A novel prolyl endopeptidase inhibitor, JTP-4819, with potential for treating Alzheimer's disease

ELSEVIER

Behavioural Brain Research 83 (1997) 147-151

BEHAVI0URAL BRAIN RESEARCH

Research report

A novel prolyl endopeptidase inhibitor, JTP-4819, with potential for treating Alzheimer's disease Katsuo Toide *, Masahiko Shinoda, Yohko Iwamoto, Takako Fujiwara, Kazuhiro Okamiya, Atsuhiro Uemura Central Pharmaceutical Research Institute, Japan Tobacco Inc., I ol, Murasaki-cho, Takatsuki, Osaka 569, Japan

Received 8 July 1995; revised 20 November 1995; accepted 22 November 1995

Abstract The pharmacological actions of JTP-4819, a new prolyl endopeptidase (PEP) inhibitor targeted for the treatment of Alzheimer's disease, are reviewed with respect to its effects on PEP activity, brain neurotransmitters, and memory-related behaviour in rats. JTP-4819 was shown to be a very potent and specific inhibitor of PEP. At nanomolar concentration, JTP-4819 inhibited the degradation of substance P, arginine-vasopressin, and thyrotropin-releasing hormone by PEP in supernatants of the rat cerebral cortex and hippocampus. Repeated administration of JTP-4819 reversed the aging-induced decrease in brain substance P-like and thyrotropin-releasing hormone-like immunoreactivity, suggesting that this drug may be able to improve the imbalance of peptidergic neuronal systems that develops with senescense by inhibiting PEP activity. JTP-4819 increased acetylcholine release from the frontal cortex and hippocampus, regions closely associated with memory, in both young and aged rats. In addition, it improved performance in several memory and learning-related tests (e.g., the Morris water maze task in aged or MCA-occluded rats and the passive avoidance test). This memory-enhancing effect of JTP-4819 may result from prevention of the metabolic degradation of brain neuropeptides by PEP as well as from the enhancement of acetylcholine release. Taken together, these unique and potent pharmacological actions of JTP-4819 suggest that it may have the potential to be used for treating Alzheimer's disease. Keywords: Prolyl endopeptidase; JTP-4819; Substance P; Arginine-vasopressin; Thyrotropin-releasinghormone; Acetylcholine;Cerebral cortex and hippocampus; Morris water maze task and passive avoidance task

1. Introduction Alzheimer's disease is a progressive neurodegenerative disorder, which is characterized clinically by progressive cognitive deterioration and pathologically by neurofibrillary tangles and senile plaques [22,29], as well as the degeneration of cortical and hippocampal cholinergic neurons [2,4,6,45]. Investigation of damage to the forebrain cholinergic system has been a major focus of attempts to understand the cognitive deficits in Alzheimer's disease and treatment has so far been primarily based on the use of cholinergic agents [27]. However, several recent studies have shown that some neuropeptide-containing neuron populations are abnormal in this disease [5,7,12,13,16,18,24,26,32], suggesting that enhancement of peptidergic neurotransmission * Correspondingauthor. Tel.: 81-726-81-9700;Fax: 81-726-81-9722.

by the inhibition of certain peptidases may possibly have therapeutic value. Many biological neuropeptides contain proline within their amino acid sequence. Prolyl endopeptidase (PEP, EC 3.4.21.26) hydrolyzes peptide bonds at the carboxyl terminus of L-proline and was first found as an oxytocin-inactivating enzyme in the human uterus 1-43]. P E P has been proposed to play a role in the metabolism of proline-containing neuropeptides like substance P (SP), arginine-vasopressin (AVP), and thyrotropin-releasing hormone (TRH) [35,46,48, 49], and there is considerable evidence that these proline-containing neuropeptides are capable of enhancing learning and memory [9,10,15,17,19,20,28,33]. On the other hand, there have been contradictory reports concerning brain levels of P E P activity in Alzheimer's disease [1,14,21]. We have developed a novel P E P inhibitor, (S)-2-{[(S)-2-(hydroxyacetyl)-lpyrrolidinyl ] carbonyl} -N-(phenylmethyl )- 1-pyrrolidi-

0166.-4328/97/$17.00Copyright © 1997 ElsevierScienceB.V. All rights reserved PH S0166-4328(96)00107-6

148

K Toide et al./Behavioural Brain Research 83 (1997) 147-151

necarboxamide (JTP-4819, Fig. 1), and have summarized in Table 1 its pharmacological actions with its potential value for the treatment of Alzheimer's disease.

