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Tacrine was withdrawn from clinical use as a drug against Alzheimer's disease in 2013, mainly due to drug-induced liver injury. The culprit of tacrine-associated hepatotoxicity is believed to be the 7-OH-tacrine metabolite, a possible precursor of quinone methide (Q meth ), which binds to intracellular -SH proteins. In our study, several different animal and human models (liver microsomes, primary hepatocytes, and liver slices) were used to investigate the biotransformation and hepatotoxicity of tacrine and its 7-substituted analogues (7-methoxy-, 7-phenoxy-, and 7-OH-tacrine). Our goal was to find the most appropriate in vitro model for studying tacrine hepatotoxicity and, through rational structure modifications, to develop derivatives of tacrine that are less prone to Q meth formation. Our results show that none of animal models tested accurately mimic human tacrine biotransformation; however, the murine model seems to be more suitable than the rat model. Tacrine metabolism was overall most accurately mimicked in three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs). In this system, tacrine and 7-methoxytacrine were hydroxylated to 7-OH-tacrine, whereas 7-phenoxytacrine formed, as expected, only trace amounts. Surprisingly, however, our study showed that 7-OH-tacrine was the least hepatotoxic (7-OH-tacrine < tacrine < 7-methoxytacrine < 7-phenoxytacrine) even after doses had been adjusted to achieve the same intracellular concentrations. The formation of Q meth -cysteine and Q meth -glutathione adducts after human liver microsome incubation was confirmed by all of the studied tacrine derivatives, but these findings were not confirmed after incubation with 3D PHH spheroids. Therefore, the presented data call into question the suggested previously hypothesized mechanism of toxicity, and the results open new avenues for chemical modifications to improve the safety of novel tacrine derivatives.

The search for tacrine derivatives, as potential Alzheimer´s disease treatment, is still being at the forefront of scientific efforts. 7-MEOTA was found to be a potent, centrally active acetylcholinesterase inhibitor free of the serious side effects observed for tacrine. Unfortunately, a relevant argumentation about pharmacokinetics and potential toxicity is incomplete; information about tacrine derivatives absorption and especially CNS penetration are still rare as well as detailed toxicological profile in vivo . Although the structural changes between these compounds are not so distinctive, differences in plasma profile and CNS targeting were found. The maximum plasma concentration were attained at 18 th min (tacrine; 38.20 ± 3.91 ng/ml and 7-MEOTA; 88.22 ± 15.19 ng/ml) after i.m. application in rats. Although the brain profiles seem to be similar; tacrine achieved 19.34 ± 0.71 ng/ml in 27 min and 7-MEOTA 15.80 ± 1.13 ng/ml in 22 min; the tacrine Kp (AUC brain /AUC plasma ) fit 1.20 and was significantly higher than 7-MEOTA Kp 0.10. Administration of tacrine and 7-MEOTA showed only mild elevation of some biochemical markers following single p.o. application in 24 hours and 7 days. Also histopathology revealed only mild-to-moderate changes following repeated p.o. administration for 14 days. It seems that small change in tacrine molecule leads to lower ability to penetrate through the biological barriers. The explanation that lower p.o. acute toxicity of 7-MEOTA depends only on differences in metabolic pathways may be now revised to newly described differences in pharmacokinetic and toxicological profiles.

Parkinson's disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons and treated with the dopamine precursor, 3,4-dihydroxy-l-phenylalanine (L-DOPA). Prolonged L-DOPA treatment is however associated with waning efficacy and the induction of L-DOPA induced dyskinesia (LID). GPR88 is an orphan G-protein Coupled Receptor (GPCR) expressed in dopaminoceptive striatal medium spiny neurons (MSNs) and their afferent corticostriatal glutamatergic neurons. Here, we studied the role of GPR88 in experimental parkinsonism and LID. Chronic L-DOPA administration to male GPR88 KO mice, subjected to unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle, resulted in more rotations than in their WT counterparts. Conversely, GPR88 KO mice had a lower abnormal involuntary movements (AIMs) score. These behavioral responses were accompanied by altered transcription of L-DOPA upregulated genes in lesioned GPR88 KO compared to WT striata. In accordance with a role for serotonin neurons in LID development, WT but not GPR88 KO striata exhibited 5-hydroxytryptamine displacement upon repeated L-DOPA treatment. Intact male GPR88 KO mice showed diminished tacrine-induced PD-like tremor and spontaneous hyperlocomotion. Dopamine and its metabolites were not increased in male GPR88 KO mice, but biosensor recordings revealed increased spontaneous/basal and evoked glutamate release in striata of male GPR88 KO mice. In conclusion, genetic deletion of GPR88 promotes l-DOPA-induced rotation and spontaneous locomotion yet suppresses the induction of LIDs and also reduces tremor. These data provide behavioral, neurochemical and molecular support that GPR88 antagonism may favour motor relief in PD patients without aggravating the induction of motor side effects., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The present study examines the effect of three adenosine receptor antagonists on tremulous jaw movements (TJMs), an animal model of tremor. Forty-five rats were pre-treated with one adenosine antagonist: caffeine (0.0, 5.0, or 10.0 mg/kg; non-selective adenosine receptor antagonist), 8-cyclopentyltheophylline (CPT; 0.0, 5.0, or 10.0 mg/kg; selective adenosine A 1 receptor antagonist), or SCH 58261 (0.0 or 8.0 mg/kg; selective adenosine A 2A receptor antagonist) followed by TJM induction with tacrine (0.0, 0.75, or 2.5 mg/kg; acetylcholinesterase inhibitor). CPT and SCH 58261 both significantly reduced TJMs while caffeine did not. Unexpectedly, both SCH 58261 and CPT reduced TJMs even in the absence of tacrine. Also, CPT showed a robust reduction of TJMs, achieved at both (5.0 mg/kg) and (10.0 mg/kg) doses and regardless of tacrine dose. In conclusion, this study shows adenosine receptor antagonism to generally suppress low-dose tacrine-induced TJMs. In concert with two prior studies, these results are suggestive of behavioral evidence for a biphasic effect of adenosine A 2A receptor antagonists (caffeine and SCH 58261) that is modulated by tacrine, and a model of this effect is proposed., (Published by Elsevier B.V.)

