Claude Garret

In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors

Abstract Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family. Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. In vitro and in vivo studies have shown that zopiclone is able to inhibit the binding of [3H] diazepam and [3H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam in vitro and to that of chlordiazepoxide in vivo . These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.

A non-peptide NK1-receptor antagonist, RP 67580, inhibits neurogenic inflammation postsynaptically.

1 The non‐peptide neurokinin NK1‐receptor antagonist, RP 67580 (3aR, 7a), a perhydroisoindolone derivative, powerfully reduced plasma extravasation in rat hind paw skin induced by local application of xylene (ID50 = 0.03 mg kg−1, i.v.) or capsaicin (ID50 = 0.06 mg kg−1, i.v.), or by i.v. injection of exogenous substance P (SP) or septide ([pGlu6,Pro9]SP(6–11)) (ID50 = 0.04–0.05 mg kg−1, i.v.). RP 67580 (1 mg kg−1, i.v.) also abolished capsaicin‐induced nasal fluid hypersecretion (by 82 ± 5%). These effects were found to be stereospecific, the enantiomer, RP 68651 (3aS, 7aS), being inactive at 1 mg kg−1, i.v. 2 In rats neonatally treated with capsaicin (50 mg kg−1, s.c.), plasma extravasation induced by SP was significantly increased (by 43 ± 7%). RP 67580 (1 mg kg−1, i.v.) completely inhibited the SP‐induced plasma extravasation in capsaicin neonatally treated‐animals, as it did in control animals. This result suggests that RP 67580 acts at the postsynaptic level for the inhibition of plasma extravasation. 3 Opioid receptor agonists, μ‐(morphine) and κ‐(PD‐117302) at 10 mg kg−1, s.c., in contrast to NK1‐receptor antagonists, did not inhibit plasma extravasation induced by exogenous SP. They were, however, partially effective against plasma extravasation induced by electrical nerve stimulation (74 ± 4% and 48 ± 9% inhibition at 10 mg kg−1, s.c. of morphine and PD‐117302, respectively, compared to 90 ± 3% inhibition obtained with RP 67580, 3 mg kg−1, s.c.). These results indicate the presynaptic action of opioid receptor agonists, in contrast to the postsynaptic action of NK1‐preceptor antagonists for the inhibition of plasma extravasation. 4 Ligature of the saphenous nerve distal to the point of electrical stimulation, local application of lignocaine to the saphenous nerve, neonatal capsaicin pretreatment, and colchicine at very low doses (120 μg kg−1 day−1 given for 3 days) were found to prevent plasma extravasation elicited by electrical nerve stimulation. 5 The foregoing results demonstrate that the non‐peptide NK1‐receptor antagonist, RP67580, is a potent inhibitor of plasma extravasation induced in skin by NK1‐receptor agonists, by local application of chemical irritants (capsaicin or xylene) or by electrical nerve stimulation. Moreover, opioid receptor agonists and colchicine inhibit plasma extravasation induced by electrical nerve stimulation but not that elicited by exogenous SP. Therefore, it is possible to inhibit neurogenic inflammation either at the presynaptic level with opioid receptor agonists and colchicine, or at the postsynaptic level with NK1‐receptor antagonists, and that the new non‐peptide NK1‐receptor antagonists may have a great potential for alleviation of inflammation in various pathological syndromes in man.

Evidence for a role of tachykinins as sensory transmitters in the activation of micturition reflex

