Tacrine sinusoidal uptake and biliary excretion in sandwich-cultured primary rat hepatocytes.

PURPOSE. The knowledge of hepatic disposition kinetics of tacrine, a first cholinesterase inhibitor was approved by FDA for the treatment of Alzheimer's disease (AD), would help to understand its hepatotoxicity, its therapeutic effect, and improve the management of patients with AD. The current study aims to characterize tacrine hepatic transport kinetics and study the role of organic cation transporters (OCTs), P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP2) in tacrine sinusoidal uptake and biliary excretion. METHODS. Modulation of tacrine hepatic uptake and efflux, biliary excretion index (BEI%), were performed in sandwich-cultured primary rat hepatocytes (SCHs) using transporters inhibitors. Conformation of the integrity of SCHs model was established by capturing images with light-contrast and fluorescence microscopy. RESULTS. Tacrine uptake in SCHs was carrier-mediated process and saturable with apparent Km of 31.5±9.6 µM and Vmax of 908±72 pmol/min/mg protein. Tetraethyl ammonium (TEA), cimetidine and verapamil significantly reduced tacrine uptake with more pronounced effect observed with verapamil which caused 3-fold reduction in tacrine uptake, indicating role for OCTs. Tacrine has a biliary excretion in SCHs with maximum BEI% value of 22.9±1.9% at 10 min of incubation. Addition of MK571 and valspodar decreased the BEI% of tacrine by 40 and 60% suggesting roles for canalicular MRP2 and P-gp, respectively. CONCLUSIONS. Our results show that in addition to metabolism, tacrine hepatic disposition is carrier-mediated process mediated by sinusoidal OCTs, and canalicular MRP2 and P-gp.

Effects of cabergoline and rotigotine on tacrine-induced tremulous jaw movements in rats.

OBJECTIVES: We examined the effects of two dopamine agonists, cabergoline and rotigotine, on tacrine-induced tremor and c-Fos expression in rats. METHODS: Rats received intraperitoneal injection of cabergoline (0.5, 1.0, or 5.0mg/kg), rotigotine (1.0, 2.5, or 10.0mg/kg), or vehicle 30min before intraperitoneal injection of tacrine (5.0mg/kg). The number of tremulous jaw movements (TJMs) after tacrine administration was counted for 5min. Animals were sacrificed 2h later under deep anesthesia, and the brain sections were immunostained in order to evaluate the c-Fos expression. RESULTS: Induction of TJMs by tacrine was dose-dependently reduced by pretreatment with cabergoline and rotigotine. The number of c-Fos-positive cells was significantly enhanced in the medial striatum, nucleus accumbens core, and nucleus accumbens shell after tacrine administration, and the enhanced expression of c-Fos in these three regions was significantly attenuated by cabergoline, while rotigotine suppressed c-Fos expression in two regions except the nucleus accumbens core. CONCLUSIONS: These results suggest that tacrine-induced TJMs would be relieved by either cabergoline or rotigotine and that anticholinesterase-induced TJMs and the ameliorating effects of dopamine agonists would relate to neuronal activation in the striatum and nucleus accumbens.

Tribuli fructus constituents protect against tacrine-induced cytotoxicity in HepG2 cells.

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.

Hepatoprotective effect of Cirsium arisanense Kitamura in tacrine-treated hepatoma Hep 3B cells and C57BL mice.

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.

Cytoprotective constituent of Hoveniae Lignum on both Hep G2 cells and rat primary hepatocytes.

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.

Phenolic constituents of galla Rhois with hepatoprotective effects on tacrine- and nitrofurantoin-induced cytotoxicity in Hep G2 cells.

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.

Influence of tacrine on dopamine-induced reactions of the gastric smooth muscle of rats.

