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Recently, we designed and synthesized a subnanomolar, reversible, dual-binding site acetylcholinesterase (AChE) inhibitor which consists of the tacrine and aroylacrylic acid phenylamide moieties, mutually linked by eight methylene units. To further investigate the process of the molecular recognition between the AChE and its inhibitor, we performed six unconstrained molecular dynamics (MD) simulations, where the compound in three possible protonation states was placed inside binding sites of two available AChE crystal structures. In all six MD trajectories, the ligand generally occupied similar space inside the AChE active site, but the pattern of the interactions between the ligand functional groups and the amino acid residues was significantly different and highly dependent upon the crystal structure used to generate initial systems for simulation. The greatest differences were observed between the trajectories obtained with different AChE crystal structures used as starting target conformations. In some trajectories, several unusual positions and dynamic behavior of the tacrine moiety were observed. Therefore, this study provides important structure-based data useful in further optimization of the reversible, dual binding AChE inhibitors, and also emphasizes the importance of the starting crystal structure used for dynamics as well as the protonation state of the reversible inhibitors.

Recently, we designed and synthesized a subnanomolar, reversible, dual-binding site acetylcholinesterase (AChE) inhibitor which consists of the tacrine and aroylacrylic acid phenylamide moieties, mutually linked by eight methylene units. To further investigate the process of the molecular recognition between the AChE and its inhibitor, we performed six unconstrained molecular dynamics (MD) simulations, where the compound in three possible protonation states was placed inside binding sites of two available AChE crystal structures. In all six MD trajectories, the ligand generally occupied similar space inside the AChE active site, but the pattern of the interactions between the ligand functional groups and the amino acid residues was significantly different and highly dependent upon the crystal structure used to generate initial systems for simulation. The greatest differences were observed between the trajectories obtained with different AChE crystal structures used as starting target conformations. In some trajectories, several unusual positions and dynamic behavior of the tacrine moiety were observed. Therefore, this study provides important structure-based data useful in further optimization of the reversible, dual binding AChE inhibitors, and also emphasizes the importance of the starting crystal structure used for dynamics as well as the protonation state of the reversible inhibitors.Communicated by Ramaswamy H. Sarma

Detailed metabolic imaging of specific brain regions in early aging may expose pathophysiological mechanisms and indicate effective neuropharmacological targets in the onset of cognitive decline. Comprehensive imaging of brain aging and drug-target effects is restricted using conventional methodology. We simultaneously visualized multiple metabolic alterations induced by normal aging in specific regions of mouse brains by integrating Fourier-transform ion cyclotron resonance mass spectrometry imaging and combined supervised and unsupervised machine learning models. We examined the interplay between aging and the response to tacrine-induced acetylcholinesterase inhibition, a well-characterized therapeutic treatment against dementia. The dipeptide carnosine (β-alanyl-l-histidine) and the vitamin α-tocopherol were significantly elevated by aging in different brain regions. l-Carnitine and acetylcholine metabolism were found to be major pathways affected by aging and tacrine administration in a brain region-specific manner, indicating altered mitochondrial function and neurotransmission. The highly interconnected hippocampus and retrosplenial cortex displayed different age-induced alterations in lipids and acylcarnitines, reflecting diverse region-specific metabolic effects. The subregional differences observed in the hippocampal formation of several lipid metabolites demonstrate the unique potential of the technique compared to standard mass spectrometry approaches. An age-induced increase of endogenous antioxidants, such as α-tocopherol, in the hippocampus was detected, suggesting an augmentation of neuroprotective mechanisms in early aging. Our comprehensive imaging approach visualized heterogeneous age-induced metabolic perturbations in mitochondrial function, neurotransmission, and lipid signaling, not always attenuated by acetylcholinesterase inhibition.

The synthesis of four tetra-tacrine clusters where the tacrine binding units are attached to a central scaffold via linkers of variable lengths is described. The multivalent inhibition potencies for the tacrine clusters were investigated for the inhibition of acetylcholinesterase. Two of the tacrine clusters displayed a small but significant multivalent inhibition potency in which the binding affinity of each of the tacrine binding units increased up to 3.2 times when they are connected to the central scaffold.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by deterioration of cognitive skills and it is the most abundant form of dementia according to the World Health Organization (WHO).1 Tacrine belongs to a class of acetylcholinesterase inhibitors (AChEIs) and it was formerly used in the treatment of AD symptoms. Albeit no longer in clinical use due to its hepatotoxicity, tacrine ring system is extensively investigated as pharmacophore in novel AChEIs, many of which showed improved pharmacological profile in the preclinical studies.2 Here we present optimized synthetic pathway and pharmacological evaluation of three novel tacrine derivatives (5, 6 and 7), possessing functionalized piperidine connected with tacrine via five methylene groups alkyl chain