2. Pharmacological studies 2.1. PEP inhibition There are several reports concerning the subcellular distribution of PEP and its specific activity appears to be higher in the cytosol than in other cellular fractions [8,11 ]. Our investigations demonstrated that JTP-4819 produced concentration-dependent inhibition (10 - t z 10 -7 M) of PEP activity in the cytosolic fraction of rat brain homogenates, with an ICso value of 0.83 nM. JTP-4819 also showed strong inhibition of PEP (ICso =0.65 nM) after incubation with rat plasma [38], suggesting its high stability in plasma. An ex vivo study showed that JTP-4819 (3mg/kg, p.o.) significantly reduced PEP activity to 30-50% in the cerebral cortex,

O

O

O

Fig. 1. Structure of JTP-4819.

hippocampus, corpus striatum, midbrain+hypothalamus, medulla oblongata, and cerebellum of rats at 1 and 3 h after administration [38], suggesting its rapid uptake into the brain. The existence of many proline-specific endopeptidases and exopeptidases has been demonstrated in the brain and other tissues [44]. JTP-4819 showed no inhibition of various proline-specific enzymes other than PEP purified from microbes, such as dipeptidyl aminopeptidase IV, proline iminopeptidase, aminopeptidase P, prolidase, and carboxypeptidase P, even at a high concentration of 100~tM [38], suggesting its marked specificity for the target enzyme.

2.2. Inhibition of neuropeptide degradation It has been suggested that PEP participates in the metabolism of SP, AVP and TRH in the brain [46]. Accordingly, we investigated whether or not JTP-4819 inhibited the degradation of these proline-containing neuropeptides by blocking PEP activity. JTP-4819 showed concentration-dependent inhibition (10 -1210 -6 M) of the in vitro degradation of SP, AVP and TRH in supernatants from rat cerebral cortex and hippocampus, with the IC50 values being 3.4 and 3.3 nM, 2.1 and 2.8 nM, and 1.4 and 1.9 nM, respectively [37]. These results suggested that JTP-4819 could inhibit the degradation of proline-containing neuropeptides in the brain secondary to the PEP inhibition.

Table 1 Summary of the pharmacological actions of JTP-4819 Parameters

Species, Brain areas

Results

Dose or concentration

Ref.

rats whole brain cerebral cortex, hippocampus, etc a microbes b

inhibition (IC5o) inhibition no effect

0.83 nM 3.0 mg/kg, p.o. 100 laM <

[38] [38] [ 38]

rats cerebral cortex and hippocampus cerebral cortex and hippocampus cerebral cortex and hippocampus

inhibition (IC~o) inhibition (IC~0) inhibition (ICso)

3.4 and 3.3 nM 2.1 and 2.8 nM 1.4 and 1.9 nM

[37] [37] [37]

aged rats cerebral cortex and hippocampus cerebral cortex and hippocampus cerebral cortex and hippocampus

increase increase no effect

1.0 mg/kg, p.o. 1.0 mg/kg, p.o. 0.3 and/or 1.0 mg/kg, p.o.

[40] [31 ] [40]

young and aged rats frontal cortex and hippocampus

increase

1.0 and/or 3.0 mg/kg, p.o.

[38]

aged rats middle cerebral artery occluded rats scopolamine-treated rats single administration coadministration of each neuropeptide

potentiation potentiation potentiation potentiation

1.0 mg/kg, p.o. 1.0 mg/kg, p.o. 1.0--10.0 mg/kg, p.o. 0.3 and 1.0 mg/kg, p.o.