A novel series of tacrine-bifendate (THA-DDB) conjugates (7a-e) were synthesized and evaluated as potential anti-Alzheimer's agents. These compounds showed potent cholinesterase and self-induced β-amyloid (Aβ) aggregation inhibitory activities. A Lineweaver-Burk plot and molecular modeling study showed that these compounds can target both catalytic active site (CAS) and peripheral anionic site (PAS) of acetylcholinesterase (AChE). The cytotoxicity of the conjugate 7d against PC12 and HepG2 cells and hepatotoxicity against human hepatocyte cell line (HL-7702) were found to be considerably less compared to THA. Moreover, treatment with 7d did not exhibit significant hepatotoxicity in mice. Finally, in vivo studies confirmed that 7d significantly ameliorates the cognitive performances of scopolamine-treated ICR mice. Therefore, 7d has high potential for the treatment of Alzheimer's disease and warrants further investigation., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: 6-chlorotacrine is a cholinesterase inhibitor showing good inhibitory potential, even better than parent compound tacrine, in vitro. Despite tacrine scaffold is broadly used for design and synthesis of novel compounds with anti-Alzheimer's potential, no in vivo effects have been investigated so far. Thus, basic toxicological and behavioural evaluation has been carried out throughout this study., Methods: Maximum tolerated dose (MTD) and median lethal dose (LD50) were assessed in BALB/c mice and Wistar rats. Behavioural effects were observed in rats performing the multiple T-maze test, the water maze test and the step-through passive avoidance test. All outcomes were compared with the effects of parent compound - tacrine., Results: The toxicity of 6-chlorotacrine was increased compared to tacrine with MTD 6.0/5.0 mg.kg-1 (i.m., male/female mice), 6.0/5.0 mg.kg-1 (i.p., male/female rats) and LD50 9.0 mg.kg-1 (male rats). At MTD doses, no histopathological changes and blood biochemistry abnormalities were observed except decreased plasma creatinine levels. 6-chlorotacrine showed good effects in the reversal of quinuclidinyl benzilate-induced amnesia. Best results were achieved at the dose of 1.8 mg.kg-1 (20% LD50) in the water maze test; the pro-cognitive effect was stronger than that of tacrine (5.2 mg.kg-1, 20% LD50). Other doses tested (0.9 mg.kg-1 and 2.7 mg.kg-1) showed similar effects as tacrine in the water maze, multiple T-maze and passive avoidance test., Conclusion: Observed effects predetermined 6-chlorotacrine as a potent parent compound for the synthesis of novel multifactorial drugs intended to the treatment of Alzheimer's disease. Even though 6- chlorotacrine showed in vivo beneficial effect with no signs of toxicity, further tests on the field of biochemistry and pharmacology are essential to disclose the exact mechanism of action, safety evaluation and the metabolic fate of the compound after the repeated administration., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The gut microbiota possesses diverse metabolic activities, but its contribution toward heterogeneous toxicological responses is poorly understood. In this study, we investigated the role of the liver-gut microbiota axis in underpinning the hepatotoxicity of tacrine. We employed an integrated strategy combining pharmacokinetics, toxicology, metabonomics, genomics, and metagenomics to elucidate and validate the mechanism of tacrine-induced hepatotoxicity in Lister hooded rats. Pharmacokinetic studies in rats demonstrated 3.3-fold higher systemic exposure to tacrine in strong responders that experienced transaminitis, revealing enhanced enterohepatic recycling of deglucuronidated tacrine in this subgroup, not attributable to variation in hepatic disposition gene expression. Metabonomic studies implicated variations in gut microbial activities that mapped onto tacrine-induced transaminitis. Metagenomics delineated greater deglucuronidation capabilities in strong responders, based on differential gut microbial composition (e.g., Lactobacillus, Bacteroides, and Enterobacteriaceae) and approximately 9% higher β-glucuronidase gene abundance compared with nonresponders. In the validation study, coadministration with oral β-glucuronidase derived from Escherichia coli and pretreatment with vancomycin and imipenem significantly modulated the susceptibility to tacrine-induced transaminitis in vivo., Conclusion: This study establishes pertinent gut microbial influences in modifying the hepatotoxicity of tacrine, providing insights for personalized medicine initiatives. (Hepatology 2018;67:282-295)., (© 2017 by the American Association for the Study of Liver Diseases.)

Background: The heterogeneity of Alzheimer disease requires the development of multitarget drugs for treating the symptoms of the disease and its progression. Both cholinergic and monoamine oxidase dysfunctions are involved in the pathological process. Thus, we hypothesized that the development of therapies focused on these targets might be effective. We have developed and assessed a new product, coded ASS234, a multipotent acetyl and butyrylcholinesterase/monoamine oxidase A-B inhibitor with a potent inhibitory effect on amyloid-β aggregation as well as antioxidant and antiapoptotic properties. But there is a need to reliably correlate in vitro and in vivo drug release data., Methods: We examined the effect of ASS234 on cognition in healthy adult C57BL/6J mice in a model of scopolamine-induced cognitive impairment that often accompanies normal and pathological aging. Also, in a characterized transgenic APPswe/PS1ΔE9 mouse model of Alzheimer disease, we examined the effects of short-term ASS234 treatment on plaque deposition and gliosis using immunohistochemistry. Toxicology of ASS234 was assessed using a quantitative high-throughput in vitro cytotoxicity screening assay following the MTT assay method in HepG2 liver cells., Results: In vivo, ASS234 significantly decreased scopolamine-induced learning deficits in C57BL/6J mice. Also, reduction of amyloid plaque burden and gliosis in the cortex and hippocampus was assessed. In vitro, ASS234 exhibited lesser toxicity than donepezil and tacrine., Limitations: The study was conducted in male mice only. Although the Alzheimer disease model does not recapitulate all features of the human disease, it exhibits progressive monoaminergic neurodegeneration., Conclusion: ASS234 is a promising alternative drug of choice to treat the cognitive decline and neurodegeneration underlying Alzheimer disease.

Novel indolotacrine analogues were designed, synthesized, and evaluated as potential drugs for the treatment of Alzheimer's disease. By using a multitarget-directed ligand approach, compounds were designed to act simultaneously as cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors. The compounds were also evaluated for antioxidant, cytotoxic, hepatotoxic, and blood-brain barrier (BBB) permeability properties. Indolotacrine 9 b (9-methoxy-2,3,4,6-tetrahydro-1H-indolo[2,3-b]quinolin-11-amine) showed the most promising results in the in vitro assessment; it is a potent inhibitor of acetylcholinesterase (AChE IC50 : 1.5 μm), butyrylcholinesterase (BChE IC50 : 2.4 μm) and MAO A (IC50 : 0.49 μm), and it is also a weak inhibitor of MAO B (IC50 : 53.9 μm). Although its cytotoxic (IC50 : 5.5±0.4 μm) and hepatotoxic (IC50 : 1.22±0.11 μm) profiles are not as good as those of the standard 7-methoxytacrine (IC50 : 63±4 and 11.50±0.77 μm, respectively), the overall improvement in the inhibitory activities and potential to cross the BBB make indolotacrine 9 b a promising lead compound for further development and investigation., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Coupling of two distinct pharmacophores, tacrine and trolox, endowed with different biological properties, afforded 21 hybrid compounds as novel multifunctional candidates against Alzheimer's disease. Several of them showed improved inhibitory properties toward acetylcholinesterase (AChE) in relation to tacrine. These hybrids also scavenged free radicals. Molecular modeling studies in tandem with kinetic analysis exhibited that these hybrids target both catalytic active site as well as peripheral anionic site of AChE. In addition, incorporation of the moiety bearing antioxidant abilities displayed negligible toxicity on human hepatic cells. This striking effect was explained by formation of nontoxic metabolites after 1 h incubation in human liver microsomes system. Finally, tacrine-trolox hybrids exhibited low in vivo toxicity after im administration in rats and potential to penetrate across blood-brain barrier. All of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7u as the lead structure worthy of further investigation.