The clinical usage of the cholinesterase inhibitor tacrine for treatment of Alzheimer's disease is accompanied by adverse effects on the gastrointestinal tract. These adverse effects are a result of the direct action of tacrine on the intestinal smooth muscles or of the modulation of certain neurotransmitters regulating gastrointestinal functions. Dopamine is a neurotransmitter that modulates gastrointestinal motility. This study was designed to examine in vitro the effects of tacrine on dopamine-induced changes in spontaneous activity of smooth muscle preparations from rat's gastric corpus. The mechanical activity was isometrically registered. Tacrine 1.10(-7)-1.10(-5) mol/l caused smooth muscle contraction, which was blocked by atropine 1.10(-6) mol/l. Tacrine 1.10(-4) mol/l provoked a relaxation resistant to atropine. Dopamine and D(2)-receptor antagonists haloperidol and R121 had no effect on tacrine-induced relaxation. Dopamine-induced contraction was concentration-dependent. It was blocked by D(2)-receptor antagonists haloperidol and R121 and by tacrine 1.10(-4) mol/l. In the presence of tacrine 1.10(-7)-10(-5) mol/l or atropine the dopamine-induced contraction was significant. The data obtained suggested that tacrine 1.10(-4) mol/l inhibited the dopamine effects on gastric corpus smooth muscles. The effect was probably not dependent on its anticholinesterase activity or not realized through direct influence on D(2)-dopamine receptors.

Sesquiterpenes with hepatoprotective activity from Cnidium monnieri on tacrine-induced cytotoxicity in Hep G2 cells.

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.

Abnormal expression of neuronal nitric oxide synthase triggers limbic seizures and hippocampal damage in rat.

Administration of tacrine (5 mg/kg ip), an anticholinesterase agent, in rats pretreated (24 h beforehand) with lithium chloride (LiCl; 12 mEq/kg ip) provides a useful experimental model to study limbic seizures and delayed hippocampal damage. Here we report Western blotting evidence demonstrating that in rat LiCl and tacrine enhance the expression of neuronal nitric oxide synthase (nNOS), but not eNOS, enzyme protein in the hippocampus during the preconvulsive period and this triggers seizures and hippocampal damage. In fact, systemic administration of 7-nitro indazole (7-NI; 50 mg/kg given ip 30 min before tacrine), a selective inhibitor of nNOS, prevented the expression of motor and electrocortical (ECoG) seizures and abolished neuronal cell death in the hippocampus. A lower dose (5 mg/kg ip) of 7-NI was ineffective. In conclusion, the present data support a role for abnormal nNOS expression in the mechanism which triggers limbic seizures and delayed excitotoxic damage in the hippocampus of rat.

Tacrine, a reversible acetylcholinesterase inhibitor, induces myopathy.

Tacrine, an acetylcholinesterase (AChE) inhibitor that has been used in the treatment of Alzheimer's disease, increases available acetylcholine (Ach) levels in the synaptic cleft thereby enhancing the activity of cholinergic pathways. However, excessive stimulation of nicotinic receptors at the neuromuscular junction results in muscle deterioration. We tested whether reversible AChE inhibitors such as tacrine may induce similar effects. In the present study, tacrine administration (7.5 mg/kg twice daily) to rats produces a 20 and 30-fold increase in the number of degenerating cells in leg and diaphragm muscle, respectively, as compared to control. This myopathy is significantly decreased by co-administration of tacrine with the nitric oxide (NO) synthase inhibitor L-NAME. These results show that tacrine can induce myopathy which may be mediated by increased NO production.

Aminotransferase levels and silymarin in de novo tacrine-treated patients with Alzheimer's disease.