[42] [ 30] [ 38] [38]

PEP activity In vitro Ex vivo PEP specificity Neuropeptide degradation SP AVP TRH Nearopeptide contents SP TRH AVP ACh release Microdialysis Memory-related behaviour Morris water maze task Passive avoidance task

aCerebral cortex, hippocampus, corpus striatum, mid brain + hypothalamus, medulla oblongata, and cerebellum. bMicrobes: F. meningosepticum, B. coaglans, E. coil, X. maltophilia.

K. Toide et al./Behavioural Brain Research 83 (1997) 147-151

2.3. Brain neuropeptide levels

Several studies of brain neuropeptides in Alzheimer's disease have revealed abnormalities [5,7,12,13,18,24, 26,32]. Based on these findings, we investigated the changes of neuropeptide levels in the cerebral cortex and hippocampus of rats with aging as well as the effects of JTP-4819. Hippocampal SP-like immunoreactivity (LI) was found to be significantly decreased by about 50% in aged rats (24 months old) compared to young rats (3 months old), whereas the cortical SP-LI content only tended to decrease. Cortical and hippocampal TRH-LI were also significantly decreased by about 60% in aged rats, but AVP-LI showed almost no change in both brain regions [31,40]. Oral administration of JTP-4819 (0.1-1 mg/kg for 21 days) produced a dose-dependent increase in brain SP-LI, and this was significant in both the cortex and hippocampus at a dose of 1 mg/kg [40]. Administration of JTP-4819 for 21 days also significantly increased cortical TRH-LI (1 mg/kg) and hippocampal TRH-LI (0.3 and 1 mg/kg) [31]. However, JTP-4819 had no significant effect on AVP-LI in both brain regions [40]. Since it has been reported that SP prevents 13-amyloidinduced neuronal death [23,47] and that TRH has a neurotrophic effect [3], the increase in brain SP-LI and TRH-LI produced by administration of JTP-4819 in aged rats may prevent the neuronal death or have a cytoprotective effect as well as enhancing memory [30,38,42]. These findings suggested that JTP-4819 may improve the imbalance of peptidergic neuronal systems that develops with senescense by inhibiting PEP activity. 2.4. Brain acetylcholine (ACh) release: Microdialysis study

Although the etiology of Alzheimer's disease is complex, the most marked and consistent neurochemical deficit is degeneration of the cholinergic system in the cerebral cortex and hippocampus [2,4,6,45]. Therefore, activation of the central cholinergic neurons by the drug could by therapeutically useful in treating Alzheimer's disease. In young rats (3 months old), JTP-4819 (1 and 3 mg/kg, p.o.) caused a significant increase in cortical ACh release (<160%) and hippocampal ACh release (<90%) at 45 min after administration. In aged rats (24 months old), JTP-4819 (3 mg/kg, p.o.) also significantly increased cortical ACh release (<40%1 and hippocampal ACh release (<60%) at 15 and 30 min after administration, respectively [38]. Thus, this drug potentiated ACh release from the frontal cortex and hippocampus, regions that are closely associated with memory [2,4,6,45], in both young and aged rats. We have previously shown