The possible involvement of tachykinin neurokinin-1 and neurokinin-2 receptors in the activation of various micturition-related reflexes was assessed by the intrathecal administration of selective neurokinin-1 or neurokinin-2 receptor antagonists at lumbosacral spinal cord level in urethane-anaesthetized rats. The effect of the glutamate N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovaleric acid, was also investigated for comparison. The effect of antagonists was investigated on: (i) the chemonociceptive vesicovesical reflex activated by topical application of capsaicin onto the urinary bladder; (ii) the distension-induced micturition reflex produced by transvesical filling with saline; (iii) distension-induced rhythmic bladder contractions in isovolumetric conditions (urethra-ligated rats); and (iv) the somatovesical excitatory reflex caused by noxious perineal pinching. The neurokinin-2 receptor selective antagonists MEN 10,376 and SR 48,968 were ineffective in the three models in all doses tested. Selective neurokinin-1 receptor antagonists blocked the chemonociceptive reflex produced by topical application of capsaicin with the rank order of potency (lowest effective dose in brackets): GR 82,334 (1 nmol/rat) > RP 67,580 (10 nmol/rat) > (+/-)CP 96,345 (100 nmol/rat). Unlike GR 82,334, RP 67,580 (10 nmol/rat) and (+/-)CP 96,345 (100 nmol/rat) were also effective on the distension-induced micturition reflex elicited by transvesical filling. Similarly, distension-induced rhythmic contractions were inhibited by RP 67,580 (10 nmol/rat) and (+/-)CP 96,345 (100 nmol/rat) whereas the effect of GR 82,334 was not significant. RP 68,651, the enantiomer of RP 67,580 devoid of neurokinin-1 receptor blocking activity, was inactive in both models. 2-Amino-5-phosphonovateric acid (250 nmol/rat) blocked the three types of vesicoexcitatory reflexes. Intravenous administration of (+/-)CP 96,345, RP 67,580 or 2-amino-5-phosphonovateric acid at the same doses proven effective after the intrathecal route, had no effect on distension-induced rhythmic contractions. To ascertain whether the effect of neurokinin-1 receptor antagonists or 2-amino-5-phosphonovaleric acid may be related to a blockade of tachykinins released from capsaicin-sensitive primary afferent neurons, the effect of RP 67,580 was investigated on the distension-evoked micturition reflex in capsaicin-pretreated rats. Capsaicin pretreatment (50 mg/kg, subcutaneously, four days before) increased bladder capacity. RP 67,580 was no longer effective in capsaicin-pretreated rats. In contrast, 2-amino-5-phosphonovateric acid produced a further increase in bladder capacity in capsaicin-pretreated rats. We conclude that tachykinin neurokinin-1 but not neurokinin-2 receptors are involved in the activation of vesicoexcitatory micturition-related reflexes in the rat spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of neurokinin B in rat spinal cord and peripheral tissues: Comparison with neurokinin A and substance P and effects of neonatal capsaicin treatment

In the present study, highly specific radioimmunoassays were developed and used to measure neurokinin B, neurokinin A and substance P in the rat spinal cord and various peripheral tissues. The results are as follows. (1) Neurokinin B and neurokinin A were distributed all along the rostrocaudal axis of the spinal cord, as is substance P, and were more concentrated in the dorsal than in the ventral region. (2) Substance P was more abundant in the central and peripheral nervous tissues than neurokinin A, while in certain peripheral organs, neurokinin A was more abundant than substance P. In the spinal cord, neurokinin B concentrations were lower than those of the other two tachykinins. (3) In contrast to neurokinin A and substance P, neurokinin B was not detected in any of the peripheral tissues examined. (4) Capsaicin treatment reduced by half neurokinin A and substance P concentrations in the dorsal region of the spinal cord, the dorsal root ganglia and the sciatic nerve, but was without effect on neurokinin B concentrations in the spinal cord. Neurokinin A, like substance P, may therefore have an important function in the transmission of sensory information, particularly in nociceptive transmission from the periphery to the spinal cord and in peripheral neurogenic inflammation. In contrast, since neurokinin B was not found in the sensory neurons, it is not likely to have these functions, but may perhaps control them.

Polyclonal Antibodies Against the Rat Nk1 Receptor - Characterization and Localization in the Spinal-cord

We have developed antibodies against the NK1 receptor and have investigated its cellular distribution. Rabbit polyclonal antibodies were generated against peptide (19-32) of the rat brain NK1 receptor. They were very specific to the NK1 site as shown by ELISA against various epitopes of NK1, NK2 and NK3 receptors and by immunoblotting of proteins from bacteria transfected with rat brain NK1 receptor cDNA and from rat cortex. Determining how immunostained NK1 receptors are distributed in the rat spinal cord made it possible to identify the cellular structures on which NK1 receptors are located and where they form synapses with SP terminals. In the superficial layers of the dorsal horn, the NK1 receptors appeared mainly of dendritic nature and were, like SP, abundant. In the deep layers of the dorsal horn and in the ventral horn, they were associated mostly with cell bodies.

Inhibition of neurogenic inflammation in the meninges by a non-peptide NK1 receptor antagonist, RP 67580

RP 67580, a non-peptide NK1 receptor antagonist, inhibited in a stereoselective and dose-dependent manner plasma extravasation caused in the dura mater by intravenous injection of capsaicin in guinea-pigs and of exogenous substance P in rats (ED50 = 35 and 2.5 micrograms/kg i.v., respectively). In the two species, RP 67580 appeared to be more effective in the dura mater than in the peripheral organs. These results indicate that selective NK1 receptor antagonists could be potentially effective for the treatment of migraine headache.