Objectives: Fucosterol is the primary sterol found in brown algae. Recently, considerable interest has been generated regarding fucosterol due to its potential antioxidant, anti-inflammatory and antidiabetic effects. The aim of this study was to investigate the protective effects of fucosterol on tert-butyl hydroperoxide (t-BHP)- and tacrine-induced oxidative stress in HepG2 cells., Methods: Fucosterol by itself exhibited no cytotoxicity at concentrations below 100 μm by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The increased intracellular reactive oxygen species (ROS) and decreased glutathione levels observed in t-BHP- and tacrine-treated HepG2 cells were ameliorated by fucosterol pretreatment, indicating that the protective effects of fucosterol are mediated by the induction of cellular defence mechanisms against oxidative stress. Moreover, elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in tacrine-treated mice were significantly reduced after oral administration of fucosterol., Key Findings: The hepatoprotective effects of fucosterol may occur via an increase in the hepatic level of glutathione and a decrease in ROS production, thereby preventing hepatic damage and the resultant increases in ALT and AST activity., Conclusion: These results suggest that fucosterol may be an effective hepatoprotective agent that could be useful for preventive therapies against oxidative stress-related hepatotoxicity., (© 2015 Royal Pharmaceutical Society.)

Previous work by our laboratory has shown that tacrine can produce long-lasting reductions in cocaine-reinforced behavior, when administered to rats as daily intravenous infusions over four days. Tacrine causes dose-related liver toxicity in different species, and its manufacture for human use was recently discontinued. This study was conducted to further characterize its actions on cocaine reward. Cocaine-experienced animals that had no contact with drug over one week resumed self-administration at levels similar to their initial baseline. When tacrine was administered over four days which were preceded and followed by washout periods to allow elimination of cocaine and tacrine respectively, subsequent cocaine self-administration was attenuated by more than one-half. Tacrine administered at 10 mg/kg-day as a chronic infusion by osmotic pump did not modify cocaine-induced increases in locomotor activity or conditioned-place preference. In rats that exhibited persistent attenuation of cocaine-self-administration after receiving tacrine, cocaine-induced reinstatement was also attenuated. No changes in plasma measures of renal or hepatic function were observed in rats receiving tacrine. In conclusion, pretreatment with tacrine can decrease cocaine-motivated behavior measured by self-administration or reinstatement, but not conditioned-place preference. Reductions in cocaine self-administration following pretreatment with tacrine do not require direct interaction with cocaine and are not secondary to either liver or kidney toxicity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The effect of ethanolic extract of Coriandrum sativum L. seeds (100, 200 mg/kg) was studied on tacrine induced orofacial dyskinesia. Tacrine (2.5 mg/kg, i.p.) treated animals were observed for vacuous chewing movements (VCM), tongue protrusions (TP) and orofacial bursts (OB) for 1 h followed by observations for locomotor changes and cognitive dysfunction. Sub-chronic administration of Coriandrum sativum L. seed extract (E-CS) (100, 200 mg/kg, p.o., for 15 days significantly (P < 0.05) decreased the tacrine induced VCM, TP and OB; and also significantly (P < 0.05), increased locomotion and cognition compared to the tacrine treated group. Biochemical analysis revealed that tacrine administration significantly (P < 0.05) decreased the levels of superoxide dismutase (SOD), Catalase (CAT), glutathione reductase (GSH) levels and also significantly (P < 0.05) increased lipid peroxidation (LPO) as an index of oxidative stress, whereas subchronic administration of E-CS significantly (P < 0.05) improved the antioxidant enzyme (i.e. SOD, CAT, and GSH) levels and also significantly (P < 0.05) decreased lipid peroxidation (LPO). The results have demonstrated the protective role of ethanolic extract of Coriandrum sativum. L against tacrine induced orofacial dyskinesia.

Although the cholinesterase inhibitor tacrine has been successfully used for the treatment of Alzheimer's disease, it is known to have hepatotoxic effects. Liquiritigenin (LQ), an active flavonoid in Glycyrrhizae radix, exerts protective effects against liver damage. This study investigated the toxic effect of tacrine on hepatocytes and the beneficial effect of LQ on tacrine intoxication in vivo and in vitro, and the underlying mechanism involved. In hepatocyte cell lines, tacrine induced cell death and oxidative stress, as indicated by decreases in cell viability and glutathione (GSH) contents, which were blocked by pretreatment with LQ. Fluorescent activated cell sorter (FACS) analysis revealed that LQ inhibited cellular H2O2 production and mitochondrial dysfunction induced by tacrine in HepG2 cells. Furthermore, LQ promoted inhibitory phosphorylation of glycogen synthase kinase-3β (GSK3β) and prevented decreases in GSK3β phosphorylation induced by tacrine. In rats treatment with tacrine at 30 mg/kg increased hepatic damage as assessed by blood biochemistry and histopathology. Administration of LQ (10 or 30 mg/kg/d, per os (p.o.)) or the hepatoprotective drug sylimarin (100 mg/kg/d) for 3 d inhibited elevations in alanine aminotransferase, aspartate aminotransferase, and histological changes induced by tacrine. These results show that LQ efficaciously protects the rat liver against tacrine-induced liver damage, and suggest that LQ is a therapeutic candidate for ameliorating the hepatotoxic effects of tacrine.

