Introduction

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AD is characterized by extensive neuronal loss and the presence of two different types of neuropathological features in the brain, extracellular deposition of amyloid and accumulation of intracellular neurofibrillary tangles (Katzman, 1986; Selkoe, 1994). It has been reported that patients suffering from AD show a remarkable loss of large cells in the nucleus basalis of Meynert's area (Whitehouse et al., 1982; Coyle et al., 1983) and a profound reduction in cortical and hippocampal ChAT activity (Davis and Maloney, 1977; Perry et al., 1977), although other neurochemical changes are also observed (Gottfries, 1985). The central cholinergic deficits found in patients with clinically diagnosed AD are reported to correlate with the degree of cognitive impairment (Perry et al., 1977, 1985). In fact, it has been shown that lesions of the basal forebrain, which has cholinergic neurons projecting to the widespread cortices and hippocampus, lead to learning impairments (Hepler et al., 1985; Miyamoto et al., 1985, 1987). Moreover, cholinergic antagonists impair learning and memory in experimental animals and humans (Gruber et al., 1967; Drachman and Leavitt, 1974; Drachman, 1977) and cholinergic drugs ameliorate learning and memory impairments in animal models of dementia (Bartus et al., 1982; Aigner et al., 1987; Miyamoto et al., 1989). Therefore, many attempts have been made to reverse the cognitive deficits by potentiating cholinergic activity, but these attempts have in general met with little success. However, it was reported by Summers et al. (1986) that oral administration of tacrine in combination with lecithin led to significant amelioration of the symptoms of AD patients. Further clinical studies have confirmed that administration of tacrine led to significant improvement, although it has side effects related to its actions on the peripheral nervous system and to hepatic toxicity (Davis et al., 1992; Holford and Reace, 1992; Knapp et al., 1994). These findings suggest that a centrally active AChE inhibitor with minimal side effects may be useful for the treatment of AD.

AChE and BuChE are present in a wide variety of tissues. The capacity of AChE to hydrolyze ACh is an important physiological function of this enzyme that terminates the effect of this neurotransmitter at the cholinergic synapse (Brimijoin, 1983). By contrast, it remains to be established whether the capacity of BuChE to hydrolyze higher choline esters, such as butyrylcholine, in preference to ACh, is an important physiological function of this enzyme or whether BuChE is involved in the hydrolysis of ACh. The different distributions of AChE and BuChE suggest that BuChE is not related to cholinergic neurotransmission in the central nervous system. Inhibition of BuChE induces peripheral side effects (Koelle et al., 1976; Dressel et al., 1980). Enhancement of the synaptic concentration of ACh in the brain is largely due to inhibition of the AChE activity. Accordingly, a selective inhibitor of AChE might be superior to nonselective inhibitors in the treatment of AD.

Tacrine has a variety of pharmacological effects other than the inhibition of AChE and BuChE; it has been shown that tacrine also inhibits action on potassium channel opening in both neuronal and cardiac tissues (Halliwell and Grove, 1989), release of neurotransmitters (Nilsson et al., 1987; Flynn and Mash, 1989) and monoamine uptake (Drukarch et al., 1988; Jossan et al., 1992). The brains of patients with AD show many neurochemical changes in addition to severe dysfunction of the basal forebrain cholinergic system, including changes in the noradrenergic (Bondareff et al., 1982; Mann et al., 1984), serotonergic (Curcio and Kemper, 1984; Yamamoto and Hirano, 1985) and peptidergic nervous systems (Davies et al., 1980, Rossor and Emson, 1982). Thus, treatment other than AChE inhibition, such as inhibition of monoamine uptake and stimulation of monoamine release, might also be beneficial for AD patients.

