Search

A dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT), RS-1259 (4-[1S)-methylamino-3-(4-nitrophenoxy)]propylphenyl N,N-dimethylcarbamate (fumaric acid)1/2salt), was newly synthesized. RS-1259 simultaneously inhibited AChE and SERT in the brain following an oral administration in mice and rats. Actual simultaneous elevation of extracellular levels of 5-HT and ACh in the rat hippocampus was confirmed by microdialysis. The compound was as effective as SERT inhibitors such as fluoxetine and fluvoxamine in a 5-hydroxytryptophan-enhancing test in mice. Spatial memory deficits in the two-platform task of a water maze in aged rats were ameliorated by RS-1259 as well as donepezil. Both RS-1259 and donepezil increased the awake episodes in the daytime electroencephalogram of rats. Although RS-1259 was weaker than donepezil in enhancing central cholinergic transmission, as observed by ACh elevation in the hippocampus and memory enhancement in aged rats, the efficacy of RS-1259 on the consciousness level, which reflects the whole activity in the brain, was almost the same as that of donepezil. These results suggest that both cholinergic and serotonergic systems are involved in maintaining brain arousal and that a dual inhibitor of AChE and SERT may be useful for the treatment of cognitive disorders associated with reduced brain activity such as in Alzheimers disease.

Highly efficient acetylcholinesterase (AChE) and serotonin transporter (SERT) dual inhibitors, (S)-4 and (R)-13 were designed and synthesized on the basis of the hypothetical model of AChE active site. Both compounds showed potent inhibitory activities against AChE and SERT. [structure: see text]

The role of NMDA receptors in the regulation of spinal motor output was studied in rats. Muscle tension of the hind limbs of decerebrate animals and spinal reflex potentials in anesthetized animals were recorded. Intrathecal injection as well as systemic or intra-4th ventricular injection of (+)-5-methyl-10-n-dihydro-5H-dibenzo[a,d]cyclohepta-5-10-imine maleate (MK-801) reduced muscle tension. Systemic MK-801 did not alter monosynaptic reflexes either in intact or spinal rats, but attenuated polysynaptic reflexes in spinal rats. Thus spinal NMDA receptors participate in spinal motor output in the presence of some specific factors such as descending facilitation and preceding segmental depolarization, which remove the Mg2+ blocking.

A newly synthesized agonist for muscarinic acetylcholine (ACh) receptors CS-932, (R)-3-(3-iso-xazoloxy)-1-azabicyclo-[2.2.2]octane hydrochloride, showed a relatively higher affinity for M1 than M2 receptors expressed in Chinese hamster ovary (CHO)-cells in comparison with ACh. CS-932 elevated the intracellular Ca2+ level only in M1-CHO cells, although ACh increased the level in both M1- and M3-CHO cells. CS-932 and ACh reduced forskolin-stimulated accumulation of cAMP in M2-CHO cells by 20% and 80%, respectively. This neurochemical profile of CS-932 indicates that the compound can activate M1-receptor-mediated functions selectively. CS-932 increased firing of cholinoceptive neurons in rat hippocampal slices, and this excitation was antagonized by pirenzepine, but not by AF-DX 116. CS-932 increased awake and decreased slow wave sleep episodes of daytime EEG in free-moving rats. It counteracted scopolamine-induced slow waves in rat cortical EEG. CS-932 also increased the power of alpha- and beta-waves, but decreased delta-wave of the cortical EEG in anesthetized monkeys. It ameliorated scopolamine-induced impairment of working memory in rats. Orally administered CS-932 had the best penetration into the brain among the muscarinic agonists tested and caused the least salivary secretion among the cholinomimetics examined. These results indicate that CS-932 has potential as a cognitive enhancer with fewer side effects in therapy for Alzheimer disease.

