AVN-322 is a Safe Orally Bio-Available Potent and Highly Selective Antagonist of 5-HT6R with Demonstrated Ability to Improve Impaired Memory in Animal Models.

BACKGROUND: In recent years, 5-hydroxytryptamine subtype 6 receptor (5-HT6 receptor, 5- HT6R) has emerged as a promising therapeutic target for the treatment of neuropathological disorders, including Alzheimer's disease (AD) and schizophrenia. 5-HT6 receptors were hypothesized to be implicated in the processes of learning, memory, and cognition with 5-HT6R antagonists being effective in animal models of cognition and memory impairment. Several selective 5-HT6R ligands are currently undergoing clinical trials for treatment of AD. METHODS: We describe results of preclinical development of a novel and highly selective and potent 5- HT6R antagonist, AVN-322, as a clinical candidate for the treatment of AD to improve concurrent debilitation of memory and cognition in the AD patients, and schizophrenia as a substance with antipsychotic effect. In the manuscript, we present its in vitro and vivo efficacy, ADME, pharmacokinetics in animals and in humans, and toxicity. RESULTS: While having high binding affinity in medium picomolar range, the lead compound demonstrates substantially better selectivity index then the reference drug candidates currently being tested in clinical studies. AVN-322 showed high oral bioavailability and favorable blood-brain barrier (BBB) penetration. In vivo testing revealed its clear cognition enhancing effect. AVN-322 significantly restored both scopolamine- and MK-801-induced cognitive dysfunction and demonstrated antipsychotic potential. CONCLUSION: Taking into account its good safety profile and favorable pharmacokinetics, AVN-322 can be reasonably considered as a novel drug candidate for the treatment of neurological disorders such as AD and/or schizophrenia.

[Substituted 2,3,4,5-tetrahydro-1H-gamma-carbolines: new antagonists of histamine H1 receptors].

The properties of 2,3,4,5-tetrahydro-1H-gamma-carbolines containing acid, ether, and amido-substituents, were assessed as potential antagonists of histamine H1 receptors (H1R), capable of blocking histamine-induced calcium fluxes in SK-N-SH cells. The structure--activity relationship for their antagonistic activity is discussed. Among the gamma-carbolines used in the study, the antihistamine activity considerably depends on the nature of substituents in positions 2, 5, and 8 of the heterocycle. The most active antagonist, ethyl 3-(2-methyl-8-fluoro-2,3,4,5-tetrahydro-1H-gamma-carboline-5-yl)propionate, with high affinity to the H1R (Ki = 6.5 nM), produces no adverse effects on motor activity of mice in doses 1-40 mg/kg, which shows the absence of a sedative effect.

Sensitivity of the brain synaptosomal membrane Mg(2+)-ATPase activity to arachidonic acid is under control of the Na+,K(+)-ATPase state.

Evidence is presented for the sensitivity of the synaptosomal plasma membrane Mg(2+)-ATPase activity to arachidonic acid being dependent on the functional state of Na+,K(+)-ATPase. An "Inversion effect" was observed at arachidonic acid concentrations exceeding 80 mumol/l when the Mg(2+)-ATPase activity (after ouabain addition) is higher than the total ATPase activity (without ouabain). The "Inversion effect" is reduced by cyclooxygenase inhibitor indomethacin or acetylsalicylic acid and restored by prostaglandin PGA2 or PGD2.

[Effect of temperature on polarity of an annular and bilayer synaptic membrane lipid]. / Vliianie temperatury na poliarnost' anuliarnogo i bisloinogo lipida sinapticheskikh membran.

Using fluorescent probe pyrene a study was carried out of temperature dependence of the annular and lipid bilayer polarity in synaptic membranes. Polarity of microenvironment was evaluated by a ratio of intensities of vibronic bands in pyrene fluorescence spectra at direct excitation of probe or through energy transfer from protein molecules. At the temperature range 10-50 degrees C the polarity of bilayer was shown to increase while that of annular lipid underwent biphasic changes. Above 20 degrees C polarity of the bilayer was higher than of the annular zone. From experiments on pyrene fluorescence quenching with potassium iodide the conclusion has been made that changes in polarity correlate with pyrene accessibility to water.