Za hlavní neurotransmiter v mozkových neuronech je považován acetylcholin (ACh), který je zodpovědný za přenos vzruchu. Je degradován dvěma enzymy, a to acetylcholinesterázou (AChE) (EC 3.1.1.7) a butyrylcholinesterázou (BChE) (EC 3.1.1.8) na cholin a acetát. Na inhibitory AChE je kladen velký důraz ve vědecké sféře, jelikož stále patří mezi hlavní léčiva při léčbě Alzheimerovy choroby či nemoci myastenia gravis. Cílem této práce bylo ověřit fotometrickou techniku při stanovení reakce AChE se substrátem na pH papírcích, stanovit kinetické parametry AChE a optimalizovat metodu pro stanovení inhibitoru - takrinu. Dále pak bylo cílem zjistit vliv vybraných matric na AChE pomocí fotometrické techniky. Pokus probíhal na pH papírcích MACHEREY-NAGEL a také na spektrofotometru metodou dle Ellmana. Použitá AChE byla z paúhoře elektrického. Bylo zjištěno, že fotometrická technika je reprodukovatelná a vhodná pro použití stanovení aktivity AChE a je vhodná pro měření skutečných vzorků. Vliv vybraných matric na tento enzym byl minimální., Acetylcholine, which is responsible for synaptic transmission, is nowadays considered to be the main neurotransmitter in the brain. It is degraded into choline and acetate by two enzymes - acetylcholinesterase (AChE) (EC 3.1.1.7) and butyrylcholinesterase (BChE) (EC 3.1.1.8) AChE inhibitors represent an interesting subject of current scientific research since they belong to the main treatments for Alzheimer's disease or myasthenia gravis. The aim of the thesis was to verify the photometric equipment while determining the reaction of AChE with the substrate on pH papers, to determine the kinetic parameters AChE, and to optimize the method for the determination of the takrin inhibitor. Furthermore, the goal was to determine the influence of selected matrices on AChE using the photometric technique. The experiment was conducted on the pH paper from MACHEREY-NAGEL and also on the spectrophotometer using the Ellman method. The AChE, which has been used, comes from an electric eel. The photometric technique was found to be reproducible and suitable for the determination of AChE activity as well as for the measurement of real samples. The influence of selected matrices on this enzyme was minimal., Fakulta chemicko-technologická, Prezentace výsledků diplomové práce. Diskuze k posudkům vedoucího a oponenta diplomové práce. Studentka zodpověděla všechny dotazy a připomínky k DP., Dokončená práce s úspěšnou obhajobou

The neurotransmitter of the cholinergic system, acetylcholine plays a major role in the brain's cognitive function and is involved in neurodegenerative disorders. Here, we present age-related alterations of acetylcholine levels after administration of the acetylcholinesterase inhibitor drug tacrine in normal mice. Using a quantitative, robust and molecular-specific mass spectrometry imaging method we found that tacrine administration significantly raised acetylcholine levels in most areas of sectioned mice brains, inter alia the striatum, hippocampus and cortical areas. However, acetylcholine levels in retrosplenial cortex were significantly lower in 14-month-old than in 12-week-old animals following its administration, indicating that normal aging affects the cholinergic system's responsivity. This small brain region is interconnected with an array of brain networks and is involved in numerous cognitive tasks. Simultaneous visualization of distributions of tacrine and its hydroxylated metabolites in the brain revealed a significant decrease in levels of the metabolites in the 14-month-old mice. The results highlight strengths of the imaging technique to simultaneously investigate multiple molecular species and the drug-target effects in specific regions of the brain. The proposed approach has high potential in studies of neuropathological conditions and responses to neuroactive treatments.

Mass spectrometry imaging (MSI) has emerged as a valuable approach for mapping multiple molecular species in sections of diverse tissues. It enables simultaneous detection of numerous compounds (from neurotransmitters to small proteins) in the brain at relatively high lateral resolution (>5 μm) on a routine basis. Matrix-assisted laser desorption/ionization (MALDI)-MSI and desorption electrospray ionization (DESI)-MSI are the most widely applied MSI techniques in tissue distribution studies. Recent advances in MSI instruments and software allow quantitative analysis of large numbers of compounds with high mass accuracy and high mass resolving power. Thus, in studies this thesis is based upon, MSI technology was used to address several challenging aspects of neuropharmacology. Restricted passage of potentially neuroactive substances into the brain, unpredictable multi-target effects, and the complexity of the central nervous system (CNS) physiology, are major obstacles in the development of efficient drugs. The simultaneous investigation of drugs’ delivery to the brain and potential effects on several CNS pathways in specific brain regions is, therefore, highly important. In addition, localization information is required for more comprehensive insights into CNS responses to both pharmaceutical agents and biological processes such as aging. MSI-based analysis of the transport of two selected drugs into the brain demonstrated effects of efflux membrane proteins on their distributions in the brain. The MDR1 substrate loperamide was found to localize specifically in the choroid plexus, indicating low brain entrance. In addition, MSI uncovered drug-drug interactions at the blood-brain barrier involving MDR1 inhibition. The technology was further used to explore neurochemical alterations induced by aging and acetylcholinesterase inhibition. First, MSI revealed that the cholinergic system’s responsivity in the retrosplenial cortex, a post-cingulate cortical area highly i

Tacrine was the first drug to be approved for Alzheimer’s disease (AD) treatment, acting as a cholinesterase inhibitor. The neuropathological hallmarks of AD are amyloid-rich senile plaques, neurofibrillary tangles, and neuronal degeneration. The portfolio of currently approved drugs for AD includes acetylcholinesterase inhibitors (AChEIs) and N-methyl-d-aspartate (NMDA) receptor antagonist. Squaric acid is a versatile structural scaffold capable to be easily transformed into amide-bearing compounds that feature both hydrogen bond donor and acceptor groups with the possibility to create multiple interactions with complementary sites. Considering the relatively simple synthesis approach and other interesting properties (rigidity, aromatic character, H-bond formation) of squaramide motif, we combined this scaffold with different tacrine-based derivatives. In this study, we developed 21 novel dimers amalgamating squaric acid with either tacrine, 6-chlorotacrine or 7-methoxytacrine representing various AChEIs. All new derivatives were evaluated for their anti-cholinesterase activities, cytotoxicity using HepG2 cell line and screened to predict their ability to cross the blood-brain barrier. In this contribution, we also report in silico studies of the most potent AChE and BChE inhibitors in the active site of these enzymes.

