eliminate the accompanying symptoms that deteriorate quality of life, encompasses diverse dosage forms for oral (tablets, film-coated tablets), sublingual (oral lyophilisate) or nasal (nasal spray) administration. Due to considerable in vitro and in vivo instability, formulation of delivery systems for biologics is primarily focused on preserving their physicochemical stability. The selection of excipients is commonly related to pH value adjustment, solubility modification and anti- adsorption/anti-agregation behaviour of proteins. As for the lyophilised forms, the focus is on cryo/lyoprotectants, collapse temperature modifiers and appropriate fillers. The selection of suitable sterilization procedure is of the utmost importance, and has resulted in the development of single use systems (SUS) technologies. Therefore, pharmacists should be familiar with optimal storage and handling conditions for these drugs, before, during and after reconstitution, as well as upon their administration. Terapija multiple skleroze obuhvata nekoliko bioloških i veći broj ne-bioloških lekova. Iako su tri rekombinantna preparata interferona-beta (IFN-β) okosnica terapije koja modifikuje prirodni tok bolesti, uvođenje pegilovane forme IFN-β-1a omogućilo bi manje frekventnu primenu leka. Za sada, natalizumab je jedino monoklonsko antitelo odobreno za terapiju relapsno-remitentne forme bolesti u našoj zemlji. Iako je terapija biološkim lekovima ograničena na parenteralni put primene, simptomatska terapija, sa ciljem ublažavanja ili eliminisanja simptoma bolesti koji smanjuju funkcionlanost i remete kvalitet života obolelih, pruža mogućnost primene različitih farmaceutskih oblika za peroralnu (tablete, film tablete), sublingvalnu (oralni liofilizat) ili nazalnu (sprej za nos) primenu. Zbog velike in vitro i in vivo nestabilnosti, formulacija farmaceutskih oblika bioloških lekova primarno je orijentisana na očuvanje njihove fizičko-hemijske stabilnosti, te je izbor ekscipijenasa obično vezan za podešavanje pH i modifikovanje rastvorljivosti i anti- adsorpciono/anti-agregaciono ponašanje proteina. Kod liofilizovanih oblika (praškova) fokus je na krio/lioprotektantima, modifikatorima temperaturnog kolapsa i prihvatljivim puniocima. Uz to, velika pažnja posvećuje se izboru adekvatnog postupka sterilizacije, pa se poslednjih godina razvijaju sistemi za aseptičnu proizvodnju sa jednokratnom upotrebom (engl. single use systems - SUS tehnologije). Od farmaceuta se očekuje poznavanje optimalnih uslova za čuvanje i rukovanje ovim lekovima, pre, tokom i nakon rekonstitucije, kao i tokom njihove primene.

Lerimazoline is a sympathomimetic drug that belongs to the imidazoline class of compounds, and is used as a nasal decongestant. Studies on lerimazoline are rare, and its pharmacological profile is not completely understood. Here, we analyzed the affinity of lerimazoline for dopamine receptor D2, serotonin 5-HT1A and 5-HT2A receptors and alpha(1)-adrenoceptor, and investigated lerimazoline contractile effects in isolated rat thoracic aorta. We also determined the effect of several antagonists on the contractile response to lerimazoline, including prazosin (alpha(1)-adrenoceptor antagonist), RX 821002 and rauwolscine (alpha(2)-adrenoceptor antagonists), JP 1302 (alpha(2C)-adrenoceptor antagonist), methiothepin (non-selective 5-HT receptor antagonist), SB 224289 (5-HT1B receptor antagonist), BRL 15572 (5-HT1D receptor antagonist), and ketanserin (5-HT2A receptor antagonist). Lerimazoline displayed high affinity for the 5-HT1A receptor (Ki = 162.5 nM), similar to the previously reported affinity for the 5-HT1D receptor. Binding affinity estimates (Ki) for alpha(1), 5-HT2A, and D-2 receptors were 6656, 4202 and 3437.5 nM, respectively (the literature reported Ki for 5-HT1B receptor is 3480 nM). Lerimazoline caused concentration-dependent contractions in 70% of preparations, varying in the range between 40% and 55% of the maximal contraction elicited by phenylephrine. While prazosin reduced the maximum contractile response to lerimazoline, rauwolscine showed a non-significant trend in reduction of the response. Both ketanserin (10 nM and 1 mu M) and methiothepin strongly suppressed the maximum response to lerimazoline. Overall, our results suggest that 5-HT2A and, less distinctly, alpha(1)-adrenergic receptors are involved in the lerimazoline-induced contractions, which makes lerimazoline an "atypical" decongestant.