BACKGROUND: Silymarin is a well-known hepatoprotective agent. Tacrine, the first drug marketed for Alzheimer's disease (AD), induces an elevation of serum liver transaminase prohibiting an effective dosage in many patients. This 12-week randomised, double-blind, placebo-controlled study was undertaken to evaluate the ability of silymarin to antagonise or prevent the hepatotoxic effects of tacrine and to analyse its action on tacrine efficacy and tolerability. METHODS: Outpatients suffering from mild-to-moderate dementia of the Alzheimer type were randomly assigned to two treatment groups: tacrine + silymarin and tacrine + placebo. The study was double-blind for silymarin and open for tacrine and was conducted in 22 French neurology and geriatric centres. Silymarin (420 mg/day) was given first (1 week) and tacrine was added at 40 mg/day for 6 weeks, then increased to 80 mg/day (6 weeks). Serum ALAT was the main evaluation criterion (> upper limit of normal, ULN). Serum ASAT as well as adverse side effects and cognitive performance assessed by MMSE and the Syndrome Kurtz test (SKT) were secondary evaluation criteria. Null hypotheses were evaluated with Fisher's exact test. FINDINGS: 222 patients were recruited and received silymarin and tacrine (110 patients) or placebo and tacrine (112 patients). 28 patients dropped out; 217 were included in the intent-to-treat analysis. No statistical difference was observed between the two groups for serum ALAT (p = 0.39). Fewer patients had ALAT levels >5 ULN in the silymarin group (-33.3%). Side effects and notably gastrointestinal disorders were much less frequent in the silymarin group. Cognitive performance remained unchanged in both groups. INTERPRETATION: Silymarin does not prevent tacrine-induced ALAT elevation but does reduce the rate of gastrointestinal and cholinergic side effects without any impact on cognitive status. As a consequence, silymarin (420 mg/day) could be co-administered with tacrine to improve tolerability in the initial phases of AD treatment.

Tremulous jaw movements produced by acute tacrine administration: possible relation to parkinsonian side effects.

Previous work has shown that cholinomimetic drugs induce "vacuous" or non-directed jaw movements in rats. In the present study, five experiments were conducted to provide a pharmacological, anatomical and behavioral characterization of tacrine-induced vacuous jaw movements. In the first experiment, tacrine produced vacuous chewing in a dose-related manner in a range of 1.25 mg/kg to 1.0 mg/kg. This effect was reduced, also in a dose-related manner, by the co-administration of the muscarinic antagonist scopolamine in a range of 0.125 to 1.0 mg/kg, but not by N-methylscopolamine. The fourth experiment examined the effect of scopolamine (2.5 to 10.0 micrograms) injected into the ventrolateral striatum on vacuous jaw movements induced by 5.0 mg/kg tacrine. Intrastriatal injections of scopolamine completely blocked tacrine-induced jaw movements. The fifth experiment utilized a slow-motion videotaping system to analyze the temporal characteristics of vacuous chewing induced by 5.0 mg/kg tacrine. The vast majority of the movements occurred in rapid "bursts," and analysis of interresponse times (i.e., the time between each jaw movement) showed that most of the jaw movements occurred within a local frequency range of 3 to 7 Hz. Thus, tacrine-induced jaw movements are reduced by antimuscarinic treatment, and most of these movements occur in the parkinsonian tremor frequency range. Tremulous jaw movements induced by tacrine in rats appear to share some characteristics with Parkinsonian tremor.

Biphasic effect of tacrine on acetylcholine release in rat brain via M1 and M2 receptors.

Rat cortical synaptosomes preloaded with [3H]choline were superfused and stimulated with K+ in order to investigate the effect of the cholinesterase inhibitor tacrine on the in vitro release of acetylcholine (ACh). Tacrine was found to biphasically both increase (10(-6) and 5 x 10(-6) M) and decrease (10(-5)-10(-4) M) the release of ACh in a concentration-dependent manner. The facilitatory effect of tacrine was prevented by atropine and the M1 antagonist pirenzepine, whereas the inhibitory effect induced by tacrine was blocked by atropine and the M2 antagonist AF-DX 116. These results indicate that tacrine causes a biphasic effect on K+ stimulated ACh release in the brain via M1 and M2 muscarinic receptors. The tacrine induced enhancement of the ACh release occurs at clinical relevant tacrine concentrations and might therefore be of importance for the treatment outcome of Alzheimer's disease.

An investigation of the interaction between halofantrine, CYP2D6 and CYP3A4: studies with human liver microsomes and heterologous enzyme expression systems.