In an attempt to find a new type of AChE inhibitor that selectively inhibits AChE in the brain, we examined the actions of potent AChE inhibitors on the central and peripheral cholinergic systems in rats. One of the compounds we examined, TAK-147 (fig. 1), inhibited apomorphine-induced circling behavior in rats with unilateral striatal lesions, at doses causing no significant changes in the general behavior of the rats (Ishihara et al., 1994). It has been shown that TAK-147 ameliorates impaired learning and memory in some animal models without affecting their general behavior and without causing behavioral depression (Miyamoto et al., 1996). In this study, we show the neurochemical effects of TAK-147 on the cholinergic and monoaminergic systems in rats.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Chemical structure of TAK-147.

	Materials and Methods

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Materials. TAK-147 was synthesized in the Pharmaceutical Research Laboratories I of Takeda Chemical Industries, Ltd. by the methods of Ishihara et al. (1994). Tacrine, imipramine hydrochloride, desipramine hydrochloride and ethopropazine hydrochloride were obtained from Sigma Chemical Co. (St. Louis, MO). The following radioligands were obtained from New England Nuclear (Boston, MA): [³H]ACh iodide, [³H]pirenzepine, [³H]AF-DX384, [³H]methylcarbamylcholine iodide, [³H]ketanserine, [³H]prazosin, [³H]rauwolscine, [³H]SCH23390, [³H]spiperone, [³H]8-hydroxy-DPAT, [³H]NA, [³H]5-HT, [¹⁴C]choline chloride and [¹⁴C] acetyl coenzyme A. All other chemical compounds used were of the highest grade available and were purchased from regular commercial sources.

Measurement of AChE activity. Male Wistar rats (Japan Clea Inc. Tokyo, Japan), each weighing 200 to 300 g, were used in these experiments. Rats were killed by decapitation and the cerebral cortices were rapidly dissected on ice and then homogenized in 10 volumes of ice-cold 0.32 M sucrose using a Polytron homogenizer (Kinematica AG Lucerne, Switzerland). The homogenate was centrifuged at 1000 × g at 4°C for 10 min and the supernatant was used as the source of AChE.

Measurement of AChE activity ex vivo. Male Wistar rats (Japan Clea Inc.), each weighing 250 to 280 g, were used in these experiments. Vehicle or TAK-147 dissolved in a 0.9% saline was administered p.o. Six animals were used per treatment condition. Animals were killed by decapitation 1 hr after TAK-147 or vehicle administration and the cerebral cortices were rapidly dissected on ice and weighed. Cerebral cortices were stored at 80°C until the measurement of AChE activity. The cerebral cortices were homogenized in seven volumes of ice-cold homogenizing buffer [114 mM Tris-HCl (pH 7.0), 0.6 M NaCl, 0.43% (v/v) Triton X-100]. Eighty µl of homogenate and 10 µl of 100 µM ethopropazine were mixed in a scintillation vial and preincubated for 15 min at room temperature. The reaction was initiated by the addition of 10 µl of [³H]ACh iodide (final concentration 2.0 mM; using nonradiolabeled compound to make approximately 70,000 dpm per assay). After incubation for 1 min at room temperature the reaction was terminated by the addition of 100 µl of stop solution. Four milliliters of scintillation cocktail was added to each reaction tube and counted by a liquid scintillation counter. Protein concentrations were measured using BCA Protein Assay Reagent (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as the standard (Smith et al., 1985).

Measurement of BuChE activity. Rat plasma was used as the enzyme source of BuChE. BuChE activities were measured using the spectrophotometric method of Ellman et al. (1961) with butyrylthiocholine iodide as the substrate. The assay mixture consisted of 80 mM Tris-HCl (pH 8.0), 3.3 mM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) and 0.2 mM butyrylthiocholine iodide. After 2 min of mixing by agitation, the reaction was monitored by the change of the absorbance at 412 nm at 25°C for 5 min. IC₅₀ values were determined by log-probit analysis of concentration-response curves with five to six concentrations.