Synthesis of ligands inactive or with low activity at alpha(1) GABA(A) receptors has become the key concept for development of novel, more tolerable benzodiazepine (BZ)-like drugs. WYS8, a remarkably (105 times) alpha(1)-subtype selective partial positive modulator, may serve as a pharmacological tool for refining the role of alpha(1) GABA(A) receptors in mediation of BZs' effects. Here, the effects of WYS8 on GABA-induced currents and on diazepam-induced potentiation of recombinant BZ-sensitive GABA(A) receptors were studied in more detail. In addition, the behavioral profile of WYS8 (0.2, 1, and 10 mg/kg i.p.), on its own and in combination with diazepam, was tested in the spontaneous locomotor activity, elevated plus maze, grip strength, rotarod, and pentylenetetrazole tests. WYS8, applied at an in vivo attainable concentration of 100 nM, reduced the stimulation of GABA currents by 1 mu M diazepam by 57 % at alpha(1)beta(3)gamma(2), but not at alpha(2)beta(3)gamma(2), alpha(3)beta(3)gamma(2), or alpha(5)beta(3)gamma(2) GABA(A) receptors. The administration of WYS8 alone induced negligible behavioral consequences. When combined with diazepam, WYS8 caused a reduction in sedation, muscle relaxation, and anticonvulsant activity, as compared with this BZ alone, whereas ataxia was preserved, and the anxiolytic effect of 2 mg/kg diazepam was unmasked. Hence, a partial instead of full activation at alpha(1) GABA(A) receptors did not necessarily result in the attenuation of the effects assumed to be mediated by activation of these receptors, or in the full preservation of the effects mediated by activation of other GABA(A) receptors. Thus, the role of alpha(1) GABA(A) receptors appears more complex than that proposed by genetic studies.

Antidepressants are one of the most widely used and criticized drug classes. Long-term effects leading to clinical efficacy of antidepressants in indications such as depression or anxiety disorders are poorly understood. The analysis of clinical trials suggests that, to a substantial degree, listening to antidepressants means hearing placebo. The precipitation of suicidal ideation is a possibility that needs to be considered when introducing antidepressants, especially in young patients. The discontinuation symptoms following cessation of treatment emerge frequently, and essentially are manifestations of withdrawal. Many, if not most, patients require multiple modifications in treatment in order to reach remission and avoid relapse or recurrence. However, these drugs are needed in clinical practice, and it is of paramount importance to precisely tailor treatment with antidepressants and adjunctive drugs to achieve optimal efficacy with minimal adverse effects. There are also hopes that novel drugs with rapid ketamine-like antidepressant actions may be introduced in the near future, thus solving the issue of acute management of severe depressions. Antidepresivi spadaju u lekove koji su u isto vreme najviše korišćeni i kritikovani. Dugotrajni efekti antidepresiva koji obezbeđuju kliničku efikasnost u indikacijama kao što su depresija ili anksiozni poremećaji slabo su razjašnjeni. Analiza rezultata kliničkih ispitivanja sugeriše da antidepresivnom dejstvu ovih lekova, u znatnom obimu, doprinosi placebo efekat. Precipitacija misli o suicidu predstavlja mogućnost koja se mora temeljno razmotriti prilikom uvođenja antidepresiva, posebno kod mladih pacijenata. Simptomi diskontinuacije nakon prekida terapije javljaju se često, i suštinski predstavljaju manifestacije obustave. Višestruke modifikacije tretmana, a u cilju postizanja remisije i izbegavanja relapsa ili recidiva, zahtevaju brojni pacijenti, ako ne i većina njih. Uprkos svemu, ovi lekovi jesu neophodni u kliničkoj praksi, i precizno prilagođavanje tretmana antidepresivima i adjuvantnim lekovima od neprocenjivog je značaja u cilju postizanja optimalne efikasnosti sa minimumom neželjenih efekata. Dodatno, postoje očekivanja da bi novi lekovi sa brzim antidepresivnim dejstvom sličnim ketaminu mogli da budu uvedeni u skorijoj budućnosti, čime bi se prevazišao problem akutnog zbrinjavanja teških depresija.

We performed a basic behavioral characterization of methanol extracts of four Balkan endemic Stachys taxa: S. anisochila (SA), S. beckeana (SB), S. plumosa (SP) and S. alpina subsp. dinarica (SAD). The behavioral activity of extracts dosed intraperitoneally in the range 100-400 mg/kg was examined in adult male Wistar rats, in the elevated plus maze, spontaneous locomotor activity, and grip strength tests, mainly predictive of anxiolytic, sedative and myorelaxant actions, respectively. All investigated Stachys extracts lacked anxiolytic or myorelaxant activities, while SB at 400 mg/kg exerted an anxiogenic-like effect. The study with the selective antagonist beta-CCt showed that the sedative effect of SAD was only partially mediated by GABAA receptors containing the alpha1-subunit. While discernible, the behavioral effects of SA and SP were not distinct. In all extracts, chlorogenic acid and verbascoside were identified. In SA, SB, and SAD the flavonoid fraction was constituted of isoscutellarein and hypolaetine glycosides, while in SP chrysoeriol and apigenin glycosides were present. The results reveal the psychotropic potential of four endemic Stachys taxa, of which SAD appeared most promising as a natural sedative.