It is well known that learning and memory ability declines with aging. Age-related long-term changes in learning and memory ability in rats were investigated with the place navigation task and the allocentric place discrimination task (APDT) in a water maze using the same animals for each task. In a working memory place navigation task, aged animals could learn the location of the platform as well as when they were young, although strategy shifts were observed. In contrast, accuracy in the APDT significantly declined from 90% to 65% with aging. This impairment was ameliorated by an acetylcholine esterase inhibitor physostigmine at 22-23 months old. No amelioration was, however, detected in the same animals tested when they further aged to 26-27 months old. These results suggest that the APDT performance is sensitive to age-related memory deficits and that this may be due to the cholinergic dysfunction.

In order to evaluate learning and memory deficits separately from and simultaneously with motivational, motor and sensory impairments in identical animals, we developed the allocentric place discrimination task test using a water maze in rats. For this assessment task, two similar, visible platforms, one was fixed and the other was floating, were simultaneously present in a pool, and the working memory of the allocentric place discrimination task was evaluated. After training, the task accuracy was high about 85% correct and animals were used repeatedly. The accuracy decreased significantly when the pool was surrounded with a black curtain. Muscarinic receptor antagonist scopolamine 0.5 mg/kg selectively impaired the accuracy. Muscle relaxant dantrolene 10 mg/kg selectively decreased swimming speed. Under low motivational condition (warm water), still time increased and swimming speed decreased, but the accuracy was not affected. Similar to warm water, opioid receptor agonist morphine 15 mg/kg increased still time and decreased swimming speed. These results suggest that the allocentric place discrimination task is useful in evaluating spatial working memory ability independently of and concurrently with also visual, motor ability and motivation in identical animals.

To date, acetylcholinesterase (AChE) inhibitors have been clinically effective drugs for the palliative treatment of Alzheimer's disease, but their clinical efficacy is limited, mainly due to their adverse effects on peripheral organs. Since patients of Alzheimer's disease often exhibit depression as well as memory impairment, dual inhibitors of AChE and serotonin transporter (SERT) would be a better therapeutic method. Anti-depressive effects based on SERT inhibition would reduce the dose-related side effects of AChE inhibitors. Such dual inhibitors were designed by the hybridization of rivastigmine and fluoxetine based on a hypothetical model of the AChE active site. Various derivatives were synthesized and evaluated for their in vitro inhibition, and then (S)-5j (RS-1259), which possessed balanced inhibitory activities of AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM), was successfully obtained. An ex vivo experiment in mice indicated that (S)-5j (RS-1259) simultaneously inhibited AChE and SERT in the brain following an oral administration. The simultaneous elevation of extracellular levels of acetylcholine and serotonin in the rat hippocampus was actually confirmed by microdialysis.

To date, acetylcholinesterase (AChE) inhibitors have been clinically effective drugs for the palliative treatment of Alzheimer's disease, but their clinical efficacy is limited, mainly due to their adverse effects on peripheral organs. Since patients of Alzheimer's disease often exhibit depression as well as memory impairment, dual inhibitors of AChE and serotonin transporter (SERT) would be a better therapeutic method. Anti-depressive effects based on SERT inhibition would reduce the dose-related side effects of AChE inhibitors. Such dual inhibitors were designed by the hybridization of rivastigmine and fluoxetine based on a hypothetical model of the AChE active site. Various derivatives were synthesized and evaluated for their in vitro inhibition, and then (S)-5j (RS-1259), which possessed balanced inhibitory activities of AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM), was successfully obtained. An ex vivo experiment in mice indicated that (S)-5j (RS-1259) simultaneously inhibited AChE and SERT in the brain following an oral administration. The simultaneous elevation of extracellular levels of acetylcholine and serotonin in the rat hippocampus was actually confirmed by microdialysis.