Tacrine was the first drug approved for the treatment of Alzheimer's disease (AD) in 1993, which was withdrawn in 2013 due to its hepatotoxicity. However, new, non-hepatotoxic tacrine derivatives have been constantly searched for. In this context, since 1997, we have prepared a number of diversely functionalized tacrines by changing the benzene ring present in tacrine to five- or six-membered aromatic ring cores that could present anticholinesterasic activity and additional pharmacological properties. The new compounds were designed as juxtaposed structures between tacrine and the well-known Ca antagonists 1,4-dihydropyridines, with the goal of obtaining multi-target directed ligands for AD. In this account, we present our results on the PyridoTacrine (PyrTac) family of tacrine analogues, resulting from the substitution of the benzene ring by a pyridine. We highlight their pharmacological profile and review similar analogues in the literature. A first set of PyrTac showed inhibitory activity of cholinesterases (ChE) and a blocking profile of voltage-gated Ca channels (VGCC). A second family with improved ChE inhibition lost VGCC blocking activity. However, the lead compound of this family (5f) presented an activating profile of the phosphatase 2A (PP2A) and showed interesting outcomes in experimental in vivo models of AD and stroke. We have identified the PyrTac ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b] [1,8]naphthyridine-3-carboxylate (5f), which presents additional pharmacological properties beyond the mere cholinergic improvement. These new properties warrant attention to 5f and its further development as a new potential therapeutic agent for AD therapy.

Tato diplomová práce se zabývá hodnocením cytotoxicity nově syntetizovaných modulátorů cholinesteras. Zvolenými látkami byly deriváty kyseliny čtvercové, takrinu a 7-metoxytakrinu. Takrin a 7-metoxytakrin jsou inhibitory acetylcholinesterasy určené pro léčbu Alzheimerovi choroby, ačkoliv v případě takrinu se již od jeho podávání ustoupilo, kvůli jeho nežádoucím účinkům. První část je zaměřena na Alzheimerovu chorobu, její patogenezi, příznaky, diagnostiku a terapii. Druhá část je věnována provedení experimentu (tzn. pasážování buněk, provedení MTT testu, apod.). V závěru práce jsou uvedeny a zhodnoceny toxikologické indexy a dávkově závislé křivky u jednotlivých testovaných sloučenin., This diploma thesis evaluates the cytotoxicity of newly synthesized modulators of cholinesterase. The chosen compounds were the derivates of squaric acid, tacrine and 7-methoxytacrine. Tacrine and 7-methoxytacrine are acetylcholinesterase inhibitors used for the treatment of Alzheimer's disease. Due to adverse effects, tacrine has been withdrawn from the market. In the first chapter, the author therefore describes Alzheimer's disease and associated pathogenesis, symptoms, diagnosis, and therapy. The second part is focused on the experiment (i. e. passaging of cells, MTT test, etc.). At the end of the work, toxicological indexes and dose-depent curves of individual test compounds are presented and evaluated., Fakulta chemicko-technologická, 1. Prezentace výsledků diplomové práce. 2. Diskuze k posudku vedoucího a oponenta diplomové práce. 3. Studentka zodpověděla všechny dotazy a připomínky k DP.

In this communication we have described the synthesis of 2-amino-6-oxo-1,6-dihydropyridine-3-carbonitrile (4), and the preparation of 5-amino-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridin-2(1H)-one (3 a), a non-hepatotoxic, antioxidant agent, showing moderate, but selective human acetylcholinesterase inhibition.

A summary of the recently published efforts on tacrine derivatives as a renewed potential therapeutic approach for the treatment of Alzheimer's disease is presented.

In the reaction of ZnCl(2) with tacrine hydrochloride in water novel tetracoordinated (C(13)H(15)N(2))(2)[ZnCl(4)]-H(2)O complex was obtained and characterized by elemental analysis, molar conductivity and X-ray analysis. The complex crystallizes in the space group P-1 of the triclinic crystal system. The structure contains two crystallographically different molecules of protonated tacrine present as counter cations, the [ZnCl(4)](2-) complex anion and one water solvent molecule. The counter cations slightly differ in the puckering of the cyclohexene ring. The molecules of protonated tacrine are involved in different intermolecular hydrogen bonds. In the crystal, the hydrogen bonding generates a 3D assembly. In the crystal, pi center dot center dot center dot pi stacking interactions between the rings of protonated tacrine were evidenced. The [ZnCl(4)](2-) complex anion has a distorted tetrahedral geometry. Three out of the four Cl atoms are involved in intermolecular hydrogen bonding. The intermolecular H-bond interactions involving the Cl atoms affect the Zn-CI bond lengths., U reakciji ZnCl2 sa takrin-hidrohloridom u vodi, dobijen je novi tetrakoordinovani (C13H15N2)2[ZnCl4]?H2O kompleks koji je okarakterisan pomoću elementalne analize, molarne provodljivosti i rendgenske strukturne analize. Kompleks kristališe u prostornoj grupi P?1 trikliničnog kristalnog sistema. Struktura sadrži dva kristalografski različita molekula protonovanog takrina koji su prisutni kao kontra-katjoni, [ZnCl4]2 kompleksni anjon i molekul kristalne vode. Molekuli katjona se neznatno razlikuju u stepenu nabiranja cikloheksenovog prstena. Molekuli protonovanog takrina su uključeni u različite intermolekulske vodonične veze. Intermolekulsko vodonično vezivanje u kristalu generiše 3D molekulski skup pi...pi interakcije između prstenova protonovanog takrina su primećene u kristalu. [ZnCl4]2- ima distorgovanu tetraedarsku geometriju. Tri od četiri Cl atoma su uključena u intermolekulske vodonične veze. Intermolekulske vodonične interakcije koje uključuju Cl atome utiču na dužinu Zn-Cl veza.