We have synthesized and characterized MP-III-022 ((R)-8-ethynyl 6 (2 fluorophenyl)-N,4-dimethyl4H-benzo[f]imidazo[1,5-alpha][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy selective positive allosteric modulator of GABA(A) receptors containing the alpha 5 subunit (alpha 5GABA(A)Rs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 110 mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of alpha 5 beta gamma 2 GABA(A) receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2'F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10-200 mg/kg), but at the price of activating non-alpha 5 GABA(A)Rs as well as the desired alpha 5GABA(A)Rs at the highest dose. At the dose of 10 mg/kg, which elicits a strong positive modulation of alpha 5GABA(A)Rs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1-10 mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of alpha 5GABA(A)Rs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism.

Classical benzodiazepines (BZs) exert anxiolytic, sedative, hypnotic, muscle relaxant, anticonvulsive, and amnesic effects through potentiation of neurotransmission at GABA(A) receptors containing alpha(1), alpha(2), alpha(3) or alpha(5) subunits. Genetic studies suggest that modulation at the alpha(1) subunit contributes to much of the adverse effects of BZs, most notably sedation, ataxia, and amnesia. Hence, BZ site ligands functionally inactive at GABA(A) receptors containing the alpha(1) subunit are considered to be promising leads for novel, anxioselective anxiolytics devoid of sedative properties. In pursuing this approach, we used two-electrode voltage clamp experiments in Xenopus oocytes expressing recombinant GABA(A) receptor subtypes to investigate functional selectivity of three newly synthesized BZ site ligands and also compared their in vivo behavioral profiles. The compounds were functionally selective for alpha(2)-, alpha(3)-, and alpha(5)-containing subtypes of GABA(A) receptors (SH-053-S-CH3 and SH-053-S-CH3-2'F) or essentially selective for alpha(5) subtypes (SH-053-R-CH3). Possible influences on behavioral measures were tested in the elevated plus maze, spontaneous locomotor activity, and rotarod test, which are considered primarily predictive of the anxiolytic, sedative, and ataxic influence of BZs, respectively. The results confirmed the substantially diminished ataxic potential of BZ site agonists devoid of alpha(1) subunit-mediated effects, with preserved anti-anxiety effects at 30 mg/kg of SH-053-S-CH3 and SH-053-S-CH3-2'F. However, all three ligands, dosed at 30 mg/kg, decreased spontaneous locomotor activity, suggesting that sedation may be partly dependent on activity mediated by alpha(5)-containing GABA(A) receptors. Hence, it could be of importance to avoid substantial agonist activity at alpha(5) receptors by candidate anxioselective anxiolytics, if clinical sedation is to be avoided.

Recent research on genetically modified mice has attributed the amnesic effect of benzodiazepines mainly to the alpha1-containing GABA(A) receptor subtypes. The pharmacological approach, using subtype selective ligands, is needed to complement genetic studies. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) (0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha1-subunit selective agonist zolpidem (0-3.0 mg/kg), and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (0-2.0 mg/kg) in the one-trial step-through passive avoidance task in rats. The compounds were administered intraperitoneally, before the acquisition test. Flumazenil and beta-CCt did not affect retention performance. Midazolam and zolpidem induced amnesia in a dose-dependent manner. The complete reversal of amnesia was unattainable. The effects of zolpidem were significantly attenuated by the both, flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg); by contrast, only flumazenil was considerably effective when combined with midazolam. DMCM exerted promnesic effects at 0.2mg/kg, in an inverted U-shape manner. Both antagonists tended to abolish this action. The results indicate that some other alpha-subunit(s), in addition to the alpha1-subunit, contribute to the amnesic actions of non-selective benzodiazepine site agonists in the passive avoidance task. On the other hand, a significant part of the DMCM-induced promnesic effect could involve the alpha1-subunit and/or other putative beta-CCt-sensitive binding site(s).