Alzheimer's disease (AD) has been treated with acetylcholinesterase (AChE) inhibitors such as donepezil. However, the clinical usefulness of AChE inhibitors is limited mainly due to their adverse peripheral effects. Depression seen in AD patients has been treated with serotonin transporter (SERT) inhibitors. We considered that combining SERT and AChE inhibition could improve the clinical usefulness of AChE inhibitors. In a previous paper, we found a potential dual inhibitor, 1, of AChE (IC50=101 nM) and SERT (IC50=42 nM), but its AChE inhibition activity was less than donepezil (IC50=10 nM). Here, we report the conformationally restricted (R)-18a considerably enhanced inhibitory activity against AChE (IC50=14 nM) and SERT (IC50=6 nM).

We have designed and synthesized a dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT) as a novel class of treatment drugs for Alzheimer's disease on the basis of a hypothetical model of the AChE active site. Dual inhibitions of AChE and SERT would bring about greater therapeutic effects than AChE inhibition alone and avoid adverse peripheral effects caused by excessive AChE inhibition. Compound ( S )- 6j exhibited potent inhibitory activities against AChE (IC 50 =101 nM) and SERT (IC 50 =42 nM). Furthermore, ( S )- 6j showed inhibitory activities of both AChE and SERT in mice brain following oral administration.

This study investigated the relationship between spatial working memory and hippocampal long-term potentiation (LTP) using the allocentric place discrimination task (APDT) in rats, in which the selection accuracy is a good index for spatial working memory. Either the selective M1 muscarinic receptor antagonist pirenzepine (50 microg) or the choline uptake inhibitor hemicholinium-3 (5 microg) impaired APDT selection accuracy, but neither affected the induction of LTP in the hippocampal CA1 region in anesthetized rats. In contrast, the selective N-methyl-D-aspartate receptor antagonist D-amino-5-phosphonopentanoate (200 nmol) did not impair APDT selection accuracy but completely blocked hippocampal CA1 LTP. These results suggest that spatial working memory is independent of hippocampal CA1 LTP and that the central cholinergic system is involved in spatial working memory, but not through the modulation of hippocampal CAI LTP.

The allocentric place discrimination task (APDT) is useful in evaluating working memory separately from and simultaneously with motivation, motor and sensory ability. Muscarinic acetylcholine receptor antagonist scopolamine has been shown to selectively impair the accuracy of APDT without changing swimming speed, distance, and still time. For further evaluation of other neurotransmitters' roles in the APDT, pharmacological manipulations were performed. Neither diazepam 3.0 mg/kg, mecamylamine 10 mg/kg, haloperidol 0.5 mg/kg, nor 8-OH DPAT 1.0 mg/kg affected accuracy of place discrimination. Two kinds of responses were observed following the administration of MK-801 0.3 mg/kg: the accuracy of rats for longer swimming distance tended to decrease, and the accuracy of rats for normal swimming distance did not change. Therefore, NM-801 did not seem to affect the working memory selectively. In addition, neither flumazenil 10 mg/kg, ondansetron 0.3 mg/kg nor R(−)-α-metylhistamine 10 mg/kg attenuated the scopolamine-induced deficits. These results suggest that the central muscarinic receptors are selectively and highly important in the APDT.

Central muscarinic cholinergic involvement in classical conditioning of eyeblink responses was determined in trace and delay paradigms. Rabbits were trained on a trace procedure in which a 250-ms tone conditioned stimulus (CS) and a 100-ms air-puff unconditioned stimulus (UCS) were presented with a 500-ms trace interval. Each training session day consisted of ten tone alone, ten air-puff alone and 80 paired CS-UCS trials. Scopolamine hydrochloride at doses of 0.03 and 0.1 mg/0.5 ml per kg, s.c. dose-dependently disrupted acquisition of conditioned responses. Rabbits that were treated with scopolamine and failed to learn showed a gradual increase in conditioned responses during an additional training period with saline injections and no transfer from earlier training. Scopolamine methyl bromide, which does not appreciably cross the blood-brain barrier, showed no effects in the trace conditioning paradigm at a dose of 0.1 mg/kg, s.c., indicating central cholinergic blockade is responsible for the suppressive effect of scopolamine. Scopolamine hydrochloride at a dose of 0.1 mg/kg, s.c. did not block acquisition in the delay procedure with a 250-ms inter-stimulus interval, although the rate of acquisition was somewhat reduced by the drug. These data are the first to demonstrate that classical conditioning of the eyeblink response in the trace procedure is highly sensitive to central cholinergic deficits.