In the reaction of ZnCl2 with tacrine hydrochloride in water novel tetracoordinated (C13H15N2)2[ZnCl4]?H2O complex was obtained and characterized by elemental analysis, molar conductivity and X-ray analysis. The complex crystallizes in the space group P?1 of the triclinic crystal system. The structure contains two crystallographically different molecules of protonated tacrine present as counter cations, the [ZnCl4]2- complex anion and one water solvent molecule. The counter cations slightly differ in the puckering of the cyclohexene ring. The molecules of protonated tacrine are involved in different intermolecular hydrogen bonds. In the crystal, the hydrogen bonding generates a 3D assembly. In the crystal, pi…pi stacking interactions between the rings of protonated tacrine were evidenced. The [ZnCl4]2- complex anion has a distorted tetrahedral geometry. Three out of the four Cl atoms are involved in intermolecular hydrogen bonding. The intermolecular H-bond interactions involving the Cl atoms affect the Zn-Cl bond lengths., U reakciji ZnCl2 sa takrin-hidrohloridom u vodi, dobijen je novi tetrakoordinovani (C13H15N2)2[ZnCl4]?H2O kompleks koji je okarakterisan pomoću elementalne analize, molarne provodljivosti i rendgenske strukturne analize. Kompleks kristališe u prostornoj grupi P?1 trikliničnog kristalnog sistema. Struktura sadrži dva kristalografski različita molekula protonovanog takrina koji su prisutni kao kontra-katjoni, [ZnCl4]2 kompleksni anjon i molekul kristalne vode. Molekuli katjona se neznatno razlikuju u stepenu nabiranja cikloheksenovog prstena. Molekuli protonovanog takrina su uključeni u različite intermolekulske vodonične veze. Intermolekulsko vodonično vezivanje u kristalu generiše 3D molekulski skup pi...pi interakcije između prstenova protonovanog takrina su primećene u kristalu. [ZnCl4]2- ima distorgovanu tetraedarsku geometriju. Tri od četiri Cl atoma su uključena u intermolekulske vodonične veze. Intermolekulske vodonične interakcije koje uključuju Cl atome utiču na dužinu Zn-Cl veza.

In the reaction of ZnCl(2) with tacrine hydrochloride in water novel tetracoordinated (C(13)H(15)N(2))(2)[ZnCl(4)]-H(2)O complex was obtained and characterized by elemental analysis, molar conductivity and X-ray analysis. The complex crystallizes in the space group P-1 of the triclinic crystal system. The structure contains two crystallographically different molecules of protonated tacrine present as counter cations, the [ZnCl(4)](2-) complex anion and one water solvent molecule. The counter cations slightly differ in the puckering of the cyclohexene ring. The molecules of protonated tacrine are involved in different intermolecular hydrogen bonds. In the crystal, the hydrogen bonding generates a 3D assembly. In the crystal, pi center dot center dot center dot pi stacking interactions between the rings of protonated tacrine were evidenced. The [ZnCl(4)](2-) complex anion has a distorted tetrahedral geometry. Three out of the four Cl atoms are involved in intermolecular hydrogen bonding. The intermolecular H-bond interactions involving the Cl atoms affect the Zn-CI bond lengths.

Effects of the cholinesterase inhibitors tacrine and bis(7)tacrine (0.25-20 mu mol/kg, s.c.) on locomotor activity and passive-avoidance response were investigated in mice treated with scopolamine (SCP, 1 or 5 mu mol/kg, i.p.), using an open-field test and step-through task with a 24-hour retention interval. Drugs were given 30 min prior to the first session. During the acquisition session, SCP treatment increased the locomotor activity (10-16\%). Tacrine, but not bis(7)-tacrine, cotreatment significantly reduced the locomotor activity by 23 or 27\%, when compared with the SCP-treated control mice. In the step-through task, tacrine or bis(7)-tacrine coadministration dose-dependently attenuated the increase in the number of footshocks (by 50 or 58\%) in SCP-treated mice. The lowest dose of tacrine and bis(7)-tacrine for prolonging the retention latency (up to 500\%) in SCP-treated mice was 5 and 1 mu mol/kg, respectively. Tacrine and bis(7)-tacrine inhibited brain acetylcholinesterase (AChE) activity 15 min (but not 30 min) after the drug administration in mice. At the same dose of 20 mu mol/kg, the bis(7)-tacrine-induced AChE inhibition in serum was 14-fold higher than that of tacrine. The results indicated that bis(7)-tacrine was less potent than tacrine in causing motor dysfunction. However, bis(7)-tacrine was more potent than tacrine in the cognitive enhancement of SCP-induced memory loss and in AChE inhibition. Copyright (C) 2009 S. Karger AG, Basel