This paper describes our preliminary results on the ADMET, synthesis, biochemical evaluation, and molecular modeling of racemic HuperTacrines (HT), new hybrids resulting from the juxtaposition of huperzine A and tacrine for the potential treatment of Alzheimer's disease (AD). The synthesis of these HT was executed by Friedländer-type reactions of 2-amino-6-oxo-1,6-dihydropyridine-3-carbonitriles, or 7-amino-2-oxo-1,2,3,4-tetrahydro-1,6-naphthyridine- 8-carbonitriles, with cyclohexanone. In the biochemical evaluation, initial and particular attention was devoted to test their toxicity on human hepatoma cells, followed by the in vitro inhibition of human cholinesterases (hAChE, and hBuChE), and the kinetics/mechanism of the inhibition of the most potent HT; simultaneous molecular modeling on the best HT provided the key binding interactions with the human cholinesterases. >From these analyses, (±)-5-amino-3-methyl- 3,4,6,7,8,9-hexahydrobenzo[b][1,8]naphthyridin-2(1H)-one (HT1) and (±)-5-amino-3-(2,6-dichlorophenyl)-3,4,6,7,8,9- hexahydrobenzo[b][1,8]naphthyridin-2(1H)-one (HT3) have emerged as characterized by extremely low liver toxicity reversible mixed-type, selective hAChE and, quite selective irreversible hBuChEIs, respectively, showing also good druglike properties for AD-targeted drugs.

Tacrine (THA), as the first approved acetylcholinesterase (AChE) inhibitors for the treatment of Alzheimer's disease (AD), has been extensively investigated in last seven decades. After dimerization of THA via a 7-carbon alkyl spacer, bis(7)-tacrine (B7T) showed much potent anti-AChE activity than THA. We here report synthesis, biological evaluation and biopharmaceutical characterization of six THA dimers referable to B7T. According to IC50 values, the in vitro anti-AChE activities of THA dimers were up to 300-fold more potent and 200-fold more selective than that of THA. In addition, the anti-AChE activities of THA dimers were found to be associated with the type and length of the linkage. All studied THA dimers showed much lower cytotoxicity than B7T, but like B7T, they demonstrated much lower absorptive permeabilities than that of THA on Caco-2 monolayer model. In addition, all THA dimers demonstrated significant efflux transport (efflux ratio >4), indicating that the limited permeability could be associated with the efflux transport during absorption process. Moreover, the dimer with higher Log P value was accompanied with higher permeability but lower aqueous solubility. A balanced consideration of activity, solubility, cytotoxicity and permeability should be conducted in selection of the potential candidates for further in vivo investigation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Tacrine (THA) is a competitive inhibitor of cholinesterase. Administration of THA for the treatment of Alzheimer's disease results in a reversible hepatotoxicity in 30-50% of patients, as indicated by elevated alanine aminotransferase levels. However, the intracellular mechanisms have not yet been elucidated. In our previous study, we found that THA induced cytotoxicity and mitochondria dysfunction by ROS generation and 8-OHdG formation in mitochondrial DNA in HepG2 cells. In this study, the mechanism underlying was further investigated. Our results demonstrated that THA induced dose-dependent apoptosis with cytochrome c release and activation of caspase-3. THA-induced apoptosis was inhibited by treating cells with a ROS inhibitor, YCG063. In addition, we observed that THA led to an early lysosomal membrane permeabilization and release of cathepsin B. Pretreatment with CA-074Me, a specific cathepsin B inhibitor resulted in a significant but not complete decrease in tacrine-induced apoptosis. These data suggest that tacrine-induced cell apoptosis involves both mitochondrial damage and lysosomal membrane destabilization, and ROS is the critical factor that integrates tacrine-induced mitochondrial and lysosomal death pathways., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The pharmacological analysis of racemic chromenotacrines (CT) 1-7, bearing the 11-amino-12-aryl-8,9,10,12-tetrahydro-7H-chromeno[2,3-b]quinolin-3-ol ring skeleton, in a series of experiments targeted to explore their potential use for the treatment of Alzheimer's disease (AD), is reported. The toxicological evaluation showed that among all these chromenotacrines, CT6 is much less hepatotoxic than tacrine in a range of concentrations from 1 to 300 μM, measured as cell viability in HepG2 cells. Moreover, CT6 did not significantly increase lactate dehydrogenase, aspartate transaminase, and alanine transaminase release in HepG2 cells. Besides, CT6 treatment exerts a high protective effect against the lipid peroxidation induced after H₂O₂-treated SH-SY5Y cells, in a concentration-dependent manner. CT6 showed an excellent antioxidant profile in the AAPH test, and protects against the decrease in cell viability induced by respiratory chain inhibitors (Oligomicyn A/Rotenone) and NO donors in neuronal cultures. This effect could be due to a mixed antiapoptotic and antinecrotic neuroprotective effect at low and intermediate CT6 concentrations, respectively. CT1-7 are potent and selective inhibitors of EeAChE in the submicromolar range. CT3 [IC₅₀ (EeAChE) = 0.007 ± 0.003 μM], and CT6 [IC₅₀ (EeAChE) = 0.041 ± 0.001 μM] are the most potent AChE inhibitors. Kinetic studies on the non-toxic chromenotacrine CT6 showed that this compound behaves as a non-competitive inhibitor (Ki = 0.047 ± 0.003 μM), indicating that CT6 binds at the peripheral anionic site, a fact confirmed by molecular modeling analysis. In silico ADMET analysis showed also that CT6 should have a moderate BBB permeability. Consequently, non-toxic chromenotacrine CT6 can be considered as an attractive multipotent molecule for the potential treatment of AD., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Long-term therapy with L-3,4-dihydroxyphenylalanine (L-DOPA), still the most effective treatment in Parkinson's disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating L-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1-D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A-CB1-D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A-CB1-D2 receptor heteromers in the striatum of both naïve and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by L-DOPA treatment. Radioligand binding data showed that A2A-CB1-D2 receptor heteromers are present in the striatum of both naïve and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of L-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in L-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with L-DOPA., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A series of tacrine-rhein hybrid compounds have been designed and synthesized as novel multifunctional potent ChE inhibitors. Most of the compounds inhibited ChEs in the nanomolar range in vitro effectively. Compound 10b was one of the most potent inhibitors and was 5-fold more active than tacrine toward AChE, and it also showed a moderate BuChE inhibition with an IC50 value of 200 nM. Kinetic and molecular modeling studies of 10b also indicated that it was a mixed-type inhibitor binding simultaneously to the active and peripheral sites of AChE. In inhibition of the AChE-induced Aβ aggregation assay, compound 10b (70.2% at 100 μM) showed the greatest inhibitory activity. In addition, 10b showed metal-chelating property and low hepatotoxicity. These results suggested that 10b might be an excellent multifunctional agent for AD treatment.

A series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster, which express AChE-1 and AChE-2 isoforms, respectively. In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE) than in B. germanica AChE (BgAChE). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about twofold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment and in silico homology modeling suggest that aromatic residues of DmAChE confer higher affinity binding, and the lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro assay data. Topical and injection assays in cockroaches found minimal toxicity of bis(n)-tacrines. Electrophysiological studies on D. melanogaster central nervous system showed that dimeric tacrines do not readily cross the blood brain barrier, explaining the observed nonlethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect, pests, and disease vectors., (© 2013 Wiley Periodicals, Inc.)