1. 1. A putative agonist for the strychnine-sensitive glycine receptor picolinic acid was tested for its anticonvulsant activities in mice and muscle-relaxant activities in rats and compared with indole-2-carboxylic acid (I2CA), an antagonist for the strychnine-insensitive glycine receptor. Their effects on segmental reflexes in the cat spinal cord were examined to elucidate their sites of action. 2. 2. Picolinic acid (200 and 400 mg/kg IP) delayed the onsets of strychnine- but not pentylenetetrazole-induced seizures. It delayed the onsets of bicuculline-induced seizures only at the higher dose. I2CA (200 and 400 mg/kg IP) delayed the onsets of these 3 kinds of seizures. Both compounds reduced muscle tone in rat decerebrate rigidity at a dose of 100 mg/kg IV. 3. 3. Picolinate methylester, a picolinate derivative with higher lipophilicity, depressed spinal reflexes in both intact and spinalized cats at cumulative doses of 25 to 200 mg/kg IV. I2CA (50 mg/kg IV) inhibited spinal reflexes only in intact preparations. 4. 4. These results suggest that the anticonvulsant and muscle-relaxant activities of picolinic acid (PA) are due to inhibition of spinal neurons, but that I2CA selectively affects supraspinal structures.

We studied the modulatory effect of (R)-4-chloro-2-(2-hydroxy-3-morpholinopropyl)-5-phenyl-4-isoxaz olin-3-one hydrochloride (CS-722), a centrally acting muscle relaxant, on synaptic transmission in the ventral horn neurons of neonatal rat lumbar spinal cord in slices using whole cell recording techniques. Pharmacologically isolated excitatory or inhibitory postsynaptic currents (EPSCs and IPSCs) were evoked by stimulating neighboring neurons. CS-722 preferentially enhanced IPSCs with little effects on EPSCs. Moreover, CS-722 reduced paired pulse facilitation of gamma-aminobutyric acid (GABA)-mediated IPSCs, suggesting its action on the presynaptic terminal. Preferential facilitatory effects on inhibitory synaptic transmission seem to be one of the mechanisms underlying muscle relaxation of this compound.

Whole-cell patch-clamp recording of GABAergic inhibitory postsynaptic currents (IPSCs) were made in ventral horn neurons of neonatal rat lumbar spinal cord in slice. In contrast to the hippocampus where paired pulse depression is reported to be observed for GABAergic IPSCs, double pulse stimulation of GABAergic inputs resulted in enhancement in the amplitude of the second IPSC in the spinal ventral horn. The facilitation ratio was decreased during enhanced synaptic transmission by increasing Ca2+ concentration in the external recording solution. Baclofen and adenosine, which are reported to depress synaptic transmission by presynaptic mechanisms, depressed IPSCs and increased the facilitation ratio. A postsynaptic manipulation such as application of bicuculline or changing the driving force did not affect the facilitation ratio. These results suggest that paired pulse facilitation of GABAergic IPSCs observed in neonatal rat spinal ventral horn appears to be based upon a mechanism similar to that underlying frequency-dependent facilitation of excitatory synaptic transmission, and is sensitive to presynaptic changes in synaptic strength.

Sites of the muscle relaxant action of CS-722 were investigated in rats. Doses injected intra-4th ventricularly (i.c.v.) or intrathecally (i.t.) were determined according to measurements of CS-722 concentration in the brain stem and the spinal cord after systemic administration. When administered i.c.v. (50 and 100 micrograms) or i.t. (200 and 400 micrograms), CS-722 reduced the radio frequency decerebrate rigidity, although the effect after i.c.v. injection was transient. These results indicate that CS-722 exerts its muscle relaxant action by affecting both the supraspinal structure and the spinal cord. Spinal reflexes were not affected by CS-722 injected i.c.v. It seems that muscle relaxation is not always accompanied by impairment of the spinal reflex.