Alzheimer's Disease (AD) is an irreversible, age-related neurodegenerative disorder which causes cognitive impairment and a wide variety of neuropsychiatric and behavioral disturbances. Acetylcholinesterase inhibitors (AChEI) are the mainstay for the treatment of AD. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acylcholinesters with a relative specificity for acetylcholine (ACh). Observation of a deficiency of cholinergic neurotransmission in AD led to the development of AChEI as the first approved treatment for dementia symptoms. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a reversible inhibitor of AChE. It was the first drug approved by the FDA for the treatment of cognitive symptoms of AD. Tacrine is now rarely prescribed as a drug for the treatment of AD due to its high hepatotoxicity in almost 50% of the patients. However, tacrine derivatives have considerable potential for the palliative treatment of AD. Synthesis of various bivalent tacrines led to the improvement in inhibitory potency and selectivity towards inhibition of AChE. Heptylene-linked bis-tacrine has especially shown immense promise to be an ideal AChEI. Thus dimerization of a lead compound seemed to be an ideal strategy where the compound can bind to both catalytic anionic site (CAS) and peripheral anionic site (PAS) on the AChE enzyme. However synthesis of N-alkyl derivatives of expanded tacrines like 12-chloro-2-methyl-6,7,8,9,10,11-hexahydrocycloocta[b]quinoline by the standard SNAr methods was unsuccessful and thus alternatives needed to be developed to synthesize N-alkylated and bivalent 12-chloro-2-methyl-6,7,8,9,10,11-hexahydrocycloocta[b]quinoline. Upon exploring the alternatives, N-arylation by Pd-catalysis seemed to be the most mild and convenient alternative over the standard SNAr procedures.

Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh). Inhibition of AChE has proven an effective treatment for the memory loss exhibited by early stage Alzheimer's disease (AD) patients; four AChE inhibitors (AChEI) have been approved by the FDA for this purpose. The first AChEI approved for the palliative treatment of AD-related memory loss was 9-amino-1,2,3,4-tetrahydroacridine (tacrine). Inhibition of AChE may present either therapeutic or toxic effects depending upon the dose administered. With the goal of discovering safe and effective pesticides to control the population of Anopheles gambiae, a malaria-transmitting mosquito indigenous to Sub-Saharan Africa, the reoptimization of the tacrine pharmacophore was undertaken. Because the optimized drug would necessarily be a poor inhibitor for human AChE (hAChE), initial ligand design focused on modification to tacrine known to negatively impact the inhibition potency for hAChE. Ultimately, an AChEI was discovered, which exhibited micromolar inhibition of Anopheles gambiae AChE (AgAChE) and essentially no potency for hAChE. Two units of this lead compound were tethered through an alkyl chain to yield a nanomolar inhibitor of AgAChE that was more than 1,100-fold selective for the mosquito enzyme over hAChE. Dimerization of an active inhibitor is an effective strategy to increase the potency and selectivity of AChEI, and many examples of tacrine hetero- and homodimers complexed to AChE can be found in the RCSB Protein Data Bank (PDB). The bond formed between the exocyclic amine moiety and the heterocyclic ring system of tacrine is analogous to an amide bond when tacrine is protonated. Therefore, the rotational profile of protonated N-alkyltacrine should exhibit a conformational profile in which dihedral angles significantly out of the plane formed by the ring system are associated with high energies relative to those when the dihe

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent metabolite resorufin. The system was tested with three P450 1A2 inhibitors, for which minimum detectable amounts (MDA) ranging from 0.7 to 9.5 ng were obtained. Analysis of a kava kava and a basil extract showed that the on-line system is applicable to complex mixtures, since in both extracts, peaks with P450 1A2 inhibiting activity were observed. © 2006 Elsevier B.V. All rights reserved.

Bis(7)-tacrine was previously demonstrated as an antagonist of gamma-aminobutyric acid type A (GABA(A)) receptors. In this study, the effects of a series of alkylene-linked tacrine dimers on GABAA receptors were examined. In radioligand binding assay, the analogues differed in binding affinity for GABAA receptors, and potency monotonically increased as the tether was shortened from nine to two methylenes. Bis(2)-tacrine, the shortest tacrine dimer, could displace [H-3]muscimol from rat brain membranes with an IC50 of 0.48 M, which was 11, 13 and 525 times more potent than the GABAA receptor antagonist (+)-bicucul line, bis(7)-tacrine and tacrine, respectively. In whole-cell patch-clamp recordings, these dimeric tacrine analogues competitively antagonized GABA-induced inward current with a rank order of potency of bis(2)-tacrine > bicuculline > bis(7)-tacrine > bis(9)-tacrine > tacrine, and the potency of bis(2)-tacrine was 11, 18 and 487 times higher than that of (+)bicuculline, bis(7)-tacrine and tacrine, respectively. Bis(2)-tacrine shifted the GABA concentration-response curve to the right in a parallel manner, and the inhibition was voltage-independent between -80 and +20 mV. It can be concluded that the shorter the alkylene linkage in tacrine dimers the stronger the binding affinity and higher the antagonistic effect on the GABAA receptor will be. (c) 2006 Elsevier Ltd. All rights reserved.