Computer simulations constitute the basis of the design and discovery of new drugs. This approach is not only significant with regards to finding new structures, but also for selecting the molecules with the highest probability of being useful in the diagnostic process and treatment of numerous diseases. In our work, we used computational software to analyze 32 new acetylcholinesterase (AChE) inhibitors and formulate ADMET predictions. To understand the influence of the structure of our derivatives on binding mode, we docked all structures to the active site of AChE and assigned some pharmacophoric features. Finally, we undertook a chemometric analysis of all the compounds on the basis of FT-IR, which gave us the possibility of performing a fast categorization of the analyzed compounds and design compounds with similar structures.

Tacrine was evaluated for its mutagenic and clastogenic activities using the Ames bacterial reverse-mutation assay and the rodent bone marrow micronucleus assay. Tacrine was tested for mutagenic potential at six different concentrations, with 1,250 µg/plate as the highest concentration, followed by five lower concentrations with 2-fold spacing. In clastogenic evaluation, tacrine was administered orally to Wistar rats for 2 days at 5, 10, and 20 mg/kg body weights to assess micronucleus induction in bone marrow erythrocytes. In the Ames assay, tacrine showed nonmutagenicity in four tester strains of Salmonella typhimurium viz. TA98, TA100, TA102, and TA1535, but it was found to be mutagenic in the TA1537 tester strain, both in the presence and absence of a metabolic activation system. Tacrine was found to be nonclastogenic on bone marrow cells of rats at all doses tested and was found to be mutagenic in only the TA1537 strain of Salmonella.

A codrug of the anti-Alzheimer drug tacrine and the natural product silibinin was synthesized. The codrug's biological and pharmacological properties were compared to an equimolar mixture of the components. The compound showed potent acetyl- and butyrylcholinesterase inhibition. In a cellular hepatotoxicity model, analyzing the influence on viability and mitochondria of hepatic stellate cells (HSC), the toxicity of the codrug was markedly reduced in comparison to that of tacrine. Using a neuronal cell line (HT-22), a neuroprotective effect against glutamate-induced toxicity could be observed that was absent for the 1:1 mixture of components. In subsequent in vivo experiments in rats, in contrast to the effects seen after tacrine treatment, after administration of the codrug no hepatotoxicity and no induction of the cytochrome P450 system were noticed. In a scopolamine-induced cognitive impairment model using Wistar rats, the codrug was as potent as tacrine in reversing memory dysfunction. The tacrine-silibinin codrug shows high AChE and BChE inhibition, neuroprotective effects, lacks tacrine's hepatotoxicity in vitro and in vivo, and shows the same pro-cognitive effects in vivo as tacrine, being superior to the physical mixture of tacrine and silibinin in all these regards.

Objectives: Tacrine was the first acetylcholinesterase inhibitor approved for the treatment of Alzheimer disease. The compound is not available for therapeutic purposes as it was withdrawn due to hepatotoxicity of its metabolites. The hepatotoxicity can be decreased by alternative ways of drug administration avoiding thus the first pass effect. The present study is aimed to investigate the influence of intramuscularly administrated tacrine on oxidative stress., Methods: Laboratory guinea pigs were exposed to tacrine at doses of 0-800 μg/kg. The animals were euthanized 1 and 24 hours after the exposure. Parameters such as ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS), carbonylated proteins, caspase 3 activity, superoxide dismutase activity and glutathione reductase activity were assessed in the frontal, temporal and occipital lobe, cerebellum, liver, spleen, heart, and kidney. Moreover, levels of glucose, total and HDL cholesterol forms, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, albumin and activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase were assessed in plasma samples., Results: Activities of the enzymatic markers, level of carbonylated proteins in organs and levels of biochemical markers in plasma were only slightly influenced by tacrine. Dose-dependent elevation of the FRAP value was recognized in the brain tissues and the liver. The TBARS value was increased in the kidney and heart 1 and 24 hours, respectively, after exposure., Conclusion: In the study, the effect of tacrine on markers of oxidative stress was proved. Possible positive effects of tacrine on the antioxidant defence in the brain tissue were discussed.

It is well established that cholinergic over-stimulation can interfere with memory processes. The aim of this study was to evaluate the effect of tacrine, an acetylcholinesterase inhibitor, on recognition memory as well as the associated hepatotoxicity in juvenile (20-day-old) and adult (100-day-old) ICR male mice. Recognition memory was assessed by open-field test and step-through task without footshocks for three sessions between 08:00 and 13:00, with a 24-hr retention interval. Tacrine (10 or 40 μmol/kg) or vehicle was administered (s.c.) 20 min. prior to the first session. During the acquisition session, tacrine suppressed the open-field behaviours, including locomotor activity, rearing, grooming and defecation (by 77-100%) in mice of both ages. During the recall (observable in both ages) and re-recall (observable in juvenile mice) session, the locomotor activity and rearing number were significantly increased, indicative of impairment in recognition memory, in mice treated with tacrine 40 μmol/kg. During the training trial, tacrine decreased the step-through number in mice of both ages. In contrast, during the retention and re-retention trials, the step-through number was increased (by 92% and 93%, respectively), indicative of impairment in step-through memory, in juvenile but not adult mice treated with tacrine 40 μmol/kg. Tacrine 40 μmol/kg elevated the serum alanine aminotransferase (ALT) activity (by 135%) in juvenile mice, but reduced the ALT activity (by 42%) in adult mice. The results indicated that 20-day-old mice seemed to be more sensitive than 100-day-old mice to tacrine-induced impairment in recognition memory and the associated liver damage., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