149

that TRH and SP [39,41] as well as AVP (unpublished data) increase ACh release from the frontal cortex and hippocampus, suggesting that this ACh-releasing effect of JTP-4819 may be mediated via the increase in TRH, SP and AVP levels secondary to PEP inhibition. 2.5. Memory-related behaviour 2.5.1. Morris water maze task in aged rats and middle cerebral artery ( M CA )-occluded rats It has been clearly demonstrated that the Morris water maze task is useful for evaluating spatial memory in rodents [25]. We found that both aged and MCAoccluded rats showed significant prolongation of escape latency in the Morris water maze task, suggesting the presence of spatial memory impairment [30,42]. Administration of JTP-4819 (lmg/kg) significantly shortened the escape latency in aged rats from day 14 onwards [42]. We previously demonstrated that central cholinergic and peptidergic neuronal function is impaired in aged rats compared to young rats [31,36,40]. Therefore, it is possible that the ameliorating effect of JTP-4819 on spatial memory impairment in aged rats may be based on the improvement of cholinergic and peptidergic neuronal function. Furthermore, administration of JTP-4819 !1 mg/kg) significantly shortened the escape latency in the MCAoccluded rats from day 10 onwards [30]. It is well documented that the MCA-occlusion model is useful for evaluating the effects of cerebral ischemia in rodents [34]. Therefore, although the improvement of spatial memory impairment by JTP-4819 in MCA-occluded rats may be based on the activation of cholinergic and peptidergic neurons, further studies are needed to clarify the actual mechanism involved. 2.5.2. Passive avoidance test In the one-trial passive avoidance test in rats with scopolamine-induced amnesia, JTP-4819 significantly improved the retention time when doses of 1 and 3 mg/kg were given 1 h before acquisition as well as when doses of 3 and 10 mg/kg were given I h before retention [38]. Based on these results, it seems important to investigate in detail how JTP-4819 potentiates the acquisition, consolidation, and retrival processes of memory. We also found that subcutaneous administration of SP (101ag/kg immediately after acquisition), AVP (10 lag/kg at 1 h before retentionl, and TRH (3 mg/kg at 1 h before retention) caused the reversal of scopolamine-induced amnesia, although each neuropeptide had a bell-shaped dose-response curve [38]. Strikingly, coadministration of oral JTP-4819 (0.3 mg/kg at 1 h before acquisition or 1 mg/kg at 1 h before retention) with subcutaneous SP (3 lag/kg), AVP (3 lag/kg), or TRH (1 mg/kg), at doses where each agent

150

K. Toide et al./Behavioural Brain Research 83 (1997) 147-151

alone had no effect, caused i m p r o v e m e n t of the retention time in scopolamine-treated rats [ 38]. Since these neuropeptides increase A C h release from the frontal cortex and h i p p o c a m p u s in rats, as m e n t i o n e d above, the antiamnesic action of JTP-4819 or the neuropeptides m a y be mediated via a n t a g o n i s m of the effect of scopolamine which is an anti-cholinergic agent. Thus, the m e m o r y - e n h a n c i n g activity of JTP-4819 m a y result from b o t h prevention of the metabolic degradation of SP, A V P and T R H by P E P as well as from the p r o m o t i o n of A C h release.

3. Summary JTP-4819 was s h o w n to strongly inhibit rat brain prolyl endopeptidase ( P E P ) b o t h in vitro ( I C s o = 0 . 8 3 n M ) and in vivo (3 m g / k g , p.o.). It exhibited a high specificity for P E P and showed g o o d stability in rat plasma. At n a n o m o l a r concentrations, JTP-4819 inhibited the degradation of substance P (SP), argininevasopressin (AVP), a n d thyrotropin-releasing h o r m o n e ( T R H ) by P E P in rat cortical and h i p p o c a m p a l supernatants. H i p p o c a m p a l SP immunoreactivity as well as cortical and h i p p o c a m p a l T R H immunoreactivity were significantly decreased in aged rats, and these changes were reversed by repeated oral administration of JTP-4819 (0.3 a n d / o r 1 mg/kg). J T P - 4 8 1 9 also caused a significant increase in A C h release from the frontal cortex and h i p p o c a m p u s at oral doses of 1 a n d / o r 3 m g / k g in b o t h y o u n g a n d aged rats. Repeated oral administration of J T P - 4 8 1 9 i m p r o v e d m e m o r y deficits s h o w n by aged rats and rats with middle cerebral artery occlusion ( b o t h at 1 m g / k g ) in the Morris water maze task, and also significantly p r o l o n g e d the retention time of rats with scopolamine-induced amnesia in the passive avoidance test ( 1-10 mg/kg). In addition, coadministration of J T P - 4 8 1 9 with SP, AVP, or T R H (at doses where each agent alone had no effect) i m p r o v e d the retention time in the passive avoidance test. These results suggest that J T P - 4 8 1 9 ameliorates m e m o r y impairment by activating cerebral peptidergic and cholinergic neurons via inhibition of P E P , thus possibly being of potential therapeutic value for Alzheimer's disease.