By means of unit recording and electrophoretic application, the effect of picolinic acid on feline spinal interneurons in situ was studied in comparison with glycine. Picolinic acid inhibited neuronal firing in 60% of neurons and in some cases the inhibitory actions were antagonized by strychnine. Inhibition of firing by glycine, which was also strychnine-sensitive, was reduced in case of concomitant administration of picolinic acid. These results suggest that picolinic acid might act as a glycine agonist at trychnine-sensitive receptors.

Recurrent inhibition of the spinal monosynaptic reflex (MSR) elicited by conditioning stimulation of the ventral root in anesthetized rats was weaker than both the recurrent inhibition of the disynaptic reflex and the inhibition of the MSR elicited by conditioning stimulation of the adjacent dorsal root. Among these 3 inhibitions, the recurrent inhibition of the MSR was enhanced to a markedly greater extent by a preceding stimulation of the medullary raphe´nucleus than were the other inhibitions. The magnitude of the enhancement of the recurrent inhibition of MSR also was much greater when the medullary stimulation was delivered 20 ms prior to the ventral root activation, as compared with a 30-ms interval. Recurrent inhibition of the MSR was enhanced by intravenous injection of lysergic acid diethylamide (LSD); however, the enhanced effect on recurrent inhibition elicited by stimulation of the raphe´nucleus was not attenuated by the drug. These results suggest that there is a non-serotonergic, descending pathway which is capable of modulating motor output solely by means of recurrent inhibition of the MSR.

1. 1. Conditioning stimulation of the nucleus raphe in the rat medulla enhanced the monosynaptic reflex (MSR) and depressed the polysynaptic reflex (PSR) in separate time courses. 2. 2. The PSR decreasing effect was reduced in preparations pretreated with reserpine, PCPA, or 5,6-DHT. The MSR increasing effect was not altered by these three pretreatments. 3. 3. Intravenously administered LSD but not methysergide reduced the PSR decreasing effect with no influence on the MSR increasing effect. These two drugs had similar effects on the unconditioned responses; the MSR was decreased and the PSR was increased. 4. 4. Thus, the PSR decreasing effect seems to be mediated via a serotonergic pathway probably descending from the nucleus raphe.

In in situ experiments on spinal rats transected at the C1 level and on non-spinal rats, muscimol (3 mg/kg, i.v.) depolarized the primary afferent fibers and diazepam (3 mg/kg, i.v.) potentiated this effect of muscimol. When diazepam was given for 14 days, the effect of muscimol was significantly reduced in non-spinal, but not in spinal rats. These findings suggest that diazepam probably interacts with GABA-ergic mechanisms more closely in supraspinal regions of the rat.

Spinal reflex and neuromuscular functions were investigated in the beagles ingesting clioquinol over a long period. The twitch tension, the refractory period, the summation of contraction and the discharge of the muscle spindles of the gastrocnemius muscle were not significantly damaged in the treated beagles, whereas the response with long latency of the spinal reflex disappeared completely during the administration not in the control but in the treated dogs. The meanings of disappearance of the spinal reflex in the treated dogs are discussed in relation to the degenerative change in the fasciculus gracilis at the cervical level of the spinal cord.