On the mechanism of shrinkage-induced potassium influx in rat and human erythrocytes.

The rates of 86Rb influx into human and rat erythrocytes were studied in media of various tonicity. At sucrose concentrations below 0.3 mol/l, the ouabain-insensitive, furosemide-inhibited component of influx increased in rat but not in human erythrocytes; this may be explained by a rise in the rate of Na+, K+, Cl-- and/or K+, Cl-cotransport. An increase in osmolarity resulted in a reduction of this as well as of the ouabain and furosemide-insensitive component in rat erythrocytes. At the same conditions a drastic inhibition of Na+, K(+)-pump occurred both in rat and human erythrocytes. We failed to observe a lag-phase in the activation of the cotransport in rat erythrocytes; i. e. the process of activation parallels the shrinkage of cells. In rat erythrocyte ghosts, the shrinkage-induced stimulation of the cotransport was lost, and the direction of their osmotic reaction (inhibition of transport pathways) was similar to that in human erythrocyte ghosts. It is suggested that the mechanism of volume regulation of ion transport in intact cells involves a step of physical amplification via a change in interactions between the protein carcass and the lipid bilayer.

[The coupling of Na,K-ATPase with the sodium channels in the plasma membranes of the brain synaptosomes of rats]. / Sopriazhenie Na,K-ATFazy s natrievymi kanalami v plazmaticheskikh membranakh sinaptosom mozga krys.

Effect of neurotoxins veratrine (100 micrograms/ml) and tetrodotoxin (1 microM) on the binding of 3H-ouabain (10(-8) M) with Na,K-ATPase of intact synaptosomes and isolated synaptic membranes was studied. The persistent opening of sodium channels in synaptosomes by veratrine results in an increase of specific binding of the labeled ligand by 20%. A similar effect was caused by Na/H exchanger monensin. Destruction of microtubules with vinblastine and colchicine has no influence on veratrine action, while depolymerization of microfilaments with cytochalasin B reverses the neurotoxin effect. In isolated synaptic membranes veratrine and tetrodotoxin stimulate ouabain binding, the absolute veratrine-induced increment being several times higher in the presence of ATP than in its absence. Since the closed vesicles of any type are not permeable to ATP and ouabain, it means that in the isolated membranes an interaction between sodium channels and Na,K-ATPase molecules takes place. In intact nerve endings such a mechanism may be operative along with the known ways of control of sodium pump and its ouabain-binding site.

[Structural reorganization of the brain synaptic membranes and aging]. / Strukturnaia reorganizatsiia sinapticheskikh membran mozga i starenie.

Results of the authors' studies and data from literature underlie the development of a notion on structural rearrangement of the brain synaptic membranes in aging. Reorganization results in conformational changes of the key membrane-bound enzymes and receptors underlying the age alterations of neuronal functions.

[Cytoskeletal regulation of the sensitivity of brain synaptosomes to the depolarizing action of veratrine]. / Reguliatsiia tsitoskeletom chuvstvitel'nosti sinaptosom mozga k depoliarizuiushchemu deistviiu veratrina.

Using a radioactive permeant cation 3H-tetraphenylphosphonium, the sensitivity of rat brain synaptosomes to depolarizing action of veratrine, which specifically opens the sodium channels, was compared before and after destruction of microtubules and microfilaments. Depolymerization of microtubules with colchicin and vinblastine decreased an apparent affinity of veratrine to its receptor in the channel, while destruction of microfilaments with cytochalasin B had the opposite effect. Colchicine did not change allosteric interactions between the receptor for veratrine and that for scorpion venom in the sodium channel evaluated by the ability of scorpion venom to facilitate veratrine-induced depolarization of synaptosomes. It is suggested that two main cytoskeleton subsystems control the state of sodium channels in the nerve ending.

[Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism]. / Transport kal'tsiia v sinaptosomakh mozga pri depoliarizatsii. Rol' potentsialzavisimykh kanalov i Na+/Ca2+-obmena.

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.

