Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening.

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.

Impact of P-Glycoprotein-Mediated Active Efflux on Drug Distribution into Lumbar Cerebrospinal Fluid in Nonhuman Primates.

Estimation of unbound drug concentration in the brain (Cu,brain) is an essential part of central nervous system (CNS) drug development. As a surrogate for Cu,brain in humans and nonhuman primates, drug concentration in cerebrospinal fluid (CCSF) collected by lumbar puncture is often used; however, the predictability of Cu,brain by lumbar CCSF is unclear, particularly for substrates of the active efflux transporter P-glycoprotein (P-gp). Here, we measured lumbar CCSF in cynomolgus monkey after single intravenous administration of 10 test compounds with varying P-gp transport activities. The in vivo lumbar cerebrospinal fluid (CSF)-to-plasma unbound drug concentration ratios (Kp,uu,lumbar CSF) of nonsubstrates or weak substrates of P-gp were in the range 0.885-1.34, whereas those of good substrates of P-gp were in the range 0.195-0.458 and were strongly negatively correlated with in vitro P-gp transport activity. Moreover, concomitant treatment with a P-gp inhibitor, zosuquidar, increased the Kp,uu,lumbar CSF values of the good P-gp substrates, indicating that P-gp-mediated active efflux contributed to the low Kp,uu,lumbar CSF values of these compounds. Compared with the drug concentrations in the cisternal CSF and interstitial fluid (ISF) that we previously determined in cynomolgus monkeys, the lumbar CCSF were more than triple for two and all of the good P-gp substrates examined, respectively. Although lumbar CCSF may overestimate cisternal CSF and ISF concentrations of good P-gp substrates, lumbar CCSF allowed discrimination of good P-gp substrates from the weak and nonsubstrates and can be used to estimate the impact of P-gp-mediated active efflux on drug CNS penetration. SIGNIFICANCE STATEMENT: This is the first study to systematically evaluate the penetration of various P-glycoprotein (P-gp) substrates into lumbar cerebrospinal fluid (CSF) in nonhuman primates. Lumbar CSF may contain >3-fold higher concentrations of good P-gp substrates than interstitial fluid (ISF) and cisternal CSF but was able to discriminate the good substrates from the weak or nonsubstrates. Because lumbar CSF is more accessible than ISF and cisternal CSF in nonhuman primates, these findings will help increase our understanding of drug central nervous system penetration at the nonclinical stage.

Resveratrol and related stilbenes: their anti-aging and anti-angiogenic properties.

Dietary stilbenes comprise a class of natural compounds that display significant biological activities of medicinal interest. Among them, their antioxidant, anti-aging and anti-angiogenesic properties are well established and subjects of numerous research endeavors. This mini-review aspires to account and present the literature reports published on research concerning various natural and synthetic stilbenes, such as trans-resveratrol. Special focus was given to most recent research findings, while the mechanisms underlying their anti-aging and anti-angiogenic effects as well as the respective signaling pathways involved were also presented and discussed.

Amurensin G induces autophagy and attenuates cellular toxicities in a rotenone model of Parkinson's disease.

Although Parkinson's disease is a common neurodegenerative disorder its cause is still unknown. Recently, several reports showed that inducers of autophagy attenuate cellular toxicities in Parkinson's disease models. In this report we screened HEK293 cells that stably express GFP-LC3, a marker of autophagy, for autophagy inducers and identified amurensin G, a compound isolated from the wild grape (Vitis amurensis). Amurensin G treatment induced punctate cytoplasmic expression of GFP-LC3 and increased the expression level of endogenous LC3-II. Incubation of human dopaminergic SH-SY5Y cells with amurensin G attenuated the cellular toxicities of rotenone in a model of Parkinson's disease. Amurensin G inhibited rotenone-induced apoptosis and interfered with rotenone-induced G2/M cell cycle arrest. In addition, knockdown of beclin1, a regulator of autophagy, abolished the effect of amurensin G. These data collectively indicate that amurensin G attenuates cellular toxicities through the induction of autophagy.

Leaf and stem of Vitis amurensis and its active components protect against amyloid ß protein (25-35)-induced neurotoxicity.

This study investigated a methanol extract from the leaf and stem of Vitis amurensis (Vitaceae) for possible neuroprotective effects on neurotoxicity induced by amyloid ß protein (Aß) (25-35) in cultured rat cortical neurons and also for antidementia activity in mice. Exposure of cultured cortical neurons to 10 µM Aß (25-35) for 36 h induced neuronal apoptotic death. At concentrations of 1-10 µg/mL, V. amurensis inhibited neuronal death, the elevation of intracellular calcium ([Ca(2+)](i)) and the generation of reactive oxygen species (ROS), all of which were induced by Aß (25-35) in primary cultures of rat cortical neurons. Memory loss induced by intracerebroventricular injection of ICR mice with 16 nmol Aß (25-35) was inhibited by chronic treatment with V. amurensis extract (50 and 100 mg/kg, p.o. for 7 days), as measured by a passive avoidance test. Amurensin G, r-2-viniferin and trans-ɛ-viniferin isolated from V. amurensis also inhibited neuronal death, the elevation of [Ca(2+)](i) and the generation of ROS induced by Aß (25-35) in cultured rat cortical neurons. These results suggest that the neuroprotective effect of V. amurensis may be partially attributable to these compounds. These results suggest that the antidementia effect of V. amurensis is due to its neuroprotective effect against Aß (25-35)-induced neurotoxicity and that the leaf and stem of V. amurensis have possible therapeutic roles for preventing the progression of Alzheimer's disease.