NA and 5-HT uptake. Male Wistar rats (Japan Clea Inc.), each weighing 200 to 250 g, were used in these experiments. Animals were killed by decapitation and the cerebral cortices and hippocampi were homogenized by five strokes at 600 rpm in five volumes of ice-cold 0.32 M sucrose using a glass-Teflon homogenizer. The homogenate was centrifuged at 1000 × g at 4°C for 10 min and the supernatant was recentrifuged at 20,000 × g at 4°C for 20 min. The pellet was resuspended in ice-cold KRB solution (116 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, 25 mM NaHCO₃, 0.1 mM EDTA-2Na and 11.1 mM D-glucose; saturated with 95% O₂/5% CO₂) containing 0.01 mM pargyline-HCl and 0.11 mM L-ascorbic acid. After preincubation of a 900-µl aliquot of this suspension with the test drug at 37°C for 5 min, 100 µl of [³H]NA (11 nM) or [³H]5-HT (10 nM) was added and the suspension was incubated further at 37°C for 5 min. The reaction was terminated by adding 4 ml of ice-cold KRB solution followed by vacuum filtration onto a Whatman GF/B filter that was then washed twice with 4 ml of ice-cold KRB solution. The radioactivity on the filter was counted by the liquid scintillation spectrophotometer. The blank was determined by the uptake of [³H]NA or [³H]5-HT at 4°C for 5 min. IC₅₀ values were determined by log-probit analysis of concentration-response curves with six to eight concentrations.

Receptor binding. The conditions used to measure ligand binding to cholinergic and monoaminergic receptors are given in table 1. K_D values were calculated from Scatchard plots. IC₅₀ values were determined by log-probit analysis of concentration-response curves with four to seven concentrations. K_i values were calculated from IC₅₀ values and K_D values (Cheng and Prusoff, 1973).

HACU. Sodium-dependent HACU into rat forebrain synaptosomes was determined according to the method of Simon et al. (1976) with modifications. The cerebral cortices and hippocampi of male Wistar rats (8- to 10-wk-old) were homogenized by five strokes at 600 rpm in 10 volumes of ice-cold 0.32 M sucrose using a glass-teflon homogenizer and then centrifuged at 1000 × g at 4°C for 10 min. The supernatant was recentrifuged at 20,000 × g at 4°C for 20 min and the resulting pellet was resuspended in KRB solution. After preincubation of a 900-µl aliquot of the suspension with the test drug at 37°C for 5 min, 100 µl of [¹⁴C]choline chloride (0.5 µM) were added and incubated further at 37°C for 5 min. The reaction was terminated by adding 4 ml of ice-cold KRB solution followed by vacuum filtration onto a Whatman GF/B filter that was then washed twice with 4 ml of ice-cold KRB solution. The radioactivity on the filter was counted by the liquid scintillation spectrophotometer. The blank was determined by the uptake of [¹⁴C]choline chloride at 4°C for 5 min. IC₅₀ values were determined by log-probit analysis of concentration-response curves with 5-7 concentrations.

ChAT activity. The supernatant prepared from rat cerebral cortex for AChE activity measurement was also used in this experiment. ChAT activity was measured by the method of Fonnum (1975) with slight modifications. Briefly, 20 µl of the enzyme solution were preincubated in a small plastic scintillation vial at 37°C for 5 min before the addition of 20 µl of a reaction mixture consisting of 50 mM sodium phosphate buffer (pH 7.4), 300 mM NaCl, 200 µM [¹⁴C]acetyl coenzyme A, 10 mM choline iodide and 200 µM physostigmine sulfate. After an incubation period of 30 min at 37°C, 500 µl of a 5:2 mixture of 0.1 mM p-chloromercuribenzoic acid in 20 mM sodium phosphate, pH 7.4 and 5 mg/ml of sodium tetraphenylborate in acetonitrile was added to stop the reaction followed by 1 ml of a scintillation cocktail. After vigorous shaking and subsequent separation of the organic and aqueous phases, [¹⁴C] radioactivity was counted by a liquid scintillation spectrophotometer. To determine the effects of AChE inhibitors on ChAT activity, physostigmine in the assay solution was substituted by TAK-147 or tacrine.