References El] Aoyagi, T., Wada, T., Nagai, M., Kojima, F., Harada, S., Takeuchi, T., Takahashi, H., Hirokawa, K. and Tsumita, T., Deficiency of kallikrein-like enzyme activities in cerebral tissue of patients with Alzheimer's disease, Experientia, 46 (1990) 94-97. [2] Araujo, D.M., Lapcliak, P.A., Robitaille, Y., Gautheir, S. and Quirion, R., Differential alteration of various cholinergic markers in cortical and subcortical regions of human brain in Alzheimer's disease, J. Neurochem., 50 (1988) 1914-1923.

[3] Banda, R.W., Means, E.D. and Scherch, H.M., Trophic effect of thyrotropin-releasing hormone in murine ventral horn neuronal cultures, Ann. NYAcad. Sci., 553 (1989) 588-589. [4] Bartus, R.T., Dean III, R.L., Beer, B. and Lippa, A.S., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408-417. [ 5] Beal, M.F. and Mazurek, M.F., Substance P-like immunoreactivity is reduced in Alzheimer's disease cerebral cortex, Neurology, 37 (1987) 1205-1209. [6] Coyle, J.T., Donald, L.P. and Delong, M.R., Alzheimer's disease; A disorder of cortical cholinergic innvervation, Science, 219 (1983) 1184-1190. [7] Crystal, H.A. and Davies, P., Cortical Substance P like immunoreactivity in case of Alzheimer's disease and senile dementia of the Alzheimer type, J. Neurochem., 38 (1982) 1781-1784. [8] Dalmaz, C., Netto, C.A., Volkmer, N., Dias, R.D. and Izquierdo, I., Distribution of proline endopeptidase activity in sub-synaptosomal fractions of rat hypothalamus. Brazilian J. Med. Biol. Res., 19 (1986) 685-690. [9] De Wied, D., Gaffori, O., Ree, J.M. and Jong, W., Central target for the behavioral effects of vasopressin neuropeptides, Nature, 308 (1984) 276-280. [10] De Wied, D., The importance of vasopressin in memory, Trends Neurosci., 7 (1984) 62-64. [ 11] Dresdner, K., Barker, L.A., Orlowski, M. and Wilk, S. Subcellular distribution of prolyt endopeptidase and cation-sensitive neutral endopeptidase in rabbit brain, J. Neurochem., 38 (1982) 1151-1154. [12] Ferrier, I.N., Cross, A.J., Johnson, J.A., Roberts, G.W., Crow, T.J., Corsellis, J.A.N., Lee, Y.C., O'Shaughnessy, D., Adrian, T.E., Mcgregor, G.P., Baracese-Hamiiton, A.J. and Bloom, S.R., Neuropeptides in Alzheimer type dementia, J. Neurol. Sci., 62 (1983) 159-170. [ 13] Fujiyoshi, K., Suga, H., Okamoto, K., Nakamura, S. and Kameyama, M., Reduction of arginine-vasopressin in the cerebral cortex in Alzheimer type senile dementia, J. Neurol. Neurosurg. Psychiatry, 50 (1987) 929-932. [14] Gibson, A., Edwardson, J.A. and Mcdermot, T., Postmortem levels of some brain peptidases in Alzheimer's disease: reduction in proline endopeptidase activity in cerebral cortex, Neurosci. Res. Commun., 9 (1991) 73-81. [ 15] G riffiths, E.C. Clinical applications of thyrotropin-releasing hormone, Clin. Sci., 73 (1987) 449-457. [ 16] Griffiths, E.C., Baris, C., Visser, T.J. and Klootwijk, W., Thyrotropin-releasing hormone inactivation by human postmortem brain, Regul. Pept., 10 (1985) 145-155. [ 17] Hasen/Shrl, R.U., Huston, J.P. and Schurman, T., Neuropeptide substance P improves water maze performance in aged rats, Psychopharmacology, 101 (1990) 23-26. [ 18] Husain, M.M. and Nemeroff, C.B., Neuropeptides and Alzheimer's disease, J. Am. Geriatr. Soc., 38 (1990) 918-925. [19] Huston, J.P. and Hasen/~hrl, R.U., The role of neuropeptides in learning: focus on the neurokinin substance P, Behat,. Brain Res., 66 11995) 117-127. [20] Huston, J.P., Hasent~hrl, R.U., Gerhaldt, P. and Schwarting, RK.W., Sequence-specific effects of neurokinin substance P on memory, reinforcement and brain dopamine activity, Psychopharmacology, 112 (1993) 147-162. [21] lchai, C., Chevallier, N., Delaere, P., Dournaud, P., Epelbaum, J., Hauw, J.J., Vincent, J.P. and Checler, F., Influence of regionspecific alterations of neuropeptidase content on the catabolic fates of neuropeptides in Alzheimer's disease, J. Neurochem., 62 (1994) 645-65. [22] Katzman, R. Alzheimer's Disease, N. Eng. J. Med., 314 (1986) 964-973. [23] KowaU, N.W., Beal, M.F., Busciglio, J., Duffy, L.K. and Yankner, B.A., An in vivo model for the neurodegenerative effects of 13