The potential of tacrine in altering cognitive/behavioral function as well as in causing toxicity was evaluated in mice of 17 and 30 days of age. Cognitive and behavioral studies were performed using a step-through passive avoidance task and a habituation open-field test with a 24-h retention interval. Tacrine was subcutaneously injected (1.25-80 mu mol/kg) 30 min prior to the first session of both tests. During the training session in step-through task, tacrine treatment dose-dependently decreased the number of footshocks, with IC50 values being 7.8 and 23.3 mu mol/kg in 17- and 30-day-old mice, respectively. Treatment with tacrine at a low dose (5 mu mol/kg) significantly prolonged the retention latency in 17-day-old mice only, but it shortened the retention latency at high doses of 20 and mu mol/kg in 17- and 30-day-old, respectively. During the acquisition session in the open-field test, tacrine treatment dose-dependently decreased the locomotor activity in 17- and 30-day-old mice, with IC50 values being 15.1 and 24.7 mu mol/kg, respectively. High doses of tacrine invariably increased the locomotor activity during the recall session. Tacrine treatment at a dose of 40 mu mol/kg caused a significant increase in serum alanine aminotransferase activity in 17- and 30-day-old mice at 6 h post-dosing, with the extent of stimulation in 30-day-old mice being more prominent. In conclusion, tacrine was more potent in enhancing/disrupting the cognitive function, inhibiting locomotor activity as well as in causing hepatotoxicity in 17-day-old than in 30-day-old mice. (c) 2006 Elsevier Inc. All rights reserved.

Alzheimer's disease (AD) and related dementing disorders having cognitive manifestations represent an increasing threat to public health. In the present study, the effects of a memory enhancing NLPR tetra-peptide (MEP), huperzine A (Hup A), or a combination of the two on the cognitive abilities of brain-lesioned mice were evaluated and compared with tacrine in the passive avoidance and Y-water maze tests for the acquisition and retention aspects of cognitive functions. MEP at mu g kg(-1) doses, and Hup A or tacrine at mg kg-1 doses significantly reversed the cognition deficits induced by scopolamine. For acquisition ability, it was observed that mice administered with MEP (4.0 mu g kg(-1)) spent less time escaping onto the platform in the water maze than those treated with tacrine (1.5 mg kg(-1)); whereas for memory retention, tacrine-administration resulted in a higher step-through latency in mice at the tested dose regime. In addition, co-administration of MEP (2.0 mu g kg(-1)) and Hup A (0.1 mg kg(-1)) exhibited an additive effect resulting in considerable improvements in both acquisition and retention abilities of brain-lesioned mice. The results demonstrated that MEP was highly efficient in the rescue of cognitive abilities of brain-lesioned mice and in particular, the effective doses of MEP were about two orders of magnitude lower than that of tacrine, a therapeutic currently used in the treatment of AD. Moreover, MEP and Hup A were effective at reduced doses when the two were co-administered, providing a rationale for their combined usage in the treatment of cognitive deficits. Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.

Intragastric administration (100-200 mumol/kg) of tacrine (THA) or bis(7)-THA could cause an acute and dose-dependent increase in plasma alanine aminotransferases activity in mice at 6 h after the drug administration. The increase in plasma enzyme activity was associated with an increase in hepatic malondialdehyde level, an indirect index of oxidative tissue damage. Pretreating mice with schisandrin B (Sch B), an active dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, at a daily dose of 0.125-0.5 mmol/kg for 3 days protected against the THA/bis(7)-THA induced hepatic oxidative damage in a dose-dependent manner. Sch B treatment (0.025-0.5 mmol/kg/day x 5) also enhanced the passive avoidance-response in mice as assessed by the step-through task experiment. The ensemble of results suggests that Sch B may be useful for reducing the potential hepatotoxicity of THA/bis(7)-THA in anti-Alzheimer's therapy.

Today, cognitive impairment can be successfully treated with acetylcholine esterase inhibitors (AChE-I) in many, but not all, patients with Alzheimer's disease (AD). To investigate the relation between tacrine treatment, inheritance of ApoE e4 alleles, and rate of progression, the differences in MMSE and CIBIC scores (efficacy parameters) after 6 and 12 months of tacrine (an AChE-I) treatment were investigated in 145 AD patients. Of these, 84 were ApoE e4-positive (ApoE4) and 61 were ApoE e4-negative (ApoE2-3). No differences were found after 6 months of treatment, but after 12 months the CIBIC scores revealed that the ApoE4 patients had declined more than the ApoE2-3 patients (p < 0.05). No differences were found for the last 6 months of treatment. The results primarily suggest a faster rate of decline in the ApoE4 AD compared to the ApoE2-3, but may also reflect that ApoE e4 genotype inheritance is a negative predictor of treatment effect of tacrine in AD patients.