Monoamine metabolism in the rat brain. Male Wistar rats (Japan Clea Inc.), 9 wk old, were given 3 mg/kg TAK-147 or 3 mg/kg tacrine p.o. The rats were killed by microwave irradiation on the head 30 min after the administration. The brains were removed and the cerebral cortex, hippocampus, striatum, diencephalon and brain stem were dissected out. Brain regions were homogenized in 0.05 N perchloric acid containing 5 mM EDTA-2Na and centrifuged at 30,000 × g for 20 min. The supernatant was adsorbed to a mixture of 30 mg alumina and 20 mg Amberlite CG-50 and eluted with 0.1 N perchloric acid. The eluent was subjected to high-performance liquid chromatography using a reverse-phase column (Nucleosil 5C-18). The mobile phase was 0.05 M citrate-acetate (pH 4.8) containing 1 to 3.5% acetonitrile and heptane sulfonic acid. Monoamines and their metabolites were detected with an electrochemical detector.

Data analysis. Data from the animal studies were analyzed using Student's t test (two-tailed). P < .05 was the threshold for statistically significant.

	Results

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Inhibition of AChE and BuChE activities in vitro. The inhibitory effects of TAK-147 on AChE and BuChE activities were compared with the effects of other AChE inhibitors, tacrine and physostigmine in vitro. TAK-147 potently inhibited the AChE activity of rat cerebral cortex extract with an IC₅₀ value of 51.2 nM (fig. 2). Inhibition of AChE activity by TAK-147 was 3.0- and 2.4-fold more potent than by tacrine or physostigmine, respectively. Kinetic analysis by Lineweaver-Burk plots showed that TAK-147 inhibition of AChE had both competitive and noncompetitive components (fig. 3). This kinetic profile was similar to that of tacrine. The interaction between TAK-147 and AChE appeared to be reversible, as TAK-147-induced inhibition of AChE was reversed by dilution (fig. 4). Tacrine also showed reversible inhibition of AChE, but the interaction between physostigmine and AChE was irreversible. Preincubation of AChE with TAK-147 or tacrine at concentrations 10-times higher than the concentrations used in the assay had little effect on the IC₅₀ values.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of TAK-147 (), tacrine () and physostigmine () on AChE activity in rat cerebral cortex. Each value shows the mean with the S.E.M. of four different experiments each done in triplicate.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Double reciprocal plots of AChE activity in the cerebral cortex in the presence of TAK-147 (A) or tacrine (B). Each value is the mean from a single experiment done in triplicate. (A) , control; , 30 nM TAK-147; , 100 nM TAK-147. (B) , control; , 100 nM tacrine; , 300 nM tacrine.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of preincubation of AChE enzyme solution with TAK-147, tacrine or physostigmine on AChE activity. The tissue extract was preincubated for 15 min at room temperature with TAK-147 (A), tacrine (B) or physostigmine (C) at 10 times the concentration used for the AChE activity measurement () or at the same concentration (). Each value is the mean from a single experiment done in triplicate.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of TAK-147 (), tacrine () and physostigmine () on BuChE activity in the rat plasma. Each value shows the mean with the S.E.M. of three different experiments each done in triplicate.

Inhibition of ex vivo AChE activities by p.o. administration of TAK-147. Administration of TAK-147 at doses of 1 to 10 mg/kg p.o. inhibited AChE activity in rat cerebral cortex in a dose-dependent manner (fig. 6). TAK-147 at the dose of 10 mg/kg reduced AChE activity in the cerebral cortex by 52.7% 1 hr after its administration. Even at 1 mg/kg TAK-147 caused a slight but significant inhibition of AChE activity (89.1% of control), when AChE activity was measured in tissue extracts of 10-times dilution. From the dose-response curve shown in figure 2, AChE activity in the cerebral cortex of TAK-147-(1 mg/kg p.o.) treated rats was estimated to be approximately 40% of that in saline-treated rats.