This study investigated the impact of benzodiazepine receptor agonist, midazolam and antagonist, flumazenil, on the rat frontal cortex slices oxygen consumption (QO(2)), in presence and absence of gamma-aminobutyric acid (GABA). QO(2) was polarographically determined, using the biological oxygen monitor. As it was previously shown, GABA on its own decreases QO(2) moderately. Midazolam decreased QO(2) at 1.0mg/kg, whereas flumazenil had no effect. In combination with per se ineffective GABA (10(-6)mol/l), flumazenil showed respiratory depressant action, presumably revealing partial agonistic activity at some of GABA(A) receptor subtypes. However, it completely antagonized effects of midazolam on QO(2), on its own and in presence of GABA. Our results show that in vivo well-established effects of midazolam on cerebral metabolic activity could be reproduced in in vitro settings. Moreover, flumazenil antagonized this action, indicating the role of GABA(A)-benzodiazepine receptor complex activation in QO(2) regulation.

This paper describes design and evaluation of parenteral lecithin-based nanoemulsions intended for brain delivery of risperidone, a poorly water-soluble psychopharmacological drug. The nanoemulsions were prepared through cold/hot high pressure homogenization and characterized regarding droplet size, polydispersity, surface charge, morphology, drug-vehicle interactions, and physical stability. To estimate the simultaneous influence of nanoemulsion formulation and preparation parameters-co-emulsifier type, aqueous phase type, homogenization temperature-on the critical quality attributes of developed nanoemulsions, a general factorial experimental design was applied. From the established design space and stability data, promising risperidone-loaded nanoemulsions (mean size about 160 nm, size distribution LT 0.15, zeta potential around -50 mV), containing sodium oleate in the aqueous phase and polysorbate 80, poloxamer 188 or Solutol (R) HS15 as co-emulsifier, were produced by hot homogenization and their ability to improve risperidone delivery to the brain was assessed in rats. Pharmacokinetic study demonstrated erratic brain profiles of risperidone following intraperitoneal administration in selected nanoemulsions, most probably due to their different droplet surface properties (different composition of the stabilizing layer). Namely, polysorbate 80-costabilized nanoemulsion showed increased (1.4-7.4-fold higher) risperidone brain availability compared to other nanoemulsions and drug solution, suggesting this nanoemulsion as a promising carrier worth exploring further for brain targeting. This is peer-reviewed version of the artcle: S.M. Đorđević, N.D. Cekić, M.M. Savić, T.M. Isailović, D. V Ranđelović, B.D. Marković, S.R. Savić, T.T. Stamenić, R. Daniels, S.D. Savić, Parenteral nanoemulsions as promising carriers for brain delivery of risperidone: Design, characterization and in vivo pharmacokinetic evaluation, Int. J. Pharm. 493 (2015) 40–54. [https://doi.org/10.1016/j.ijpharm.2015.07.007] Published version: [http://cer.ihtm.bg.ac.rs/handle/123456789/1697]

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2′F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors.

The complex clinical picture of mono-neuropathy following injury of peripheral nerve is often accompanied by changes in the patients’ affective states. It has been shown that positive allosteric modulators of GABAA receptors can decrease nociceptive transmission in animal pain models. However, recent fi ndings suggest a possibility that at least some of the antinociceptive effects of benzodiazepines, related to neuropathic painful stimuli, may to a signifi cant degree be a consequence of their anxiolytic action. In this study we evaluated the possible delayed effects of SH–I–048A, a newly– synthesized high–effi cacy nonselective positive modulator of GABAA receptors, on anxiety–like behavior and locomotor activity in Wistar rats with a previously induced peripheral nerve injury. Assessment of behavioral parameters, using spontaneous locomotor activity and elevated plus maze tests, was performed 48h after completion of single-day threei.p. injections treatment consisting of zero, one, two or three doses of SH–I–048А (10 mg/kg). In general, rats’ behavior observed 72 h after a moderate peripheral nerve injury did not indicate the persistence of sequelae of mono-neuropathic pain. The rats treated with three doses of SH–I–048А displayed an enhanced immobility time in the locomotor activity test, without concomitant decrease of the total distance traveled. On the other hand, in the group treated with two doses of SH– I–048А, a decrease in the emotional reactivity in the elevated plus maze test was observed. Subtle changes in the regimen of dosing of SH–I–048А affect locomotor activity and anxiety-related behavior in animals with peripheral nerve injury. Kompleksna klinička slika neuropatije nakon povrede perifernog nerva često je praćena i promenama afektivnog stanja pacijenata. U animalnim modelima bola pokazano je da pozitivni alosterni modulatori GABAA receptora mogu da smanje nociceptivnu transmisiju. Međutim, skorašnji nalazi ukazuju i na mogućnost da je prividni Obradović et al. 199 antinociceptivni efekat benzodiazepina nakon delovanja akutnog bolnog stimulusa u značajnoj meri posledica anksiolitičnog efekta. U ovoj studiji ispitani su mogući odloženi efekti novosintetisanog, neselektivnog liganda GABAA receptora (SH–I–048A) na lokomotornu aktivnost i ponašanje u vezi sa anksioznošću kod pacova soja Wistar prethodno podvrgnutih povredi perifernog nerva. Procena bihejvioralnih parametara sprovedena je testovima spontane lokomotorne aktivnosti i uzdignutog plus lavirinta 48 sati nakon što su životinje u periodu od 24 sata primile rastvarač, jednu, dve ili tri doze SH–I–048A (10 mg/kg). Generalno, ponašanje pacova praćeno 72 sata nakon umerene povrede perifernog nerva nije ukazivalo na prisustvo perzistentnih posledica neuropatskog bola. Pacovi prethodno tretirani sa tri doze SH–I–048A ispoljili su povećano vreme imobilnosti u testu lokomotorne aktivnosti, bez značajnog smanjenja ukupnog pređenog puta. S druge strane, u grupi koja je tretirana sa dve doze SH–I–048A zapažena je smanjena emocionalna reaktivnost u uzdignutom plus lavirintu. Kod životinja sa povredom perifernog nerva, suptilne razlike u režimu doziranja pozitivnog modulatora GABAA, mogu značajno da utiču na ponašanje vezano za lomotornu aktivnost i nivo anksioznosti.