The purpose of this investigation was to explore the potentiality of a novel animal model to be used for the in vivo evaluation of the ability of a drug delivery system to promote the passage through the blood-brain barrier (BBB) and/or to improve the brain localization of a bioactive compound. A Tween 80-coated poly-L-lactid acid nanoparticles was used as a model of colloidal drug delivery system, able to trespass the BBB. Tacrine, administered in LiCl pre-treated rats, induces electrocorticographic seizures and delayed hippocampal damage. The toxic effects of tacrine-loaded poly-L-lactid acid nanoparticles (5mg/kg), a saline solution of tacrine (5mg/kg) and an empty colloidal nanoparticle suspension were compared following i.p. administration in LiCl-pre-treated Wistar rats. All the animals treated with tacrine-loaded nanoparticles showed an earlier outcome of CNS adverse symptoms, i.e. epileptic onset, with respect to those animals treated with the free compound (10 min vs. 22 min respectively). In addition, tacrine-loaded nanoparticles administration induced damage of neuronal cells in CA1 field of the hippocampus in all treated animals, while the saline solution of tacrine only in 60% of animals. Empty nanoparticles provided similar results to control (saline-treated) group of animals. In conclusion, the evaluation of time-to-onset of symptoms and the severity of neurodegenerative processes induced by the tacrine-lithium model of epilepsy in the rat, could be used to evaluate preliminarily the capability of a drug delivery system to trespass (or not) the BBB in vivo., ((c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Tacrine (CAS 321-64-2) is a reversible acetylcholine esterase inhibitor that, despite exerting beneficial effects on Alzheimer's disease (AD), displays marked hepatotoxicity. Searching for safer drugs and taking into account the multi-pathogenesis of AD, two tacrine-NO donor hybrid molecules (FL16, FL38) as well as a tacrine-ferulic acid hybrid (FL67) were synthesized. NO donors coupled to the tacrine moiety may exert an additional beneficial effect on AD via an increased blood supply to the brain and by reducing inflammation. Ferulic acid is an antioxidant. To investigate the hepatotoxicity and effects on the cytochrome P450 (CYP) system of the liver, female rats were treated with the highest tolerated dose of tacrine or equimolar doses of the novel compounds 24 or 36 h, respectively, before sacrifice. Tacrine caused pericentral necrosis and fatty degeneration of the hepatocytes, loss of liver glycogen and (indicating oxidative stress) induction of heme oxygenase (HO)-1. No histopathological changes were observed with the hybrids, but a glycogen deficit and an elevation of HO-1 was noticed after FL38 or FL67 treatment. Both tacrine and the hybrids, but mainly FL38 and FL67, caused an induction of CYP1A1, 2B1 and 3A2 expression. CYP activity was noticeably increased after treatment with FL38 and FL67 only. Thus, since it displays much less hepatotoxicity and interaction potential at the CYP system than tacrine and the other hybrids, FL16 is a good candidate for further investigations.

A new phenolic amide, tribulusimide D (4-hydroxy-N-[3-(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-1-yl]-3-methoxybenzamide) (1), together with a known phenolic amide, terrestriamide ((E)-3-(4-hydroxy-3-methoxyphenyl)-N-[2-(4-hydroxyphenyl)-2-oxoethyl]-prop-2-enamide) (2) and a flavonol glycoside, quercetin-3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (3) were isolated from the H2O extract of Tribuli Fructus. Compounds 1 and 3 showed significant hepatoprotective activities, with EC50 values of 13.46 +/- 0.2 and 7.06 +/- 0.7 microM, respectively, against tacrine-induced cytotoxicity in HepG2 cells.

Bioassay-guided fractionation of the MeOH extract of Suaeda glauca yielded four phenolic compounds, methyl 3,5-di-O-caffeoyl quinate (1) and 3,5-di-O-caffeoyl quinic acid (2), isorhamnetin 3-O-beta-D-galactoside (3), and quercetin 3-O-beta-D-galactoside (4). Compounds 1 and 2 were hepatoprotective against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells with the EC(50) values of 72.7+/-6.2 and 117.2+/-10.5 microM, respectively. Silybin as a positive control showed an EC(50) value of 82.4+/-4.1 microM.

Tacrine belongs to the group of acetylcholinesterse (AChE) inhibitors used as drugs for treatment of Alzheimer's disease (AD). The formation of hydroxyderivatives of tacrine is well-established step in the metabolization of this drug in liver by microsomal cytochrome P450 enzymes family. Genetic polymorphism of cytochrome P450 enzymes is probably responsible for balance between a number of stable and non-toxic metabolites and highly protein-reactive and toxic ones. By this manner may be explained why the hepatotoxicity of tacrine was observed only in the part of persons and why not every patient with AD responds to the treatment by this drug.

In search of safer anti-Alzheimer drugs, 14 NO-donor-tacrine hybrids (1- 14) were synthesized and evaluated for their ability to inhibit cholinesterases and for vasorelaxation effects. Compounds 1- 13 showed good cholinesterases inhibitory activities in vitro, while 14, particularly, was highly selective, preferring butyrylcholinesterase rather than acetylcholinesterase. Four selected compounds (1, 9, 11, and 14) moderately relaxed the porcine pulmonary arteries in organ bath. In the hepatotoxicity study, significant hepatotoxicity was caused by tacrine but not by 9.

Objects: To study the effect of zonisamide on experimental tremors in rats., Methods: Effect of zonisamide on harmaline- or oxotreorine-induced tremors, and tacrine-induced tremulous jaw movements (TJMs) was studied., Results: Zonisamide significantly suppressed both harmaline- and oxotremorine-induced tremors dose-dependently. Zonisamide also significantly suppressed tacrine-induced TJMs, and this effect was not lost under conditions of monoamine-depletion or dopaminergic blockade., Conclusion: The anti-tremor effects of zonisamide may be achieved by a non-dopaminergic mechanism. Since it effectively suppressed tremors that are based on different kinds of tremors, we propose a novel perspective of clinical potential of zonisamide as a non-specific, anti-tremor drug.

Cirsium arisanense Kitamura (Compositae) has been used for hundreds of years in Taiwan as a folk medicine for hepatoprotection. However, no scientific research has demonstrated this effect. In the present study, we extracted the phenol-containing aqueous components of C. arisanense roots (CaR) and leaves/stem (CaL), and then assessed their hepatoprotective activities in both human hepatocellular carcinoma Hep 3B cells and C57BL/6 mice strain. High performance liquid chromatography (HPLC) analysis revealed that the components of CaR and CaL differed from those of the positive control silymarin. CaR exhibited a higher phenolic content and antioxidant capacity than CaL. Hep 3B cells treated with silymarin (0-200 microg/ml) demonstrated a concentration-dependent decrease in viability; however, both CaR and CaL did not exhibit any apparent cytotoxicity. Silymarin at 100 microg/ml, as well as CaR and CaL, not only protect Hep 3B cells from tacrine-induced hepatotoxicity but also decrease the expression of hepatitis B surface antigen (HBsAg). Moreover, an animal experiment demonstrated that CaR, CaL, and silymarin have hepatoprotective effects in C57BL/6 mice injected with tacrine, and they significantly decrease the levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST). These effects of CaR and silymarin, but not of CaL, may occur via an increase in the hepatic glutathione level and the elimination of the nitric oxide production. In conclusion, the phenol-containing aqueous components from C. arisanense have potential in hepatoprotection.