K. Toide et al./Behavioural Brain Research 83 (1997) 147-151

[24]

[25] [26]

[27] [28]

[29] [30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

amyloid and protection by Substance P, Proc. Natl. Acad. Sci. USA, 88 ( 1991 ) 7247-7251. Mazurek, M.F., Beal, M.F., Bird, E.D. and Martin, J.B., Vasopressin in Alzheimer's disease: a study of postmortem brain concentrations, Ann. Neurol., 20 (1986) 665-670. Morris, R.G.M., Spatial localization does not require the presence of local cues, Learn. Motiv.. 12 (1981) 239-260. Rossor, M.N., Emson, P.C., Dawbarn, D., Mountjoy, C.Q. and Roth, M., Neuropeptides and dementia, Prog. Brain Res., 66 (1986) 143-159. Sarter, M., Taking stock of cognition enhancers, Trends Pharmacol. Sci., 12 ( 1991 ) 456-461. Schlesinger, K., Lipsitz, D.U., Peck, P.L., Pelleymounter, M.A., Stewart, J.M. and Chase, T.N., Substance P enhancement of passive and active avoidance conditioning in mice, Pharmacol. Biochem. Behav., 19 (1983) 655-661. Selkoe, D.J., Deciphering Alzheimer's disease: the amyloid precursor protein yields new clues, Science, 248 (1990) 1058-1060. Shinoda, M., Matsuo, A. and Toide, K., Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on memory related behaviors and brain regional neuropeptides contents in MCA-occluded rats, Jpn. J. Psychopharmacol. (Suppl.), (1995) pp. 81. Shinoda, M., Okamiya, K. and Toide, K., Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on thyrotropin-releasing hormone-like immunoreactivity in the cerebral cortex and hippocampus of aged rats, Jpn. J. Pharmacol., 69 (1995) 273-276. Sorensen, P.S., Hammer, M., Vorstrup, S. and Gjerris, F., CSF and plasma vasopressin concentrations in dementia, J. Neurol. Neurosurg. Psychiatry, 46 (1983) 911-916. Taira, K. and Kaneto, H., Experimental models for studying the avoidance response in mice and the anti-amnesic effect of prolyl endopeptidase inhibitors, Folia Pharmacol. Japon, 89 (1987) 243-252. Tamura, A., Graham, D.I., Mcculloch, J. and Teasdale, G.M., Focal cerebral ischemia in the rat. 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion, J. Cereb. Blood Flow Metab., 1 ( 1981 ) 53-60. Taylor. W.L. and Dixon, J.E., Catabolism of neuropeptides by a brain proline endopeptidase, Biochem. Biophys. Res. Commun., 94 (1980) 9-15. Toide, K., Fujiwara, T., Iwamoto, Y., Okamiya, K. and Shinoda, M., Effect of JTP-4819, a novel prolyl endopeptidase (PEP) inhibitor, on central cholinergic neurons of aged rats, Jpn. J. Psychopharmacol.. 14 (1994) 497. Toide, K., Fujiwara, T., lwamoto, Y., Shinoda, M., Okamiya, K. and Kato. T.. Effect of a novel prolyl endopeptidase inhibitor,