Amurensin G, a potent natural SIRT1 inhibitor, rescues doxorubicin responsiveness via down-regulation of multidrug resistance 1.

The transition from a chemotherapy-responsive cancer to a chemotherapy-resistant one is accompanied by increased expression of multidrug resistance 1 (MDR1, p-glycoprotein), which plays an important role in the efflux from the target cell of many anticancer agents. We recently showed that a Forkhead box-containing protein of the O subfamily 1 (FoxO1) is a key regulator of MDR1 gene transcription. Because nuclear localization of FoxO1 is regulated by silent information regulator two ortholog 1 (SIRT1) deacetylase, we wondered whether SIRT1 dominates MDR1 gene expression in breast cancer cells. Overexpression of SIRT1 enhanced both FoxO reporter activity and nuclear levels of FoxO1. Protein expression of MDR1 and gene transcriptional activity were also up-regulated by SIRT1 overexpression. In addition, SIRT1 inhibition reduced both nuclear FoxO1 levels and MDR1 expression in doxorubicin-resistant breast cancer cells (MCF-7/ADR) cells. A potent SIRT1 inhibitor, amurensin G (from Vitis amurensis), was identified by screening plant extracts and bioassay-guided fractionation. The compound suppressed FoxO1 activity and MDR1 expression in MCF-7/ADR cells. Moreover, pretreatment of MCF-7/ADR cells with 1 µg/ml amurensin G for 24 h increased cellular uptake of doxorubicin and restored the responsiveness of MCF-7/ADR cells to doxorubicin. In xenograft studies, injection of 10 mg/kg i.p. amurensin G substantially restored the ability of doxorubicin to inhibit MCF-7/ADR-induced tumor growth. These results suggest that SIRT1 is a potential therapeutic target of MDR1-mediated chemoresistance and that it may be possible to develop amurensin G as a useful agent for chemoresistance reversal.

New bioactive prenylflavonoids and dibenzocycloheptene derivative from roots of Dendrolobium lanceolatum.

Two new flavanones (1 and 2), a new flavan (3), and a new rare dibenzocycloheptene derivative (4) together with a known flavan, 4'-hydroxy-2' ',2' 'dimethyl-pyranoflavan (5), were isolated from the roots of Dendrolobium lanceolatum. Their structures were established on the basis of spectral evidence, and an X-ray analysis was performed to confirm the structure of 4. Compounds 1-3 exhibited antimalarial activity with IC50 values of 2.6, 3.3, and 3.1 microg/mL, respectively. Compounds 1-5 showed moderate antimycobacterial activity with MIC values of 6.3, 12.5, 25, 25, and 50 microg/mL, respectively. In addition, 1 showed strong cytotoxicity against cancer cell lines KB, BC, and NCI-H187 with IC50 values of 1.2, 1.6, and 0.6 microg/mL, respectively, while 2 showed moderate cytotoxicity against the NCI-H187 cell line with an IC50 value of 8.1 microg/ mL.

Anti-immobility activity of different antidepressant drugs using the tail suspension test in normal or reserpinized mice.

The tail suspension test is a screening procedure recently used in mice to detect antidepressant activity of drugs. The ability of amine re-uptake inhibitors to decrease immobility in non-reserpinized and in reserpinized mice was studied. Reserpine (4 mg/kg ip) was injected 4 h previously. Anti-depressants were administered ip, 60 min before tail suspension. Animal activity was recorded for 6 min. Preferential serotonin re-uptake blockers (fluoxetine, fluvoxamine, clomipramine) were poorly active in non-reserpinized mice and inactive in reserpine-treated mice. Noradrenergic drugs (desipramine, demexiptiline, viloxazine) were more efficient in reserpinized than in non-reserpinized mice. The mixed serotonin-noradrenaline re-uptake inhibitor (imipramine) shows an activity which should be considered between serotonin re-uptake inhibitors and noradrenaline re-uptake inhibitors. DA re-uptake inhibitors (amineptine, GBR 12909) exhibited the highest anti-immobility effect in non reserpinized animals but were of low efficacy after reserpine treatment. Amphetamine differed from dopamine re-uptake inhibitors by its better activity in reserpinized animals. Moreover, it was the only drug showing an equal anti-immobility effect in non reserpinized and reserpinized mice because the dose of 8 mg/kg of amphetamine reduced immobility in reserpinized mice with the same intensity as the dose of 4 mg/kg in non reserpinized mice whereas no other drugs tested in this study achieved the same effect. Comparison of anti-immobility activities of putative anti-depressants in non-pre-treated and in reserpine-pre-treated mice, using the tail suspension test, may be useful to discriminate amphetamines from antidepressant drugs and to differentiate between categories of amine re-uptake blockers.