Background: Tacrine, an anticholinesterase used to treat Alzheimer's disease (AD), leads to an increase in serum alanine aminotransferase (ALT) levels. The factors determining individual susceptibility are largely unknown. The purpose of this study was to investigate genetic predisposition., Methods: Rats were administered single dose tacrine (3-40 mg/kg). After 6 and 24 h, hepatic gene expression was determined using the affymetrix rat U34A microarray. On the basis of the gene expression data, the IL6 gene was identified as a potential candidate for tacrine transaminitis susceptibility. Sixty-nine patients with AD on tacrine with or without transaminitis were genotyped for 17 IL6 polymorphisms., Results: Serum aspartate aminotransferase levels in rats increased after tacrine (40 mg/kg) administration. Forty-six and 29 genes showed significant upregulation at 6 and 24 h, respectively, after administration, including the IL-6-regulated acute phase response genes alpha2-macroglobulin, fibronectin and haptoglobin. Five of the 17 IL6 polymorphisms studied in AD patients showed an association (P<0.05) with transaminitis [ALT>2 x upper limit of normal (ULN)]. An association existed between maximum ALT and area under curve for ALT over 15 weeks and an intronic polymorphism (P<0.01) and a 3'-variable nucleotide tandem repeat (P<0.05). Multilocus haplotype analysis showed one haplotype (which included the -597A, -572G, -174G and variable nucleotide tandem repeat-D alleles) had a frequency of 0.1 in patients with ALT values >2 x ULN, whereas it was absent in patients with ALT less than 2 x ULN (P=0.0093, Pcorrected=0.049)., Conclusion: The IL6 genotype may act as a predisposing factor for tacrine transaminitis. This, however, requires further confirmatory functional studies. The role of acute dosing rodent models in identifying candidate genes associated with drug-induced liver injury in man deserves further study.

A phytochemical investigation of the EtOH extract of Hoveniae Lignum yielded four phenolic compounds, phloretin (1), 5-(4'-hydroxyphenyl)-gamma-valerolactone (2), (-)-epiafzelechin (3), and maesopsin (4). Compound 1 was hepatoprotective against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells with an EC50 value of 37.55 +/- 0.42 microM. Compound 1 (0.4-200 microM) also significantly reduced tert-butyl hydroperoxide-induced cytotoxicity in rat primary hepatocytes as measured by the cellular leakage of lactate dehydrogenase and the level of aspartate transaminase.

Bis(7)-tacrine was evaluated for efficacy on memory retention in mice 17 days of age and 30 days of age. The tests used were a passive-avoidance response test and a measure of spontaneous motor activity. Also, possible drug-induced hepatotoxicity and acute drug toxicity were evaluated. Behavioral studies were performed using a step-through task and an open-field test with a 24-h interval between training and evaluation tests. Bis(7)-tacrine (0.06-20 micromol/kg) was subcutaneously injected 30 min prior to the first session of both test types. During the training session of the step-through task, bis(7)-tacrine treatment reduced (by 46%, P<0.01) the number of avoidable electric shocks (footshocks) only at a high dose of 20 micromol/kg in mice 17 days of age, but dose-dependently decreased the number of footshocks (10-56%, P<0.001) in mice 30 days of age. Bis(7)-tacrine treatment at all doses tested did not produce any detectable changes in retention latency in mice 17 days of age, but the drug significantly prolonged retention latency at low doses (1.25 and 2.50 micromol/kg), and not high doses (5-20 micromol/kg), in mice 30 days of age. In the open-field test, bis(7)-tacrine decreased spontaneous motor activity in the acquisition session only at a high dose of 20 micromol/kg in mice 17 days of age and 30 days of age (by 28 and 45%, respectively), but did not affect spontaneous motor activity in the recall session. Bis(7)-tacrine treatment at a dose of 20 micromol/kg produced a more potent hepatotoxic effect in mice 30 days of age than in mice 17 days of age, (P<0.05), and the drug caused acute toxicity with comparable potencies in mice of both age groups. In conclusion, mice 30 days of age seemed to be more sensitive than mice 17 days of age to bis(7)-tacrine-induced cognitive function enhancement and hepatotoxicity. Bis(7)-tacrine appears to be more potent and more selective as a cognitive function-enhancing agent than tacrine.

Controversial results about the involvement of CYP 1A2 and oxidative stress in tacrine-induced hepatotoxicity have been described by the different research groups. We suggested that different expression levels of CYP 1A2 in cell lines and primary hepatocytes in vitro may be the cause of the controversial results above. Therefore, this paper re-evaluated the toxicity of tacrine by using gel entrapment culture of rat hepatocytes. The toxic effect of tacrine on hepatocytes was assayed by the reduction of methyl thiazolyl tetrazolium (MTT) and intracellular glutathione (GSH), as well as by albumin synthesis. It was found that the detectable hepatotoxic dose of tacrine is much lower in hepatocytes entrapped in gel than in those in monolayer cultures. The fact that fluvoxamine, a potent cytochrome P450 (CYP) 1A2 inhibitor, reduced tacrine toxicity and the expression of the CYP 1A2 gene was maintained in gel entrapped hepatocytes, but not in hepatocyte monolayers, could illustrate a close association between CYP 1A2 expression levels and tacrine toxicity. Glycyrrhetinic acid (GA), a free radical scavenger, protected gel entrapped hepatocytes from tacrine toxicity, but was ineffective in hepatocyte monolayers. Hence, gel entrapped hepatocytes could reflect higher tacrine hepatotoxicity in vivo than hepatocyte monolayers.

Acetylcholinesterase inhibitors are commonly used as cognitive enhancers for dementia in aged people. Among them, tacrine (THA) but not galanthamine, was shown to exhibit hepatotoxicity which reduces its clinical use. PMS777, both a PAF antagonist and a new potent acetylcholinesterase inhibitor was recently demonstrated to reverse scopolamine-induced amnesia in mice without toxicity. In the present study, the effects of THA, galanthamine and PMS777 were compared in HepG2 cells on the oxidative parameters involved in the reported hepatotoxicity of THA. THA (> or = 10 microM) induced an oxidative stress as shown by elevated ROS and MDA production and by a decrease in GSH level. Moreover, mitochondrial membrane potential and redox status were decreased. At low concentrations (< or =10 microM), there was no significant disturbance. None of the oxidative stress markers was affected by PMS777 up to the maximum concentration tested and it is suggested that PMS777 is not cytotoxic for HepG2 cells. Galanthamine was also without cytotoxicity. Our results suggest that the toxic effect of THA above 10 microM may be caused by drug-induced mitochondrial energization impairment and destabilisation of membrane phospholipids associated with an oxidative stress. In contrast by preventing these dysfunctions, PMS777 could be safer than THA.

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 micro 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 IC(50) values being 7.8 and 23.3 micro mol/kg in 17- and 30-day-old mice, respectively. Treatment with tacrine at a low dose (5 micro 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 40 micro 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 IC(50) values being 15.1 and 24.7 micro mol/kg, respectively. High doses of tacrine invariably increased the locomotor activity during the recall session. Tacrine treatment at a dose of 40 micro 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.