AIM: To examine the antagonism of tacrine on the amnesic effects of scopolamine (Sco), anisodine (AT(3)), atropine (Atr), and anisodamine (Ani). METHODS: Cognitive functions and locomotor activities were determined using two sessions of step-through and open-field tests, respectively. Mice were injected with one of the belladonna alkaloids (0.05 - 50 mu mol . kg(-1), ip) and tacrine (50 mu mol . kg(-1), sc) 30 min before the first session. RESULTS: Tacrine completely blocked the avoidance-learning deficit caused by Sco 0.5 mu mol . kg(-1), AT(3) and Atr 5 mu mol . kg(-1), or Ani 50 mu mol . kg(-1). But tacrine partly antagonized the learning deficit induced by Sco 5 - 50 mu mol . kg(-1) or Atr and AT3 50 mu mol . kg(-1). The avoidance-memory deficit caused by Sco 0.05 - 5 mu mol . kg(-1) or Atr 5 mu mol . kg(-1) was completely or partly attenuated by tacrine, which did not antagonize the memory deficit elicited by Sco and Atr 50 mu mol . kg(-1), AT(3) 5 and 50 mu mol . kg(-1), and Ani 50 mu mol . kg(-1). During the acquisition, the locomotor activity of the mice was inhibited by tacrine. This reduction was completely antagonized by Sco 0.5 - 50 mu mol . kg(-1) AT(3) 5 - 50 mu mol . kg(-1), Atr 5 - 50 mu mol . kg(-1), and only partly antagonized by AT(3) and Atr 0.5 mu mol . kg(-1) or Ani 50 mu mol . kg(-1). CONCLUSION: Compared with the avoidance-memory deficit, the avoidance-learning deficit caused by belladonna alkaloids is more preferentially attenuated by tacrine.

The present study investigates the effects of bis(7)-tacrine, a novel dimeric acetylcholinesterase inhibitor, on hydrogen peroxide(H2O2)-induced cell injury with comparison to the corresponding monomer, tacrine. Exposure of rat pheochromocytoma line PC12 cells to H2O2 induced significant cell damage. This reagent also caused redox desequilibrium as indicated by a decrease in activities of intracellular antioxidant enzymes such as glutathione peroxidase as well as catalase and an accumulation of malondialdehyde, a product of lipid peroxidation. Pretreatment of cells with bis(7)tacrine or tacrine attenuated H2O2-induced cell toxicity, and bis(7)-tacrine demonstrated higher potency than tacrine in improving redox desequilibrium. These results suggest that bis(7)-tacrine and tacrine significantly protect against H2O2 insult, which might be beneficial for their potential usage in the prevention and treatment of Alzheimer's disease. (C) 2000 Elsevier Science Ireland Ltd. Ail rights reserved.

Alzheimer disease (AD) involves neuronal degeneration with impaired cholinergic transmission in the cerebral cortex and hippocampus in areas of the brain particularly associated with memory and higher intellectual functioning. Other neurotransmitter deficits also occur, but the mechanisms underlying the widespread impairment of synaptic functions remain uncertain. Research on the molecular basis of AD has elucidated a pathogenic pathway from which a range of rational pharmacological interventions has emerged. Although at least 3 cholinesterase inhibitors (tacrine hydrochloride, donepezil, and rivastigmine tartrate) are now available and provide patients with modest relief, the most promising strategy involves approaches to retarding, halting, or preventing the formation or accumulation of beta-amyloid (Abeta) plaques. Estrogen is believed to have antioxidant or other anti-Abeta effects, as hormonal replacement therapy in women with menopause is associated with a reduced risk or delayed onset of AD. The association between nonsteroidal anti-inflammatory drugs and a reduced risk of AD has not yet been confirmed, but these agents may protect the brain from the reactive glial and microglial responses associated with Abeta deposition. Also, recent studies suggested that antioxidants, such as vitamin E taken alone or in combination with selegiline hydrochloride, can delay the progression of AD. Despite these encouraging results, no current therapy has been shown to halt or reverse the underlying disease process. The proof of the principle that anti-Abeta drugs will work in the transgenic models of AD is eagerly awaited with the expectation that they will eventually prove successful in humans.

HEPTYLENE-LINKED bis-(9-amino-1,2,3,4-tetrahydroacridine) (bis(7)-tacrine) is a potential palliative therapeutic agent for Alzheimer's disease (AD), on the basis of its superior acetylcholinesterase (AChE) inhibition and memory-enhancing potency relative to tacrine. In this study we report that bis(7)-tacrine exhibits a potentially complementary central nervous system action, antagonism of GABA(A) receptor function. Bis(7)tacrine displaced [H-3]muscimol from rat brain membranes with an apparent K-i of 6.0 mu M; tacrine and physostigmine were shown to be 18 and 170 times less potent, respectively. In whole-cell patch-clamp recordings, bis(7)-tacrine inhibited GABA-induced inward current with an IC50 of 5.6 mu M, and shifted the GABA concentration-response curve to the right in a parallel manner. These results suggest that bis(7)-tacrine is a competitive antagonist of the GABA(A) receptor. NeuroReport 10:795-800 (C) 1999 Lippincott Williams & Wilkins.

HupA, a novel alkaloid isolated from the Chinese medicinal herb Huperzia serrata, is a reversible, potent, and selective inhibitor of AChE. Compared with other well-known AChEIs, such as physostigmine, galanthamine, tacrine, and even donepezil, HupA has better penetration through the blood-brain barrier, higher oral bioavailability, and longer duration of AChE inhibitory action. HupA exhibited memory-enhancing efficacy in a broad range of animal models of cognitive impairment. Double-blind and placebo-controlled clinical trials have demonstrated that HupA produced significant improvements in memory deficiencies in aged patients and patients with AD. Furthermore, both animal and clinical safety testings showed that HupA was devoid of unexpected toxicity, particularly the dose-limiting hepatotoxicity induced by tacrine. These encouraging preclinical and clinical findings suggest that HupA is a highly promising candidate for clinical development as a symptomatic treatment for AD and other memory disorders related to a central cholinergic deficiency.