Long-term use of benzodiazepine-type drugs may lead to physical dependence, manifested by withdrawal syndrome after abrupt cessation of treatment. The aim of the present study was to investigate the influence of duration of treatment, as well as the role of alpha(1)-containing GABA(A) receptors, in development of physical dependence to diazepam, assessed through the level of anxiety and susceptibility to pentylenetetrazole (PTZ)-induced seizures, 24 h after withdrawal from protracted treatment in rats. Withdrawal of 2 mg/kg diazepam after 28, but not after 14 or 21 days of administration led to an anxiety-like behavior in the elevated plus maze. Antagonism of the diazepam effects at alpha(1)-containing GABA(A) receptors, achieved by daily administration of the neutral modulator beta CCt (5 mg/kg), did not affect the anxiety level during withdrawal. An increased susceptibility to PTZ-induced seizures was observed during diazepam withdrawal after 21 and 28 days of treatment. Daily co-administration of beta CCt further decreased the PTZ-seizure threshold after 21 days of treatment, whilst it prevented the diazepam withdrawal-elicited decrease of the PTZ threshold after 28 days of treatment. In conclusion, the current study suggests that the role of alpha(1)-containing GABA(A) receptors in mediating the development of physical dependence may vary based on the effect being studied and duration of protracted treatment. Moreover, the present data supports previous findings that the lack of activity at alpha(1)-containing GABA(A) receptors is not sufficient to eliminate physical dependence liability of ligands of the benzodiazepine type.

Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2 mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10 mg/kg dose of the novel ligand and 2 mg/kg diazepam; however, SH-I-048A was relatively more active at α1- and α5-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24 h after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands.

Considering the importance of hydantoin derivatives in treatment of status epilepticus, four 5-phenylhydantoins, whose lipophilicities were estimated to be similar to that of phenytoin, were synthesized. Evaluation of their anticonvulsant activities was performed on rats by subcutaneous pentylenetetrazol seizure test and intravenous pentylenetetrazol threshold test, and spontaneous locomotor activity test was used to assess possible sedative effects. X-ray analysis of three compounds suggested that certain analogies might be drawn between interactions in crystal packing and biological interactions responsible for their anticonvulsant activity. It was found that 5-ethyl-5-phenyl-3-propylhydantoin exhibits the most favorable pharmacological properties among the synthesized compounds, i.e., anticonvulsant activity comparable to phenytoin with lower liability for induction of sedation in rats.

The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABA(A) receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABA(A) receptors containing alpha(1) subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential alpha(1)-subunit selective antagonist beta CCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10 mg/kg) or beta CCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABA(A) receptors may reverse the withdrawal-induced anxiety involves the alpha(1) subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABA(A) receptors.

Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABA A receptors, containing the α 1 , α 2 , α 3 or α 5 subunit, factual substrates of BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α 1 -subunit affinity-selective antagonist Β-CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α 1 -subunit selective ligand-WYS8 (0.2, 1 and 10mg/kg), on its own and in combination with the non-selective agonist DZP (2mg/kg) or Β-CCt (5mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α 1 -subtype selective weak partial positive modulator (40% potentiation at 100nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se, did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with Β-CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α 1 subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition.