The bioassay-guided fractionation of the MeOH extract of the root barks of Cudrania tricuspidata furnished three hepatoprotective compounds, gerontoxanthone A (2), cudraflavone B (4), gericudranin E (5), together with four prenylated xanthones, cudraxanthone B (3), isocudraxanthone K (6), cudraxanthone C (7), cudraxanthone H (8), and a prenylated flavanone, euchrestaflavanone C (1). Compounds 4 and 5 showed significant hepatoprotective effects with the EC50 values of 37.39+/-0.4 and 39.87+/-0.7 microM, respectively, and compound 2 showed moderate hepatoprotective effect with an EC50 value of 125.9+/-1.5 microM, against tacrine-induced cytotoxicity in Hep G2 cells. Silybin as positive control showed an EC50 value of 84.76+/-0.5 microM. Isocudraxanthone K (6) is a new compound.

New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized as new potent drugs that may simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting the beta-amyloid (A beta) peptide aggregation through binding to both catalytic and peripheral sites of the enzyme. Particularly, compounds 5 and 6 emerged as the most potent heterodimers reported so far, displaying IC50 values for AChE inhibition of 20 and 60 pM, respectively. More importantly, these dual AChE inhibitors inhibit the AChE-induced A beta peptide aggregation with IC50 values 1 order of magnitude lower than that of propidium, thus being the most potent derivatives with this activity reported up to date. We therefore conclude that these compounds are very promising disease-modifying agents for the treatment of Alzheimer's disease (AD).

The bioassay-guided fractionation of the MeOH extract of Galla Rhois furnished two hepatoprotective compounds, an equilibrium mixture of 3-galloyl-gallic acid and 4-galloyl-gallic acid isomers (3), 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (4), and two inactive phenolic compounds, gallic acid methyl ester (1) and gallic acid (2). Compounds 3 and 4 showed significant hepatoprotective effects with EC50 values of 70.39+/-5.4 and 29.51+/-0.7 microM, respectively, against tacrine-induced cytotoxicity, and 150.9+/-6.4 and 23.81+/-0.5 microM, respectively, against nitrofurantoin-induced cytotoxicity in Hep G2 cells.

Phytochemical investigation of the aqueous extract of the roots of Agrimonia pilosa Ledeb. (Rosaceae), as guided by hepatoprotective activity in vitro, furnished two isocoumarins, agrimonolide (1) and agrimonolide 6-O-beta-D-glucoside (3), and (+)-catechin (2). Compound 1 showed hepatoprotective effects on both tacrine-induced cytotoxicity in human liver-derived Hep G2 cells and tert-butyl hydroperoxide-induced cytotoxicity in rat primary hepatocytes with EC50 values of 88.2 +/- 2.8 and 37.7 +/- 1.6 microM, respectively.

The antioxidative and hepatoprotective potentials of two anthraquinones, alaternin (2-hydroxyemodin) and emodin, to scavenge and/or inhibit hydroxyl radicals generated by the Fenton reaction and to protect tacrine-induced cytotoxicity in human liver derived HepG2 cells were evaluated, respectively. The inhibitory activity on hydroxyl radical generated in a cell-free chemical system (FeSO4/H2O2) was investigated by a fluorescence spectrophotometer using a highly fluorescent probe, 2',7'-dichlorofluorescein. The hydroxyl radical scavenging activity was determined by electron spin resonance spectroscopy using 5,5-dimethy-1-pyrroline-N-oxide as hydroxyl radicals trapping agents. Tacrine-induced HepG2 cell toxicity was determined by a 3-[4,5-dimethylthiazole-2yl]-2,5-diphenyltertrazolium bromide assay. Although the scavenging activity of alaternin on hydroxyl radical was similar to that of emodin in dose-dependent patterns, the inhibitory activity exhibited by the former on hydroxyl radical generation was stronger than that of the latter, with IC50 values of 3.05 +/- 0.26 microM and 13.29 +/- 3.20 microM, respectively. In addition, the two anthraquinones, alaternin and emodin showed their hepatoprotective activities on tacrine-induced cytotoxicity, and the EC50 values were 4.02 microM and 2.37 microM, respectively. Silymarin, an antihepatotoxic agent used as a positive control exhibited the EC50 value of 2.00 microM. These results demonstrated that both alaternin and emodin had the simultaneous antioxidant and hepatoprotective activities.

Bioassay-guided fractionation of the EtOH extract of Cnidium monnieri (Apiaceae) furnished two hepatoprotective sesquiterpenes, torilin (1) and torilolone (2), together with a new derivative, 1-hydroxytorilin (3). Compounds 1 and 2 showed hepatoprotective effects on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. The EC50 values of compounds 1 and 2 were 20.6 +/- 1.86 (P < 0.01) and 3.6 +/- 0.1 (P < 0.01) microM, respectively. Silybin as a positive control showed an EC50 value of 69.0 +/- 3.4 microM.

Intragastric administration (100-200 micromol/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.

(1) The reference symptomatic treatment for mild to moderate Alzheimer's disease is a cholinesterase inhibitor such as donepezil, but efficacy is only moderate and only about 10% of those patients treated actually benefit. (2) Galantamine is the fourth cholinesterase inhibitor to be marketed in France for Alzheimer's disease. The clinical file contains data from five double-blind placebo-controlled trials lasting 3-6 months, but no data comparing galantamine with other drugs. (3) These trials show that about 5-13% of patients treated with galantamine may be improved. (4) Adverse effects are very frequent, and are similar to those of other cholinesterase inhibitors, i.e. nausea, vomiting, diarrhoea, abdominal pain, dyspepsia, etc. (5) For patients who are eligible for drug therapy, the reference treatment is still donepezil, for want of anything better.

The only therapeutic drugs for combating dementia disease are acetylcholine esterase inhibitors (AChEI). However, the use of tacrine, the first AChEI to be launched as an Alzheimer's disease (AD) drug, has been limited by serious side effects. Therefore, efforts to search for more potent and selective inhibitors of AChE still remain highly significant in the therapeutic treatment of AD. In this work we modified the cyclohexyl ring of velnacrine, a less toxic analogue of tacrine, by synthesizing a series of thiopyranoquinolines in which the C-3 methylene unit was replaced by a sulphur atom. The anti-AChE data show that the activity was maintained with the bioisosteric substitution carried out. The introduction of a chlorine atom at different positions of the aromatic ring resulted in an array of different activities. In an attempt to understand the different behaviours displayed by the chlorine-substituted derivatives, a molecular docking study was performed.