Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.     

Chlorinated tacrine analogs: Design,synthesis and biological evaluation of their anti-cholinesterase activity as potential treatment for Alzheimer’s disease

In search of potent acetyl cholinesterase inhibitors with low hepatotoxicity for the treatment of Alzheimer’s disease, introduction of a chloro substitution to tacrine and some of its analogs has proven to be beneficial in maintaining or potentiating the cholinesterase inhibitory activity. Furthermore, it was found to be able to reduce the hepatotoxicity of the synthesized compounds, which is the main target of the study. Accordingly, a series of new 4-(chlorophenyl)tetrahydroquinoline derivatives, was synthesized and characterized. The synthesized compounds were evaluated for their in vitro and in vivo anti-cholinesterase activity using tacrine as a reference standard. Furthermore, they were investigated for their hepatotoxicity compared to tacrine. The obtained biological results revealed that all synthesized compounds displayed equivalent or significantly higher anti-cholinesterase activity and lower hepatotoxicity in comparison to tacrine. In addition, in silico drug-likeness of the synthesized compounds were predicted and their practical logP were assessed indicating that all synthesized compounds can be considered as promising hits/leads. Furthermore, docking study of the compound showing the highest in vitro anticholinesterase activity was performed and its binding mode was compared to that of tacrine.     

Tacrine and its analogues impair mitochondrial function and bioenergetics: a lipidomic analysis in rat brain

Tacrine is an acetylcholinesterase (AChE) inhibitor used as a cognitive enhancer in the treatment of Alzheimer's disease (AD). However, its low therapeutic efficiency and a high incidence of side effects have limited its clinical use. In this study, the molecular mechanisms underlying the impact on brain activity of tacrine and two novel tacrine analogues (T1, T2) were approached by focusing on three aspects: (i) their effects on brain cholinesterase activity; (ii) perturbations on electron transport chain enzymes activities of non-synaptic brain mitochondria; and (iii) the role of mitochondrial lipidome changes induced by these compounds on mitochondrial bioenergetics. Brain effects were evaluated 18 h after the administration of a single dose (75.6 μmol/kg) of tacrine or tacrine analogues. The three compounds promoted a significant reduction in brain AChE and butyrylcholinesterase (BuChE) activities. Additionally, tacrine was shown to be more efficient in brain AChE inhibition than T2 tacrine analogue and less active than T1 tacrine analogue, whereas BuChE inhibition followed the order: T1 > T2 > tacrine. The studies using non-synaptic brain mitochondria show that all the compounds studied disturbed brain mitochondrial bioenergetics mainly via the inhibition of complex I activity. Furthermore, the activity of complex IV is also affected by tacrine and T1 treatments while FoF(1) -ATPase is only affected by tacrine. Therefore, the compounds' toxicity as regards brain mitochondria, which follows the order: tacrine > T1 > T2, does not correlate with their ability to inhibit brain cholinesterase enzymes. Lipidomics approaches show that phosphatidylethanolamine (PE) is the most abundant phospholipids (PL) class in non-synaptic brain mitochondria and cardiolipin (CL) present the greatest diversity of molecular species. Tacrine induced significant perturbations in the mitochondrial PL profile, which were detected by means of changes in the relative abundance of phosphatidylcholine (PC), PE, phosphatidylinositol (PI) and CL and by the presence of oxidized phosphatidylserines. Additionally, in both the T1 and T2 groups, the lipid content and molecular composition of brain mitochondria PL are perturbed to a lesser extent than in the tacrine group. Abnormalities in CL content and the amount of oxidized phosphatidylserines were associated with significant reductions in mitochondrial enzymes activities, mainly complex I. These results indicate that tacrine and its analogues impair mitochondrial function and bioenergetics, thus compromising the activity of brain cells.     

Effect of reversible inhibitors on the thermal denaturation of cholinesterases]

The capacity of reversible inhibitors--galanthamine and tacrine--to protect the cholinesterase of rat and mouse brain from the thermal denaturation with the action of the temperature of 56 and 58 degrees C was revealed. The protective action was also noted when the reversible inhibitors decreased the activity of the enzyme. It appeared that with galanthamine the cholinesterase resistance to the thermal action was greater than with the action of tacrine.     

Design,synthesis and evaluation of tacrine–flurbiprofen–nitrate trihybrids as novel anti-Alzheimer’s disease agents

To search for multifunctional anti-Alzheimer’s disease (AD) agents with good safety, the previously synthesized tacrine–flurbiprofen hybrids 1a and 1b were modified into tacrine–flurbiprofen–nitrate trihybrids 3a–h. These compounds displayed comparable or higher cholinesterase inhibitory activity relative to the bivalent hybrids. Compound 3a was the most potent, which released moderate NO, exerted blood vessel relaxative activity, and showed significant Aβ inhibitory effects whereas tacrine and flurbiprofen did not exhibit any Aβ inhibitory activity at the same dose. In addition, 3a was active in improving memory impairment in vivo. More importantly, the hepatotoxicity study showed that 3a was much safer than tacrine, suggesting it might be a promising anti-AD agent for further investigation.     