[Regulation of mechanical stability of erythrocytes by serum immunoglobulins]. / Reguliatsiia mekhanicheskoi prochnosti éritrotsitov immunoglobulinami plazmy krovi.

Autologic plasma protects human erythrocytes from hemolysis induced by their suction through glass microfiber filters. The protective effect is related to the protein fraction with molecular mass above 100 kD and reproduced by gamma-globulin. The action of proteins is abolished after heating the erythrocytes above 45 degrees C as well as in the presence of galactose and ribose but not glucose, mannose and lactose. It is suggested that an increase in mechanical stability of erythrocytes is caused by interaction of immunoglobulins with glycoproteid and glycolipid membrane receptors and mediated by the changes of structural state of the cytoskeleton.

[Free calcium concentrations in the nerve endings in the brain of rats with spontaneous hypertension]. / Kontsentratsiia svobodnogo kal'tsiia v nervnykh okonchaniiakh golovnogo mozga krys so spontannoi gipertenziei.

Fluorescent indicator Quin-2 was used for the determination of free calcium (Ca2+in) in synaptosomes incubated in the normal medium and media where sodium is replaced by potassium or choline. At external calcium concentration of 1 mM, Ca2+in in all three media was 20-30% higher in synaptosomes of spontaneously hypertensive rats (SHR) than in control animals. At external calcium concentration of 5 mM, the increase in Ca2+in values induced by K+-depolarization in sodium- or choline-containing media was 50-80% higher in synaptosomes of SHR. These differences are suggested to be the basis for the mechanism of increased peripheral chain activity in the sympathetic nervous system in primary hypertension.

[Effect of microenvironment on the conformation of m-cholinoreceptors in brain synaptic membranes]. / Vliianie mikrookruzheniia na konformatsionnoe sostoianie m-kholinoretseptorov sinapticheskikh membran mozga.

Sensitivity of m-cholinergic receptor of synaptic membranes to SH-reagents (PChMB and NEM) was compared before and after its solubilization with detergents. PChMB blocked specific binding of m-antagonist QNB to both forms of the receptor in equal extent, while NEM was effective only after solubilization. After the membranes modification with butanol or arachidonic acid the membrane-bound receptor became sensitive to NEM. The solubilized receptor lost its ability to allosteric inhibition by acid. It is suggested that the receptor conformation is under the control of the membrane microenvironment.

[Erythrocyte hemolysis by detergents--a mathematical model and analysis of the concentration and kinetic curves]. / Gemoliz éritrotsitov detergentami--matematicheskaia model' i analiz kontsentratsionnykh i kineticheskikh krivykh.

A mathematical model of erythrocyte lysis by detergents is developed which takes into consideration the kinetics of detergent binding to plasma membrane. Experimentally obtained sigmoidal kinetic and concentration curves of hemolysis are well described by the model. A comparative study is carried out in terms of the model of hemolytic action for five detergents: Triton X-100, sodium dodecylsulfate, sodium deoxycholate, cetyltrimethylammonium bromide, and cetylpyridinium chloride. The amount of detergent which should be bound to an erythrocyte membrane to induce lysis was found to be roughly the same for all detergents studied. However, detergents vary in their affinity to the membrane. Cetylpyridinium displays the highest affinity (and consequently the highest hemolytic activity), whereas deoxycholate has the least one.

[Effect of the general anesthetic ketamine on the potassium permeability of plasma membranes of brain synaptosomes]. / Vliianie obshchego anestetika ketamina na kalievuiu pronitsaemost' plazmaticheskikh membran sinaptosom mozga.

General anesthetic ketamine (1 mM) decreases relative sodium permeability (PNa+/PK+) of synaptosomes measured by transmembrane potential--external potassium concentration curves. Since tetrodotoxin (10(-7) M) does not affect the value PNa+/PK+ it is concluded that potassium permeability of the membrane increases in the presence of ketamine.

[Characteristics of the heterogeneity of the physical properties of the lipid phase of synaptic membranes from the rat brain using the fluorescent probe pyrene]. / Kharakteristika geterogennosti fizicheskikh svoistv lipidnoi fazy sinapticheskikh membran iz mozga krys s pomoshch'iu fluorestsentnogo zonda pirena.