View larger version (64K): [in this window] [in a new window]   Fig. 6.   Effects of TAK-147 administered p.o. on ex vivo AChE activities of cerebral cortex of the rat. TAK-147 at 1 to 10 mg/kg or vehicle was administered p.o. 1 hr before decapitation. Each value shows the mean with the S.E.M. of six animals. *P < .05, **P < .01, compared with the saline control group (Student's t test).

Cholinergic receptor binding profile. A summary of the cholinergic receptor radioligand binding results is given in table 3. TAK-147 had the highest affinity for [³H]pirenzepine and [³H]AF-DX 384 labeled muscarinic M₁ and M₂ receptors in the rat cerebral cortex; the K_i values were 234 and 340 nM, respectively. Tacrine also showed high affinity for muscarinic M₁ and M₂ receptors with IC₅₀ values of 264 and 598 nM, respectively. The inhibitory activities of TAK-147 and tacrine for muscarinic M₁ and M₂ receptors were much lower than those of atropine which had K_i values of 0.628 and 0.834 nM, respectively. Physostigmine had only low affinity for both muscarinic receptor types. In contrast to the muscarinic receptors, TAK-147 and tacrine showed very low affinity for [³H]methylcarbamylcholine-labeled nicotinic receptors with IC₅₀ values of 32,600 ± 18,100 (S.E.M.) and 32,200 ± 11,600 nM, respectively. The inhibitory actions of these compounds at nicotinic receptors were not competitive at the [³H]methylcarbamylcholine binding site because the calculated Hill coefficients of these compounds were 0.286 ± 0.053 and 0.438 ± 0.086, respectively, whereas the IC₅₀ value and Hill coefficient of nicotine were 476 ± 5 and 0.976 ± 0.041 nM, respectively. Thus TAK-147 and tacrine possibly inhibit [³H]methylcarbamylcholine binding by interacting at other sites than ligand binding.

HACU and ChAT. The effects of TAK-147, tacrine and physostigmine on the HACU and ChAT were examined. TAK-147 showed very weak inhibition of HACU with an IC₅₀ of 17,400 ± 3,100 nM. Tacrine also showed weak inhibition of HACU with an IC₅₀ of 9770 ± 840 nM. Physostigmine showed no effect on HACU, even at 100 µM. However, TAK-147, tacrine and physostigmine showed no effect on the ChAT activity of rat cerebral cortex extracts at concentrations of 100 µM.

NA and 5-HT uptake. TAK-147 inhibited NA and 5-HT uptake into the synaptosomal fraction in a dose-dependent fashion. The IC₅₀ values for TAK-147 inhibition of NA and 5-HT uptake were 4020 and 1350 nM, respectively (table 4). TAK-147 inhibition of 5-HT uptake was 12 times weaker than that of imipramine which had IC₅₀ value of 117 nM. Tacrine showed less potent inhibition of NA and 5-HT uptake than TAK-147 with IC₅₀ values of 7120 and 8220 nM, respectively. Physostigmine showed no inhibitory action on NA and 5-HT uptake into the synaptosomal fraction at concentrations of 100 µM.

Monoaminergic receptor binding profile. TAK-147 showed moderate inhibition of binding for the monoamine receptors tested (table 5). The K_i values of TAK-147 for alpha-1, alpha-2, D₁, D₂, 5-HT_1A and 5-HT₂ receptor binding were 324, 2,330, 27,200, 12,400, 6,690 and 3,510 nM, respectively. Tacrine showed similar but weaker inhibition of NA and 5-HT receptor binding compared to TAK-147 whereas tacrine exhibited no inhibition of DA receptors. Physostigmine showed no inhibition of binding for the monoamine receptors tested in this experiment.