Inverse agonism at the benzodiazepine site of alpha(5) subunit-containing GABA(A) receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for alpha(5) GABA(A) receptors have been synthesized and tested in a few animal models. PWZ-029 is an alpha(5) GABA(A) selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2,5 and 10 mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2 mg/kg) also successfully reversed the 0.3 mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5,10 and 15 mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10 mg/kg) also proved to be ineffective in the reversal of the 1 mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective as GABA(A) inverse agonists.

Benzodiazepines negatively affect motor coordination and balance and produce myorelaxation. The aim of the present study was to examine the extent to which populations of gamma-aminobutyric acid A (GABA(A)) receptors containing alpha(1) and alpha(5) subunits contribute to these motor-impairing effects in rats. We used the nonselective agonist diazepam and the alpha(1)-selective agonist zolpidem, as well as nonselective, alpha(1)-subunit and alpha(5)-subunit-selective antagonists flumazenil, beta CCt, and XLi093, respectively. Ataxia and muscle relaxation were assessed by rotarod and grip strength tests performed 20 min after intraperitoneal treatment. Diazepam (2 mg/kg) induced significant ataxia and muscle relaxation, which were completely prevented by pretreatment with flumazenil (10mg/kg) and beta CCt (20 mg/kg). XLi093 antagonized the myorelaxant, but not the ataxic actions of diazepam. All three doses of zolpidem (1, 2, and 5 mg/kg) produced ataxia, but only the highest dose (5 mg/kg) significantly decreased the grip strength. These effects of zolpidem were reversed by beta CCt at doses of 5 and 10 mg/kg, respectively. The present study demonstrates that alpha(1) GABA(A) receptors mediate ataxia and indirectly contribute to myorelaxation in rats, whereas alpha(5) GABA(A) receptors contribute significantly, although not dominantly, to muscle relaxation but not ataxia. Behavioural Pharmacology 23:191-197

Considering the importance of hydantoin derivatives in treatment of status epilepticus, four 5-phenylhydantoins, whose lipophilicities were estimated to be similar to that of phenytoin, were synthesized. Evaluation of their anticonvulsant activities was performed on rats by subcutaneous pentylenetetrazol seizure test and intravenous pentylenetetrazol threshold test, and spontaneous locomotor activity test was used to assess possible sedative effects. X-ray analysis of three compounds suggested that certain analogies might be drawn between interactions in crystal packing and biological interactions responsible for their anticonvulsant activity. It was found that 5-ethyl-5-phenyl-3-propylhydantoin exhibits the most favorable pharmacological properties among the synthesized compounds, i.e., anticonvulsant activity comparable to phenytoin with lower liability for induction of sedation in rats.

Over the last years, genetic studies have greatly improved our knowledge on the receptor subtypes mediating various pharmacological effects of positive allosteric modulators at GABA(A) receptors. This stimulated the development of new benzodiazepine (BZ)-like ligands, especially those inactive/low-active at GABA(A) receptors containing the alpha(1) subunit, with the aim of generating more selective drugs. Hereby, the affinity and efficacy of four recently synthesized BZ site ligands: SH-053-2'N, SH-053-S-CH3-2'F, SH-053-R-CH3-2'F and JY-XHe-053 were assessed. They were also studied in behavioral tests of spontaneous locomotor activity, elevated plus maze, and water maze in rats, which are considered predictive of, respectively, the sedative, anxiolytic, and amnesic influence of BZs. The novel ligands had moderately low to low affinity and mild to partial agonistic efficacy at GABA(A) receptors containing the alpha(1) subunit, with variable, but more pronounced efficacy at other BZ-sensitive binding sites. While presumably alpha(1) receptor-mediated sedative effects of GABA(A) modulation were not fully eliminated with any of the ligands tested, only SH-053-2'N and SH-053-S-CH3-2'F, both dosed at 30 mg/kg, exerted anxiolytic effects. The lack of clear anxiolytic-like activity of JY-XHe-053, despite its efficacy at alpha(2)- and alpha(3)-GABA(A) receptors, may have been partly connected with its preferential affinity at alpha(5)-GABA(A) receptors coupled with weak agonist activity at alpha(1)-containing subtypes. The memory impairment in water-maze experiments, generally reported with BZ site agonists, was completely circumvented with all four ligands. The results suggest that a substantial amount of activity at alpha(1) GABA(A) receptors is needed for affecting spatial learning and memory impairments, while much weaker activity at alpha(1)- and alpha(5)-GABA(A) receptors is sufficient for eliciting sedation.