A theoretical analysis of the dependence of pyrene excimerization on its concentration in biological membranes was carried out. It was shown that in synaptic membranes the concentration dependencies of pyrene excimerization parameter upon direct stimulation appear as nonlinear, thus being reflective of the heterogeneity of physical properties of the lipid phase. Upon pyrene excitation at the expense of the energy transfer from tryptophanyl residues the dependence is linear, which points to the homogeneity of the anular lipid pool. It was assumed that a comparison of microviscosity of the anular and bilayer lipids requires independent measurements of the coefficient of the probe distribution between the lipids or of the anular lipid content in the membrane.

Inactivation of muscarinic acetylcholine receptors in brain synaptic membranes by free fatty acids. Evaluation of the role of lipid phase.

Arachidonic, linolenic and linoleic acids decreased the binding of the m-cholinergic antagonist [3H] QNB and did not affect the ratio of high to low affinity binding sites to the agonist carbamoylcholine in rat brain synaptic membranes. In the presence of arachidonic acid, SH-reagent N-ethylmaleimide acquired the ability to block QNB binding to receptor. Lipids in the bilayer and annular regions were probed by fluorescence of 1,6-diphenyl-1, 3, 5-hexatriene and pyrene. A microviscosity drop induced by increasing temperature from 10 to 37 degrees C did not affect the level of QNB equilibrium binding, whereas arachidonic acid strongly inhibited the binding at concentrations inducing the same drop in microviscosity as that induced by heating. For various unsaturated fatty acids an equal extent of receptor blocking was reached at quite different degrees of bilayer fluidization, the state of annular lipid being not changed under these conditions. It is suggested that the effect of unsaturated acids is reached through their direct interaction with the receptor, which undergoes a conformational change, rather than by an alteration of the physical state of the lipid phase of the membrane.

[Effect of arachidonic acid on the physical properties of bilayer and annular lipids of synaptic membranes]. / Vliianie arakhidonovoi kisloty na fizicheskie svoistva bisloinykh i pribelkovykh lipidov sinapticheskikh membran.

It has been found by fluorescence analysis of pyrene excimerization that arachidonic acid while decreasing microviscosity of lipid bilayer increases it in the annular lipid zone. After incorporation of fatty acid into the membrane the annular lipid acquires the properties of a cooperative system manifested in the appearance of thermal transition near 25 degrees C.

[Determination of electrical characteristics of synaptosomal membranes of the brain]. / Opredelenie élektricheskikh kharakteristik sinaptosomal'nykh membran mozga.

A correct method for evaluating the potential differences across plasma membranes of synaptosomes from the brain is presented. It takes into consideration the multicompartment synaptosome organization and is based on the accumulation of the radioactive permeant cation [3H]tetraphenylphosphonium. It is shown that upon potassium depolarization of the synaptosomes to about -5 mv there is a sharp decrease in the ion selectivity of the synaptic membranes.

[Muscarinic cholinoreceptors of brain synaptic membranes from animals of various ages]. / Issledovanie muskarinovykh kholinoretseptorov sinapticheskikh membran mozga zhivotnykh raznogo vozrasta.

In old rats in contrast to young and mature ones the treatment of synaptic membranes with SH-reagent N-ethylmaleimide, cholinergic agonist carbamylcholine or both of them decreases the specific binding of labeled muscarinic antagonist 3H-quinuclidinylbenzylate. The age related change of receptor conformation with exposure of thiol groups responsible for antagonist binding is suggested.

[Age changes of structural state of synaptic membranes of brain]. / Vozrastnye izmeneniia strukutrnogo sostoianiia sinapticheskikh membran mozga.

In the brain synaptic membranes from old rats (24-26 months) in comparison with the mature ones (6-7 months) an increase was shown in disintegration rate with DDS Na, mobility of 5-, 16-doxyl-stearate spin probes and in thermosensitivity of acetylcholinesterase. The total number of SH-groups decreased. While protein composition remained constant lysophosphatidylcholine and saturated fatty acid content in old animals increased. The age-dependent structural rearrangement of synaptic membranes is suggested.