1. We have assessed the interaction of the antimalarial halofantrine with cytochrome P450 (CYP) enzymes in vitro, with the use of microsomes from human liver and recombinant cell lines. 2. Rac-halofantrine was a potent inhibitor (IC50 = 1.06 microM, Ki = 4.3 microM) of the 1-hydroxylation of bufuralol, a marker for CYP2D6 activity. Of a group of structurally related antimalarials tested, only quinidine (IC50 = 0.04 microM) was more potent. 3. Microsomes prepared from recombinant CYP2D6 and CYP3A4 cell lines were shown to catalyse halofantrine N-debutylation. 4. The metabolism of halofantrine to its N-desbutyl metabolite by human liver microsomes showed no correlation with CYP2D6 genotypic or phenotypic status and there was no consistent inhibition by quinidine. 5. The rate of halofantrine metabolism showed a significant correlation with both CYP3A4 protein levels (r = 0.88, P = 0.01) and the rate of felodipine metabolism (r = 0.86, P = 0.013), a marker substrate for CYP3A4 activity. Inhibition studies showed that ketoconazole is a potent inhibitor of halofantrine metabolism (IC50 = 1.57 microM). 6. In conclusion, we have demonstrated that halofantrine is a potent inhibitor of CYP2D6 in vitro and can also be metabolised by the enzyme. However, in human liver microsomes it appears to be metabolised largely by CYP3A4.

Hypothermia induced by cholinomimetic drugs is blocked by galanin: possible involvement of ATP-sensitive K+ channels.

Central administration of galanin in the mouse dose-dependently blocked the hypothermia induced by the muscarinic receptor agonist, 2-ethyl 8-methyl-2,8-diazospiro[4,5]decan-1,3-dion hydrobromide, RS86 (minimum effective dose, MED = 3 nmol) and the acetylcholinesterase inhibitor tetrahydroaminoacridine, (MED = 3 nmol). This inhibitory effect was reversed over the dose range (0.1, 0.3, 1, 3 nmol) by the galanin receptor antagonist galantide (MED = 0.3 nmol). Furthermore, the ATP-sensitive K+ channel blockers glibenclamide (MED = 1 nmol) and gliquidone (10 nmol) both prevented the inhibitory effects of galanin on RS86 induced hypothermia. Glibenclamide (10 nmol) also reversed the inhibitory effects of galanin on tetrahydroaminoacridine induced hypothermia. Preincubation of rat cortical membranes with galanin (10 nM, 1000 nM) in vitro had no effect on binding affinity, receptor number or pharmacology of the rat cortical muscarinic receptor. In contrast to the high affinity of glibenclamide, galanin only weakly displaced [3H]glibenclamide binding in mouse whole brain homogenates (36% at 10 microM). These studies suggest that the inhibitory effect of galanin on cholinergically mediated hypothermia induced by RS86 and tetrahydroaminoacridine may be exerted via an action at ATP-sensitive K+ channels but is unlikely to be acting directly at the site labelled by [3H]glibenclamide.

Effects of tetrahydro-9-aminoacridine on the electrocorticogram of rats with a unilateral lesion of the nucleus basalis magnocellularis.

The effects of tetrahydro-9-aminoacridine (THA) were studied on the electrocortical activity of control rats and of rats with a unilateral lesion of the nucleus basalis magnocellularis (NBM) produced by alpha-amino-3-hydroxy-4-isoxozole propionic acid (AMPA). This lesion almost completely deprived the lesioned hemisphere of its cholinergic innervation. In control rats, THA (10 mg/kg i.p.) increased the amplitude of the slow components of the electrocorticogram (less than 9 Hz). These effects were antagonised by atropine (5 mg/kg i.p.). Lesion of the NBM alone decreased the amplitude of frequencies in the 12-16 Hz frequency band but did not significantly affect the slower frequencies. THA (10 mg/kg) restored the amplitude of the 12-16 Hz activity to the level seen in control rats before THA but did not affect activity in the other frequency bands. The results suggest that THA requires some residual cholinergic innervation in order to exert its effect.