Inhibition of two different cholinesterases by tacrine

Kinetic parameters of the effect of tacrine as a cholinesterase inhibitor have been studied in two different sources: snake venom (Bungarus sindanus) acetylcholinesterase (AChE) and human serum butyrylcholinesterase (BChE). Tacrine inhibited both venom acetylcholinesterase (AChE) as well as human serum butyrylcholinesterase (BChE) in a concentration-dependent manner. Kinetic studies indicated that the nature of inhibition was mixed for both enzymes, i.e. Km values increase and Vmax decrease with the increase of the tacrine concentration. The calculated IC50 for snake venom and for human serum were 31 and 25.6 nM, respectively. Ki was observed to be 13 nM for venom acetylcholinesterase (AChE) and 12 nM for serum butyrylcholinesterase (BChE). KI (constant of AChE-ASCh-tacrine complex into AChE-ASCh complex and tacrine) was estimated to be 20 nM for venom and 10 nM for serum butyrylcholinesterase (BChE), while the gammaKm (dissociation constant of AChE-ASCh-tacrine complex into AChE-tacrine complex and ASCh) were 0.086 and 0.147 mM for snake venom AChE and serum BChE, respectively. The present results suggest that this therapeutic agent used for the treatment of Alzheimer's disease can also be considered an inhibitor of snake venom and human serum butyrylcholinesterase. Values of Ki and KI show that tacrine had more affinity with these enzymes as compared with other cholinesterases from the literature.     

The new approach in development of anti-Alzheimer's disease drugs via the cholinergic hypothesis

A wide range of evidences show that cholinesterase (ChE) inhibitors can interfere with the progression of Alzheimer's disease (AD). The earliest known ChE inhibitors, namely, physostigmine and tacrine, showed modest improvement in the cognitive function of AD patients. However, clinical studies show that physostigmine has poor oral activity, brain penetration and pharmacokinetic parameters while tacrine has hepatotoxic liability. Studies were then focused on finding a new type of acetylcholinesterase (AChE) inhibitor that would overcome the disadvantages of these two compounds. During the study, by chance we found a seed compound. We then conducted a structure-activity relationship (SAR) study of this compound. After four years of exploratory research, we found donepezil hydrochloride (donepezil). Recently, acetylcholinesterase inhibitors (AChEIs) have been studied for other mechanisms of action, such as neuroprotective action and lowering of beta-amyloid (beta-amyloid). Donepezil also reduced beta-amyloid plaque in in vitro. The amyloid hypothesis is believed to be the most promising approach in the development of anti-AD drugs. We speculate the mechanism of lowering beta-amyloid by donepezil implicate alpha-secretase (alpha-secretase) enhancer.     

Effect of anti-dementia drugs on LPS induced neuroinflammation in mice

Inflammation has been recently implicated in pathogenesis of dementia disorders. Effect of anti-dementia (Acetylcholinesterase inhibitor) drugs tacrine, rivastigmine and donepezil were studied on neuroinflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) in mice. Interleukin-2 (IL-2) and isoforms of acetylcholinesterase (AChE) were estimated in different brain areas as marker for neuroinflammation and cholinergic activity respectively. LPS significantly increased the level of IL-2 in all the brain areas while enhancement of AChE activity varied in brain areas. It was found that administration of tacrine, rivastigmine and donepezil in mice significantly attenuated the LPS induced increased levels of IL-2 along with the significant reduction of AChE activity predominantly in salt soluble (SS) fraction as compared to the detergent soluble (DS) fraction in a dose dependent manner. In vitro effect of LPS was also studied in different brain areas. LPS significantly increased the AChE activity in SS fractions but the significant increase was not found in DS fractions. The present study indicate that cholinesterase inhibitor anti-dementia drugs are effective against LPS induced neuroinflammation that may be linked to enhanced cholinergic activity.     

Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity

The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer’s disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC₅₀ = 33.0 µM; 2, IC₅₀ = 8.5 µM) compared to tacrine (CYP1A2 IC₅₀ = 1.5 µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species.     

