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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]

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.

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.

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.

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.

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.

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.

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.