[38]

[39]

[40]

[41]

[42]

[43]

[44] [45]

[46] [47]

[48]

[49]

151

JTP-4819, on neuropeptide metabolism in the rat brain, NaunynSchmied. Arch. Pharm., 353 (1996) 355-362. Toide, K., lwamoto, Y., Fujiwara, T. and Abe, H., JTP-4819: A novel prolyl endopeptidase inhibitor with potential as a cognitive enhancer, J. Pharmacol. Exp. Ther., 274 (1995) 1370-1378. Toide, K., lwamoto, Y., Fujiwara, T. and Okamiya, K., A novel prolyl endopeptidase (PEP) inhibitor, JTP-4819, activates peptidergic-cholinergic neurotransmission as a cognitive enhancer: Implications and mechanism of action. In A. Louilot, T. Durkin, U. Spampinato and M. Cador (Eds.), Proceedings of the 6th International Conference in Neurosci., Monitoring Molecules in Neuroscience, 6th International Conference in Neurosci., Acheve d'imprimer sur les presses, 1994, pp. 273-274. Toide, K., Okamiya, K., Iwamoto, Y. and Kato, T., Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on prolyl endopeptidase activity and substance P- and arginine-vasopressin-like immunoreactivity in the brains of aged rats, J. Neurochem., 65 ( 1995 ) 234-240. Toide, K., Shinoda, M., Takase, M., Iwata, K. and Yoshida, H., Effects of a novel thyrotropin-releasing hormone analogue, JTP-2942, on extracellular acetylcholine and choline levels in the rat frontal cortex and hippocampus, Eur. J. Pharmacol., 233 (1993) 21-28. Toide, K., Shinoda, M. and Uemura, A., Effect of JTP-4819, a novel prolyl endopeptidase (PEP) inhibitor, on passive avoidance and Morris water maze task, Jpn. J. Psychopharmacol., 14, 454 (1994). Walter, R., Shlank, H., Glass, J.D., Schwartz, I.L and Kerenyi, T.D., Leucylglycinamide released from oxytocin by human enzyme, Science, 173 (1971) 827-829. Walter, R., Simmons, W.H. and Yoshimoto, T., Proline specific endo- and exopeptidases, Mol. Cell. Biochem., 30 (1980) 111-127. Whitehouse, P.J., Price, D.L., Struble, R.G., Clark, A.W., Coyle, J.T. and Delong, M.R., Alzheimer's disease and senile dementia: Loss of neurons in the basal forebrain, Science (Wash. DC), 215 (1982) 1237-1239. W i l l S., Mini review prolyl endopeptidase, Life Sci., 33 (1983) 2149-2157. Yankner, B.A., Duffy, L.K. and Kirschner, D.A., Neurotropic and neurotoxic effects of Amyloid ~ protein: reversal by tachykinin neuropeptides. Science, 250 (1990) 279-282. Yoshimoto, T., Nishimura, T., Kita, T. and Tsuru, D., Post-proline cleaving enzyme (Prolyl endopeptidase) from bovine brain, J. Biochem., 94 (1983) 1179-1190. Yoshimoto. T., Ogita. K., Walter, R., Koida, M. and Tsuru, D., Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man, Biochim. Biophys. Acta, 569 (1979) 184-192.