Interaction of reversible inhibitors with the catalytic centers and allosteric sites of cholinesterases]

The kinetics of inhibition of the human red blood cell cholinesterase with galanthamine tacrine and oxazyl (ambenonium) and the effect of these drugs on chick, mouse, cat and rat blood plasma enzyme activity was studied. Galanthamine proved to bind with acetylcholinesterase in the anionic areas of the catalytic centres, oxazyl interacted in the area of the allosteric anionic site, and tacrin interacted with the hydrophobic areas of the enzyme.     

Conjugates of Tacrine and Its Cyclic Homologues with <Emphasis Type="Italic">p</Emphasis>-Toluenesulfonamide as Novel Acetylcholinesterase and Butyrylcholinesterase Inhibitors

Using the acylation reaction with tosyl chloride of N-aminopropyl analogues of tacrine and its cyclic homologues with different size of the aliphatic cycle (5–8), we synthesized a number of new derivatives of p-toluenesulfonamide. It is shown that the synthesized hybrid compounds of tacrine and p-toluenesulfonamide are effective inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with the preferential inhibition of BChE. They also displace propidium from the peripheral anionic site of the electric eel AChE (Electrophorus electricus). The characteristics of the efficiency and selectivity of cholinesterase inhibition by the test compounds were confirmed by the results of molecular docking.     

Invited Review Cholinesterase inhibitors for Alzheimer’s disease therapy: from tacrine to future applications

This review starts with an historical background of the pharmacological development of tacrine almost fifty years ago (1949). Tacrine is the first drug to be tested, clinically, on a large scale and to be registered (1993) for treatment of Alzheimer’s disease. For the first time, clinical results of four second generation cholinesterase inhibitors (ChEI) (donepezil, ENA 713, eptastigmine and metrifonate) are reviewed and compared with other ChEI such as tacrine, physostigmine and galanthamine. Data based on more than 6000 patients show that second generation drugs are well tolerated and show evidence of clinical efficacy. Differences are mainly due to frequency of side effects, number of drop outs and percentage of improved patients. These results also demonstrate the presence of clinical efficacy for all ChEI tested so far. Clinical mechanism of action, levels of efficacy and differences among various ChEI are discussed. Future potential indications are suggested. The present data indicate that optimization of effects prolongation and maintenance of clinical gains will depend on further knowledge of the compounds pharmacodynamic properties.     

Molecular perception of interactions between bis(7)tacrine and cystamine-tacrine dimer with cholinesterases as the promising proposed agents for the treatment of Alzheimer’s disease

The infamous chronic neurodegenerative disease, Alzheimer’s, that starts with short-term memory loss and eventually leads to gradual bodily function decline which has been attributed to the deficiency in brain neurotransmitters, acetylcholine, and butylcholine. As a matter of fact, design of compounds that can inhibit cholinesterases activities (acetylcholinesterase and butylcholinesterase) has been introduced as an efficient method to treat Alzheimer’s. Among proposed compounds, bis(7)tacrine (B7T) is recognized as a noteworthy suppressor for Alzheimer’s disease. Recently a new analog of B7T, cystamine-tacrine dimer is offered as an agent to detain Alzheimer’s complications, even better than the parent compound. In this study, classical molecular dynamic simulations have been employed to take a closer look into the modes of interactions between the mentioned ligands and both cholinesterase enzymes. According to our obtained results, the structural differences in the target enzymes active sites result in different modes of interactions and inhibition potencies of the ligands against both enzymes. The obtained information can help to investigate those favorable fragments in the studied ligands skeletons that have raised the potency of the analog in comparison with the parent compound to design more potent multi target ligands to heal Alzheimer’s disease.     

Syntheses of 1,2-diamino and 1,2-aminoalcohol derivatives in the piperidine and pyrrolidine series as anti-amnesic agents.

Tacrine, one of the drugs available for Alzheimer's disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine has been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research is a continuation of our efforts in the area of 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) (cf. ref9). A serendipitous discovery led us to the biologically active open chain analogue 9, and we proceeded to elaborate on this molecule. Overall, the compounds we prepared were poor inhibitors of acetylcholinesterase as compared to tacrine. The single exception was compound 20 which exhibited an effect comparable to that of tacrine, but only at a dose in the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 9, this compound was found to be an effective antiamnesic agent.     

Dual functional cholinesterase and PDE4D inhibitors for the treatment of Alzheimer’s disease: Design,synthesis and evaluation of tacrine-pyrazolo[3,4-b]pyridine hybrids

A series of tacrine-pyrazolo[3,4-b]pyridine hybrids were synthesised and evaluated as dual cholinesterase (ChE) and phosphodiesterase 4D (PDE4D) inhibitors for the treatment of Alzheimer’s disease (AD). Compound 10j, which is tacrine linked with pyrazolo[3,4-b]pyridine moiety by a six-carbon spacer, was the most potent acetylcholinesterase (AChE) with IC₅₀ value of 0.125 μM. Moreover, compound 10j provided a desired balance of AChE and butylcholinesterase (BuChE) and PDE4D inhibition activities, with IC₅₀ value of 0.449 and 0.271 μM, respectively. The above results indicated that this hybrid was a promising dual functional agent for the treatment of AD.     