Action of the palmitic ester of pipotiazine on dopamine metabolism in the nigro-striatal, meso-limbic and meso-cortical systems

SummaryThe effects of a single injection of the palmitic ester of pipotiazine on dopamine synthesis were examined in slices of the striatum, the olfactory tubercule + nucleus accumbens and the frontal and anterior cingulate parts of the cerebral cortex in the rat. In the two first structures, dopamine synthesis was determined by measuring the rate of formation of 3H−H2O during the conversion of l-3,5-3H-tyrosine into 3H-Dopa. Newly synthesized 3H-dopamine and 3H-noradrenaline were measured in the cerebral cortex. In some experiments, the specific activity of tyrosine in the tissues was determined in order to calculate the conversion index of tyrosine into dopamine.Marked differences were seen in the three structures examined. The palmitic ester of pipotiazine had a biphasic effect on dopamine synthesis in the striatum; an acceleration occurring for the first 5 days was followed by a marked reduction which lasted for several weeks. A biphasic effect was also seen in the olfactory tubercule + nucleus accumbens. However the first phase of acceleration of dopamine synthesis was of longer duration (about 21 days) and the subsequent inhibitory phase was less pronounced and shorter than that observed in the striatum. The cerebral cortex reacted differently since only a single phase of increased dopamine synthesis was seen. This phase lasted for at least 35 days.The overall durations of the various changes in the rate of dopamine synthesis and in the antiamphetamine (stereotypies) activity of the ester were closely correlated.These results are discussed in relation to the antipsychotic and extrapyramidal side-effects of neuroleptics.

Comparison in different tissue preparations of the in vitro pharmacological profile of RP 67580, a new non-peptide substance P antagonist

We describe the effects of RP 67580, a new non-peptide substance P (SP) antagonist, on tachykinin-induced contractions of guinea-pig ileum, trachea and urinary bladder, rabbit pulmonary artery and rat portal vein. All NK1 agonists tested (SP, Septide, SPOMe and [Pro9]SP) contracted guinea-pig ileum, trachea and urinary bladder (pD2 = 7.5 to 9.1), but they had no effect on rabbit pulmonary artery or rat portal vein (pD2 < 6). RP 67580 inhibited these effects: guinea-pig ileum, pA2 = 7.1 to 7.6; guinea-pig trachea and urinary bladder, pKB = 6.3 to 6.8. The difference in RP 67580 activity in these tissues might be due to the existence of subtypes of NK1 receptors. RP 67580 (1 microns) did not affect the contractions induced by the two NK2 agonists, NKA and [Lys5, MeLeu9, Nle10]NKA(4-10) (pA2 < 6), except in guinea-pig ileum (pA2 = 7.3-7.5) where these two NK2 agonists interact apparently with NK1 receptors. In the tissue preparations used, RP 67580 (1 micron) was without effect on contractions induced by the NK3 agonists: NKB and senktide. These results indicate the high selectivity for NK1 receptors of RP 67580. In all cases, similar results were obtained with another non-peptide SP antagonist, (+/-) CP-96,345. The present work provides further evidence that RP 67580 and (+/-) CP-96,345 exert in vitro a potent, selective and competitive antagonistic action on NK1 receptors and suggests the existence of at least two distinct NK1 receptor subtypes in some guinea-pig peripheral organs.

Species differences between [3h] substance P binding in rat and guinea-pig shown by the use of peptide agonists and antagonists

The affinities of various substance P agonists and antagonists for NK1 receptors in rat and guinea-pig tissues were compared. Striking species differences were observed. Both septide and [D-Pro4,D-Trp7,9]SP-(4-11) possessed much higher affinity for sites in the guinea-pig (brain and ileum) than for sites in the rat brain. These results could be explained by differences in the structure of the NK1 receptor according to the species, although the existence of various subtypes of NK1 binding sites in the two species cannot be excluded.

Substance P and neurokinin A variations throughout the rat estrous cycle; comparison with ovariectomized and male rats: II. Trigeminal nucleus and cervical spinal cord

Substance P and neurokinin A were assayed in the trigeminal nucleus and cervical spinal cord of 4‐day cycling female, ovariectomized, and male rats. During the estrous cycle, levels were largely stable in the trigeminal nucleus. In ovariectomized rats, the levels differed from those on any day of the estrous cycle suggesting a weak effect of ovarian steroids. In males, the variations in the substance P and neurokinin A contents in the trigeminal nucleus were not similar to those in either cyclic or ovariectomized rats.