Effects of TAK-147 and tacrine on monoamine metabolism in the rat brain. The effect of p.o. administration of TAK-147 and tacrine on monoamine turnover in the rat brain was examined (table 6). We used the maximal doses of TAK-147 and tacrine without peripheral side effects (Miyamoto et al., 1996). TAK-147 increased monoamine turnover in several brain regions; treatment with TAK-147 (3 mg/kg p.o.) significantly increased NA turnover in the hippocampus and striatum, DA turnover in the hippocampus and diencephalon and 5-HT turnover in the cerebral cortex and diencephalon. TAK-147 also caused small, statistically nonsignificant increases in DA turnover in the striatum and cerebral cortex, and in 5-HT turnover in the hippocampus and striatum. Tacrine also increased monoamine turnover in a similar pattern to TAK-147. Tacrine (3 mg/kg p.o.) significantly increased NA turnover in the hippocampus and striatum, DA turnover in the hippocampus and 5-HT turnover in the cerebral cortex and hippocampus.

	Discussion

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In vitro studies showed that TAK-147 inhibits AChE activity potently and reversibly. Inhibition by TAK-147 was strongly selective for AChE over BuChE, in excess of three orders of magnitude, whereas neither tacrine nor physostigmine had such selective activity. Although both AChE and BuChE hydrolyze ACh in vitro, AChE shows higher affinity for ACh than BuChE. BuChE prefers to hydrolyze higher choline esters, such as butyrylcholine. Studies of subjects with mutation of the human BuChE gene have suggested that the enzyme at most plays only an auxiliary role in the nervous system (Soreq and Zakut, 1990). Its presence in the liver and plasma, as well as the population distribution of human BuChE mutations, suggests that one function of BuChE is to catalyze the hydrolysis of dietary esters and to detoxicate poisons that are eaten or inhaled (Soreq and Zakut, 1990; Jbilo et al., 1994). Inhibition of BuChE might disrupt detoxication of toxic compounds and cause peripheral toxicity such as hepatic toxicity. Moreover, animals or humans with low BuChE activity, which is caused by specific BuChE inhibitors or some genetic mutations, exhibit high sensitivity for nonselective AChE inhibitors (Gupta and Dettbarn, 1987; Prody et al., 1989; Loewenstein-Lightenstein et al., 1995). Loewenstein-Lightenstein et al. (1995) also suggested that the interaction of AChE inhibitors with BuChE might shorten their half-life and weaken their ability to arrest the breakdown of ACh in cholinergic nerve endings. Thus, tacrine and physostigmine, both of which have been used in multiple clinical studies, seem to be less favorable than TAK-147, because tacrine and physostigmine inhibit both AChE and BuChE at the same concentration. Recently, there are some other new selective AChE inhibitors, such as E2020 (Sugimoto et al., 1995) and galanthamine (Harvey, 1995), in clinical trials and these compounds showed some advantages over tacrine and physostigmine in the treatment of AD. Thus selective AChE inhibitors might be better drugs for the treatment of Alzheimer's disease than nonselective ones.

It has been reported that TAK-147 causes dose-dependent behavioral changes in rats; miosis and fasciculation were induced at relatively low doses (ED₅₀ values of 5.8 and 10.2 mg/kg p.o., respectively) and lacrimation and salivation are induced at relatively high doses (ED₅₀ values of 92.3 and 169.3 mg/kg p.o., respectively; Miyamoto et al., 1996). TAK-147 also ameliorates impaired learning and memory in some animal models of dementia at relatively low doses without affecting the general behavior (Miyamoto et al., 1996). Consistent with this report, our ex vivo studies showed that p.o. administration of TAK-147 at doses of 1 to 10 mg/kg significantly inhibited AChE activity in the cerebral cortex of rats without affecting their general behavior. The concentration of TAK-147 that caused inhibition of AChE was the lowest concentration examined in this experiment. These results suggest that the ability of TAK-147 to ameliorate impaired learning and memory in animal models is due to its ability to inhibit AChE activity in the brain.