Synthesis and in vitro evaluation of N-alkyl-7-methoxytacrine hydrochlorides as potential cholinesterase inhibitors in Alzheimer disease

All approved drugs for Alzheimer disease (AD) in clinical practice ameliorate the symptoms of the disease. Among them, acetylcholinesterase inhibitors (AChEIs) are used to increase the cholinergic activity. Among new AChEI, tacrine compounds were found to be more toxic compared to 7-MEOTA (9-amino-7-methoxy-1,2,3,4-tetrahydroacridine). In this Letter, series of 7-MEOTA analogues (N-alkyl-7-methoxytacrine) were synthesized. Their inhibitory ability was evaluated on recombinant human acetylcholinesterase (AChE) and plasmatic human butyrylcholinesterase (BChE). Three novel compounds showed promising results towards hAChE better to THA or 7-MEOTA. Three compounds resulted as potent inhibitors of hBChE. The SAR findings highlighted the C₆–C₇ N-alkyl chains for cholinesterase inhibition.     

Syntheses of benzoquinolizidine and benzoindolizidine derivatives as anti-amnesic agents.

Tacrine, one of the drugs available for Alzheimer's disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine have been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research was undertaken to produce an acetylcholinesterase inhibitor by employing 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) as templates. Thus, we aimed to achieve three goals relative to tacrine: eliminate the pyridine and quinoline moieties and render the molecule less flat. Overall, the compounds we prepared were poorer inhibitors of acetylcholinesterase compared to tacrine. The single exception was compound 6f which exhibited an effect comparable to that of tacrine, but only at a dose of the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 6b, this compound proved to be an effective anti-amnesic agent at 45 mg/kg dose.     

Selective blockade of the delayed rectifier potassium current by tacrine in Drosophila

Tetrahydroaminoacridine (tacrine) is an anticholinesterase agent used in the treatment of Alzheimer's disease. Its effectiveness against dementia is attributed to its inhibition of acetylcholine breakdown in the synaptic cleft. Tacrine has also been shown to block ionic currents, including many types of potassium (K⁺) currents, calcium currents, and sodium currents. However, the physiologic significance of this blockade, especially with respect to its effectiveness against Alzheimer's disease, is not clear because of relatively high (several hundred micromolar to millimolar) concentrations of tacrine employed in many studies of channel blockade, and because it blocks several types of currents. A complete mutational and pharmacologic resolution of ionic currents in the larval muscles of Drosophila allowed us to examine the selectivity of tacrine's effects at very low concentrations. At concentrations as low as 10 μM, tacrine selectively blocked the delayed rectifier K⁺ current without affecting the three other K⁺ currents or the calcium channel current in these cells. It also increased the duration of the action potentials significantly. An interesting aspect of tacrine's selectivity is that the current blocked by it is the quinidine-sensitive delayed rectifier K⁺ current rather than the 4-aminopyridine (4-AP)-sensitive transient K⁺ current. This is in contrast to the generally emphasized structural relationship between tacrine and 4-AP. Since tacrine is structurally related to quinidine as well, these observations suggest a structural basis for the selectivity of tacrine, 4-AP, and quinidine for specific K⁺ channels. Furthermore, the data are consistent with the possibility of increased neurotransmitter release, due to prolonged presynaptic action potentials, acting synergistically with the anticholinesterase activity of tacrine to increase its therapeutic effectiveness. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 1–10, 1997.     

The Cognitive Psychopharmacology of Alzheimer's Disease: Focus on Cholinergic Systems

The primary pathology in Alzheimer's disease (DAT) occurs in the basal forebrain cholinergic system (BFCS), which provides the major cholinergic innervation to the neocortex, hippocampus and amygdala. Consistent with the 'cholinergic hypothesis' of dementia in DAT, the most effective treatments so far developed for DAT are drugs which act to boost the functions of the BFCS. These include the centrally acting cholinesterase inhibitor tacrine, and the cholinergic agonist nicotine, acute administration of which leads to an improvement in attentional functions, in line with recent animal studies of the role of the BFCS in cognition. We conclude that future research should include the development of more potent, longer-lasting, less toxic cholinergic agents, which appear to be the best candidates for alleviating the cognitive symptomatology of DAT. Such drugs may also be useful in the treatment of a number of other cognitive disorders, including Lewy body dementia, attention deficit/hyperactivity disorder, and schizophrenia.