THE anticholinesterase effects of bis(7)-tacrine were compared with tacrine in vitro and in vivo. Based on IC50 ratios, the dimeric analog bis(7)-tacrine was, in a reversible manner, up to 150-fold more potent and 250-fold more selective than tacrine for acetylcholinesterase (AChE) over butyrylcholinesterase (BChE). Following a single oral administration, both bis(7)-tacrine and tacrine produced dose-dependent inhibitions of AChE in rat brain, but bis(7)-tacrine exhibited higher efficacy and AChE/BChE selectivity than tacrine. The anti-AChE efficacy of bis(7)-tacrine was quite similar following an oral or i.p. administration, but tacrine showed much lower efficacy when administered orally than when given i.p. These findings suggest bis(7)-tacrine, a highly potent and selective inhibitor of AChE, can probably be used as an improved drug in the palliative treatment of AD. NeuroReport 10:789-793 (C) 1999 Lippincott Williams & Wilkins.

AIM: To study the effects of 1, 7-N-heptylene-bis-9, 9'-amino-1,2, 3, 4-tetrahydroacridine [bis (7)-tacrine], a novel dimeric acetylcholinesterase inhibitor (AChEI) derived from 9-amino-1,2,3, 4-tetrahydroaminoacridine (tacrine), on scopolamine-induced spatial memory impairment. METHODS: The effects of bis(7)-tacrine were investigated on the 5-d performance of young adult rats in the Morris water maze. The latency to find the platform in the water maze was measured to evaluate performance. Tacrine was used as a reference drug. RESULTS: Scopolamine (0.3 mg.kg(-1), ip) resulted in an increase in latency period ( >100\% increase) as compared with saline treated controls. Both bis (7)-tacrine and tacrine lessened the increased latency induced by scopolamine to the level of saline control group. The relative potency of bis (7)-tacrine (0.35 mu mol.kg(-1), ig or ip) to shorten the escape latency was 24 or 12 times of tacrine (8.52 mu mol.kg(-1) ig, 4.26 mu mol. kg(-1) ip) following ig or ip administration, respectively. There appeared to be an inverse bell-shape dose-dependent effect for both compounds tested. CONCLUSION: Bis (7)-tacrine is a more potent and orally active AChEI than tacrine, and has potential for the palliative treatment of Alzheimer disease.

To describe the use of tacrine in nursing home residents using data from a clinically based resident assessment instrument used by all US nursing homes.

Research has been focused on the human cholinesterase genes, their variant protein products and the biological roles of these proteins in different tissue - and cell types. To elucidate the biochemical properties and biological functions of this large array of ChE variants, we employed microinjected oocytes and transiently transgenic embryos of Xenopus laevis. This has led to the demonstration that the C-terminal peptide characteristic of the brain and muscle form of acetyicholinesterase (AChE) and butyrylcholinesterase (BuChE) leads to synaptic targeting of these enzymes. Moreover, we were able to show that AChE exerts a morphogenic action on synapse development. The biochemical and biological functions of the BCHE gene and its BuChE protein product were approached by a combination of Xenopus oocyte expression and molecular genetics. Substitution of Asp at position 70 in BuChE by Gly creates an enzyme incapable of hydrolyzing succinylcholine, unlike the native enzyme. This structure-function relationship explained the known clinical syndrome of "succinylcholine apnea". The next step was to replace the BuChE peptide harboring Asp70 with that of AChE. This introduced charge modification in the resultant active chimera and explained many of the differences between AChE and BuChE with respect to drug and poison sensitivities. A population diversity study revealed 11% heterozygotes for Asp/Gly70 among Israelis and shed new light on individual variabilities noted for the newly tested anti-AChE drugs, examined for their potential in Alzheimer's therapy. Our current efforts are to extend these studies to elucidate the protective roles of these proteins against organophosphate intoxication, using transgenic mice overexpressing the human ACHE gene.

BackgroundIn Alzheimer's disease, there is a marked decline in the function of cholinergic neurons in the brain. However, studies of treatment with cholinesterase inhibitors have produced conflicting results. We conducted a multicenter trial to evaluate whether the cholinesterase inhibitor tacrine (1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate) could improve cognition in patients with Alzheimer's disease.MethodsOf 632 eligible patients with probable Alzheimer's disease, 215 improved while receiving tacrine during a preliminary crossover phase to determine responsiveness and the best dose. The 215 patients were randomly assigned to receive either placebo or their best dose of tacrine (10 or 20 mg four times a day) in a six-week, double-blind trial. The primary measures of efficacy were the cognitive subscale of the Alzheimer's Disease Assessment Scale and the Clinical Global Impression of Change scale; the secondary measures included the Mini-Mental State Examination and the assessment of the activities of daily living.ResultsAt the end of the six-week trial, the patients receiving tacrine had a mean adjusted cognitive-subscale score of 30.3 (Alzheimer's Disease Assessment Scale) as compared with 32.7 in patients receiving placebo. This represents a smaller decline (by 2.4 points) in cognitive performance in the tacrine group (P < 0.001). There were no differences between the groups in their global-rating scores. The tacrine group had a significantly smaller decline in the activities of daily living. The results of the Mini-Mental State Examination favored tacrine, but the differences were small and not statistically significant (a score of 16.0 with tacrine vs. 15.3 with placebo; P = 0.08). Gastrointestinal symptoms, elevation of aminotransferase levels, and headache were the most frequent side effects; all could be reversed by reducing the dose or discontinuing treatment.ConclusionsIn this short-term study in patients with Alzheimer's disease w