Neuronal cell losses in AD are not restricted to the medial basal forebrain cholinergic complex. The locus coeruleus (Bondareff et al., 1982; Mann et al., 1984; Marcyniuk et al., 1986), which provides NA innervation, and the raphe nucleus (Curcio and Kemper, 1984; Yamamoto and Hirano, 1985), which provides 5-HT innervation, also show severe neuronal dropout in AD and senile dementia. Dysfunction of both the NA and 5-HT nervous systems occurs in patients with AD, so drugs for treating AD should have both cholinergic and monoaminergic characteristics. TAK-147 moderately inhibited NA and 5-HT uptake into synaptosomal fractions of the rat brain, with IC₅₀ values of 4020 and 1350 nM, respectively. The activity of TAK-147 for the inhibition of 5-HT uptake was six times weaker than that of imipramine. However, if TAK-147 were administered at higher doses or repeatedly, the monoaminergic nervous systems might be activated by the antidepressant-like profile of TAK-147, such as monoamine uptake inhibition. Indeed, treatment with TAK-147 (3 mg/kg p.o.) significantly increased NA turnover in the hippocampus and striatum, DA turnover in the hippocampus and diencephalon and 5-HT turnover in the cerebral cortex and diencephalon (table 6). At this dose, the pharmacological actions of TAK-147 are mainly due to the increase in the extracellular ACh concentration caused by arresting the degradation of ACh. Tacrine (3 mg/kg p.o.) also increased monoamine turnover in a similar pattern to TAK-147. In this study, we used the maximal doses of TAK-147 and tacrine that cause no peripheral side effects (Miyamoto et al., 1996). At this dose, TAK-147 is likely to inhibit AChE activities in the brain more potently than tacrine, thus the regional difference of the effects on monoamine metabolism may be due to the difference of regional distribution of these compounds.

The specific M₁ muscarinic receptor agonist xanomeline increases DA turnover in the striatum, nucleus accumbens and hippocampus, but does not alter the levels of DA, NA, 5-HT and 5-hydroxyindoleacetic acid in the rat brain (Bymaster et al., 1994). In addition, physostigmine stimulates DA synthesis by stimulating muscarinic receptors in the striatum and by stimulating both muscarinic and nicotinic receptors in the limbic areas (Grenhoff and Svensson, 1992). The effects of TAK-147 on monoamine turnover are thought to be secondary effects caused by activation of the cholinergic nervous system via muscarinic and nicotinic receptors. In the ligand binding studies, both muscarinic and nicotinic binding sites show considerable regional variation; the number of [³H]quinuclidinylbenzilate binding sites (muscarinic sites) is high in the striatum, cerebral cortex and hippocampus (Kobayashi et al., 1978), whereas the number of [³H]--bungarotoxin binding sites (nicotinic sites) is high in the hypothalamus, hippocampus and cerebral cortex (Segal et al., 1978). The effects of TAK-147 on monoamine turnover are marked in the diencephalon, hippocampus and cerebral cortex, but weak in the striatum, suggest that its effects on the monoaminergic nervous systems are mainly mediated by the stimulation of nicotinic receptors rather than muscarinic receptors. It has been shown that TAK-147 antagonizes reserpine-induced behavioral depression, and does not accelerate behavioral despair in the forced swimming test (Miyamoto et al., 1996). Furthermore, TAK-147 does not impair lever press operant behavior even at high doses of 10 to 30 mg/kg p.o. (Miyamoto et al., 1996). These pharmacological effects of TAK-147 may be closely related to its stimulative action on the monoaminergic system. Thus, AChE inhibitors, such as TAK-147, which activate both muscarinic and nicotinic receptors, might be of greater benefit in the treatment of the early stage of AD than muscarinic or nicotinic agonists.

In conclusion, TAK-147 inhibited AChE activity in the rat cerebral cortex potently and reversibly, but did not affect BuChE activity in the rat plasma. TAK-147 showed moderate to weak effects on cholinergic and monoaminergic systems other than its effect on AChE. TAK-147 induced a statistically significant decrease in the AChE activity of the cerebral cortex and significantly accelerated the turnover rates of DA, NA and 5-HT in rat brain, without producing behavioral changes, via activation of peripheral cholinergic receptors. These results suggest that TAK-147 is a central selective AChE inhibitor with moderate activation of monoaminergic systems, and it may be useful for the treatment of AD.