Background: The potential for use of chitosan-treated alginate microparticles as a vehicle for oral phenytoin delivery has not been thoroughly exploited. Aim: We studied the influence of preparation procedure and chitosan type on physicochemical properties and release behavior of alginate-chitosan microparticles. Method: The total number of 24 microparticles formulations prepared by varying contents of calcium gelling ions and varying contents and type of chitosan was examined. As an additional variable, two different hardening times (1 and 24 hours) were employed. Possible interactions of components, surface morphology of microparticles as well as release profile of phenytoin were studied. Results: Both series of formulations with regard to hardening times, irrespective of the chitosan type and/or concentration employed appeared to be highly loaded with the model drug (above 90%). The drug release studies showed that the kinetics of phenytoin cannot be straightforwardly predicted based on the molecular weight of chitosan alone. On the other hand, prolonging the hardening time from 1 to 24 hours had significantly improved phenytoin kinetics, and gave rise to a formulation with the liberation half-time of about 2.5 hours. Conclusion: This study showed that the latter formulation is eligible for further modifications aimed at improving the regularity of phenytoin absorption.

The clinical use of benzodiazepines (BZs) is hampered by sedation and cognitive deterioration. Although genetic and pharmacological studies suggest that alpha(1)- and alpha(5)-containing GABA(A) receptors mediate and/or modulate these effects, their molecular substrate is not fully elucidated. By the use of two selective ligands: the alpha(1)-subunit affinity-selective antagonist beta-CCt, and the alpha(5)-subunit affinity- and efficacy-selective antagonist XLi093, we examined the mechanisms of behavioural effects of diazepam in the tests of spontaneous locomotor activity and water-maze acquisition and recall, the two paradigms indicative of sedative- and cognition-impairing effects of BZs, respectively. The locomotor-activity decreasing propensity of diazepam (significant at 1.5 and 5 mg/kg) was antagonized by beta-CCt (5 and 15 mg/kg), while it tended to be potentiated by XLi093 in doses of 10 mg/kg, and especially 20 mg/kg. Diazepam decreased acquisition and recall in the water maze, with a minimum effective dose of 1.5 mg/kg. Both antagonists reversed the thigmotaxis induced by 2 mg/kg diazepam throughout the test, suggesting that both GABA(A) receptor subtypes participate in BZ effects on the procedural component of the task. Diazepam-induced impairment in the declarative component of the task, as assessed by path efficiency, the latency and distance before finding the platform across acquisition trials, and also by the spatial parameters in the probe trial, was partially prevented by both, 15 mg/kg beta-CCt and 10 mg/kg XLi093. Combining a BZ with beta-CCt results in the near to control level of performance of a cognitive task, without sedation, and may be worth testing on human subjects.

Benzodiazepine (BZ) site ligands affect vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity through modulation of neurotransmission at GABA(A) receptors containing alpha 1, alpha 2, alpha 3 or alpha 5 subunits, and may have numerous experimental and clinical applications. The ability of non-selective BZ site inverse agonists to enhance cognition, documented in animal models and human studies, is clinically not feasible due to potentially unacceptable psychomotor effects. Most investigations to date have proposed the alpha 1 and/or alpha 5 subunit-containing GABA(A) receptors as comprising the memory-modulating population of these receptors. The novel ligand PWZ-029, which we synthesized and characterized electrophysiologically, possesses in vitro binding selectivity and moderate inverse agonist functional selectivity at alpha 5-containing GABA(A) receptors. This ligand has also been examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of the effects on the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively. The improvement of task learning was detected at the dose of 5 mg/kg in the passive, but not active avoidance test. The inverse agonist PWZ-029 had no effect on anxiety or muscle tone, whereas at higher doses (10 and 20 mg/kg) it decreased locomotor activity. This effect was antagonized by flumazenil. and also by the lower (but not the higher) dose of an agonist (SH-053-R-CH3-2'F) selective for GABA(A) receptors containing the alpha 5 subunit. The hypolocomotor effect of PWZ-029 was not antagonized by the antagonist beta-CCt exhibiting a preferential affinity for alpha 1-subunit-containing receptors. These data suggest that moderate negative modulation at GABA(A) receptors containing the alpha 5 subunit is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory, while the influence of higher doses of modulators at these receptors on motor activity shows an intricate pattern whose relevance and mechanism await to be defined.