Gas chromatographic procedure for the determination of sodium cyclamate was studied. According to recent studies, it has been reported that sodium cyclamate reacts with nitrite and produces cyclohexanol, cyclohexanone and cyclohexene. The author's experiment showed that the major product of the reaction was cyclohexyl nitrite, together with some cyclohexanol and slight cyclohexene in the presence of excess nitrite at temp. below 5°C.Using the blended liquid phase column consisting of DC-550 and DEGS (10% of each) on Gaschrom P (60/80mesh) at 60°C, carrier gas nitrogen (60ml/min) and a flame ionization detector, cyclohexyl nitrite and cyclohexanol were investigated. The results are given in Fig. 1- (C) and 2.Sodium cyclamate was determined on the basis of the cyclohexyl nitrite. The cyclohexyl nitrite was prepared by treating varying amounts of sodium cyclamate with 5ml of 10% sodium nitrite and 4ml of 10% sulfuric acid in ice water and it was extracted with 10ml of n-hexane containing 0.25 v/v% n-decane as internal standard and then injected to the Shimazu gas chromatograph.The relation between peak heights of cyclohexyl nitrite and amounts of sodium cyclamate was linear in the range of 20-600mg of the latter, and in this case, cyclohexanol did not appeared. But, when excessive amounts (>800mg) of sodium cyclamate were present, the yield of cyclohexyl nitrite diminished and that of cyclohexanol increased. The minimum limit of detection was approximately 0.1μg as sodium cyclamate.

Effects of drugs affecting the central nervous system on the spinal reflexes and the descending modulations of the reflexes were simultaneously evaluated in rats. Mono- and polysynaptic reflexes were, respectively, increased and decreased by conditioning stimulation of the nucleus raphe in the medulla, with a deflection in resting dorsal root potential being evoked by the stimulation. Baclofen exclusively depressed the segmental responses without affecting the descending modulatory systems. On the other hand, KW-6629 (7-chloro-N,N,3-trimethylbenzo[b]furan-2-carboxamide) significantly depressed the descending modulations without affecting the segmental responses which were sensitive to baclofen. Diazepam and suriclone reduced the descending influence without affecting the ventral root reflexes. Tolperisone and chlorphenesin carbamate as well as tizanidine depressed not only the segmental reflexes but also the descending modulations. Thus, sites of drug actions were estimated.

It was examined whether the anticonvulsant activity of valproic acid (di-n-propylacetic acid, DPA) was exerted via the inhibitory GABA system in the central nervous systems (CNS). Dorsal root reflexes (DR-DRR) were not augmented but intensely suppressed following the administration of DPA (50 and 200 mg/kg, i.v.). DPA did not depolarize the resting dorsal root potentials. These electrophysiological findings may indicate that DPA does not potentiate GABA system in the spinal cord of rats. DPA (1 and 10 mM) neither affected the binding of 2 or 40 nM of [3H]muscimol nor the binding of [3H]diazepam in the rat brain membranes; the same concentrations of DPA did not affect the enhancement of binding of [3H]diazepam induced by the addition of GABA (100 microM). The result obtained from binding assays indicates that DPA does not interact with the GABA-benzodiazepine receptor complex. The findings in the present study do not support the proposal that the anticonvulsant action of DPA is exerted by potentiation of the GABA system in the CNS.

Intraspinal microinjection into the lumbar spinal segment (L5 was attempted to evaluate the direct effects of drugs or transmitters on the spinal reflexes. The micropipettes (tip diameter 40-50 μm) filled with drug solution were inserted into the ventral or dorsal horn, and the volume of 0.1-0.2 μl was injected. Control solution of high pH, low pH or high osmolarity had no effect on the monosynaptic reflex (MSR) or polysynaptic reflex (PSR). L-Glutamic acid microinjected into the ventral horn depolarized the resting ventral root potential, and GABA microinjected into the dorsal horn depolarized the resting dorsal root potential. Using this method, we investigated the role of serotonin in the spinal cord. Serotonin (2nM) microinjected into the ventral horn had no influence on MSR, but facilitated PSR. This effect was suppressed by methysergide (s.c.) and LSD (i.v.). Serotonin microinjected into the dorsal horn had no influence on MSR, but suppressed PSR. This effect was reduced by methysergide, but not influenced by LSD. It was thus suggested that serotonin had no direct effect on motoneurons and that there are at least two different serotonergic mechanisms in the interneurons of the rat spinal cord.