The pharmacological approach, using subtype selective ligands, complements genetic studies on the specific contribution of individual receptor subtypes to the various effects of benzodiazepines.The aim of this study was to examine the relative significance of alpha1-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site on anxiety and memory processes.We tested the effects of the nonselective antagonist flumazenil, the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt), the nonselective agonist midazolam, the preferential alpha1-subunit selective agonist zolpidem, and the nonselective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in a two-way active avoidance task in rats. The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of the two agonists were also examined. In the schedule 2 x 30 trials, drugs were administered i.p. 20 min before the training session. Avoidance responses in the training session are an anxiety-mediated behavior, whereas performance in the retention session relates to the effects on memory.Flumazenil and beta-CCt did not affect behavior. Midazolam (2.0 mg/kg) facilitated acquisition performance, while DMCM (1.0 and 2.0 mg/kg) induced the opposite effect. Flumazenil antagonized both effects. Beta-CCt potentiated the effect of midazolam, and partly antagonized the effect of DMCM. Midazolam (0.5 and 1.0 mg/kg) and zolpidem (1.0-3.0 mg/kg) impaired, while DMCM (0.1 mg/kg) facilitated the subjects' performance in the retention test. The amnesic effects were attenuated but not fully reversed, while the effect of DMCM was counteracted by both antagonists.The results indicate the alpha1-subunit interferes with the anxiolytic effect of a benzodiazepine site agonist and may contribute to the DMCM-induced anxiogenic effect. It is also substantially involved in the bidirectional memory processing in the active avoidance paradigm.

Recent research using genetically modified mice has pointed to the specific contribution of individual receptor subtypes to the various effects of benzodiazepines. The aim of this study was to examine the relative significance of alpha(1)-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site in the elevated plus-maze (EPM) under dim red light in rats. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha(1)-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt, 0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha(1)-subunit selective agonist zolpidem (0-2.0 mg/kg) and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0-2.0 mg/kg). The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of both kinds of agonists were also examined. The standard spatio-temporal parameters reflecting anxiety (percentage of open arm entries and time) and locomotion (closed and total arm entries) were analyzed. beta-CCt did not affect behavior, while flumazenil at the highest dose (20.0 mg/kg) decreased indices of open arm activity and total arm entries. Midazolam at the dose of 1.0 mg/kg significantly increased the percentage of open arm time, whereas at 2.0 mg/kg both anxiety-related parameters were increased. In contrast to the open arm entries, the open arm time was independent of the decreased closed arm entries, observed at 2.0 mg/kg. Flumazenil abolished these effects, whereas beta-CCt partially potentiated the anxiolytic actions of midazolam. Zolpidem significantly increased both open-arm indices at 1.0 mg/kg, but the effect was dependent on the decreased closed arm entries. The selectivity of the anxiolytic-like effects of zolpidem was further checked under brighter white illumination. In these settings, the influence on anxiety-related, but not activity-related parameters, was absent. All of the activity-related effects of midazolam and zolpidem were mainly counteracted by both antagonists. DMCM produced significant anxiogenic effects at 1.0 mg/kg (open arm time) and 2.0 mg/kg (both parameters). beta-CCt (30.0 mg/kg) and flumazenil at higher dose (20.0 mg/kg) antagonized the effects of DMCM. The results indicate the anxiolytic effects of a non-selective benzodiazepine site agonist involve a predominant role of subunits other than alpha(1), whereas the behavioral indices of the anxiolytic-like properties of an alpha(1)-selective ligand, if observed, depend on the experimental settings and the changes in locomotor activity, and hence were behaviorally non-specific. The present results generally correspond well to the behavioral findings with the genetically modified mice. On the other hand, the relative significance of the alpha(1)-subunit in the anxiogenic effects of DMCM could not be clearly deduced.

The purpose of the present study was to examine the influence of midazolam on the retrieval and acquisition rate of two-way active avoidance in rats. In the schedule 2 x 100 trials, the effects of midazolam (0.5-5.0 mg/kg), benzodiazepine binding site antagonist flumazenil (2.5-10.0 mg/kg), specific antagonist of GABA(A) receptor, bicuculline (0.5-4.0 mg/kg), and the blocker of GABA(A) receptor containing Cl(-) channels, picrotoxin (1.0-5.0 mg/kg), on the second day retrieval of avoidance performance were investigated, as well as the influence of the used blockers of GABA(A) receptor function on midazolam effects. Furthermore, the effect of midazolam (1.0 mg/kg) on acquisition rate in the 5 x 50 schedule, as well as the effects of third day treatment changing in that paradigm, was examined. Throughout the study, drugs were given intraperitoneally, 30 min before testing. Midazolam at the dose of 1.0 mg/kg facilitated avoidance retrieval, whereas flumazenil and bicuculline did not significantly change behavior. Picrotoxin (5.0 mg/kg) diminished performance. All three kinds of blockers used abolished facilitatory action of midazolam, confirming GABAergic mediation of the effect of benzodiazepine. Midazolam (1.0 mg/kg) increased acquisition rate during five consecutive days relative to saline, but without significant effect on the first day acquisition. In the case of third day changing of treatments, the intersection of regression rate lines was detected. Results from active avoidance paradigm experimentally support the findings from human studies that in certain circumstances, benzodiazepines, potentiating GABAergic neurotransmission, could produce retrieval-enhancing effects in memory tasks.