Maturation of the hypothalamic control of pulsatile gonadotropin-releasing hormone secretion at onset of puberty. I. Increased activation of N-methyl-D-aspartate receptors.

In the male rat the timing of puberty can be estimated by the rapid increase in testicular weight occurring between 25-50 days of age. We found that elongated spermatids, the most mature germ cells identified using flow cytometry, were first seen at 25 days (4% of the testicular cells), while an adult proportion (63%) was attained by 45 days of age. We have shown previously that hypothalamic explants could release GnRH in a pulsatile fashion at a frequency increasing around the age of 25 days, thus consistent with the time of onset of puberty. Since pulsatile GnRH secretion could be suppressed by MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor activation, we postulated that an increased activation of those receptors could be involved in the neuroendocrine mechanism that activates pulsatile GnRH secretion at the onset of puberty. Such a concept was supported by the NMDA-induced release of GnRH, which was observed using 1 mM NMDA at 25 days, while a dose of 20-50 mM was required at 15 or 50 days of age. MK-801 could provide an index of NMDA receptor activation, since the antagonistic effect of MK-801 is use dependent. This particular property was confirmed by the inability of MK-801 (5 pM) to block the depolarization (veratridine)-induced release of GnRH in the presence of 0.001 mM NMDA, while partial or complete suppression was obtained in the presence of 0.1 and 10 mM NMDA, respectively. Using explants obtained at 5, 10, 15, 20, 25, 30, 35, and 50 days of age, the lowest concentrations of MK-801 that blocked the veratridine-induced release of GnRH were, respectively, 10(7), 10(7), 10(7), 10(3), 10, 10(2), 10(4), and 10(8) pM. In contrast, there was no age-related difference in sensitivity to the inhibitory effect of Mg2+, a noncompetitive NMDA receptor antagonist which is not use dependent. The pulsatile secretion of GnRH occurred at a similar frequency at 25 and 50 days of age (4.7 and 5.4 pulses/3.5 h, respectively) but it was suppressed by a lower MK-801 concentration at 25 days (10(4) pM) than at 50 days (10(8) pM). These data indicate that the NMDA receptors involved in the control of pulsatile GnRH secretion are markedly and transiently activated around the time of onset of puberty in the male rat.

Intracellular and extracellular changes of [Ca2+] in hypoxia and ischemia in rat brain in vivo.

Changes in intra- and extracellular free calcium concentration were evaluated with ion-selective microelectrodes during periods of anoxia and ischemia in three different regions of intact rat brain. Recordings stable for at least 2 min and in most cases for 4-6 min were chosen for analysis. Under normoxic conditions neuronal [Ca2+]i varied between less than 10(-8) and 10(-7) M from cell to cell but no systematic regional differences were observed. Elimination of O2 or interruption in blood flow caused, within 30-60 s, slight intracellular alkalinization followed by a small rise in [Ca2+]i, a mild degree of hyperpolarization, and disappearance of electrical activity in the cortex, in that order. It is postulated that a decline in cellular energy levels, as manifested by H+ uptake associated with creatine phosphate hydrolysis, leads to an increase in [Ca2+]i, which activates Ca2(+)-dependent K+ channels and consequently enhances gK. 2-4 min later there was a sudden, large rise in [K+]e, a fall in [Ca2+]e and a rapid elevation of [Ca2+]i. The magnitude of the latter was greatest in a high proportion of hippocampal neurons in area CA1 and some cortical cells, while it was smallest and relatively delayed in thalamic neurons. In the hippocampus area CA1 increases in [Ca2+]i to as much as 6-8 x 10(-4) were observed; some of these could be reversed when O2 or blood flow were restored to normal. Pretreatment of animals with ketamine and MK-801, antagonists of excitatory amino acid transmitters, markedly slowed and decreased the rises in [Ca2+]i. The effects of the two agents were most pronounced in the hippocampus. It is concluded that the receptor-operated channels are largely responsible for Ca2+ entry into certain cells during hypoxia/ischemia. This pathway may be of primary importance in parts of the hippocampus and cortex, regions of the brain that are particularly vulnerable to O2 deprivation and which receive high glutamatergic input and have an abundance of excitatory amino acid receptors.

MK801 induces c-fos protein in thalamic and neocortical neurons of rat brain.

MK801, a non-competitive NMDA receptor antagonist, leads to a dramatic induction of c-fos-like protein in neurons in deep layers of the neocortex, in dorsal and ventral midline thalamic nuclei and in neurons in the central grey of rat brain. This induction of c-fos by MK801 is dose- and time-dependent occurring within 2 h and dissipating by 24 h after injection (0.5-8.0mg/kg, i.p.). The mechanism of this paradoxical induction of c-fos by MK801 is unclear, however the pattern of induction appears to follow the distribution of the antagonist-preferring NMDA receptor site.

Serum and depolarizing agents cause acute neurotoxicity in cultured cerebellar granule cells: role of the glutamate receptor responsive to N-methyl-D-aspartate.

The life span of neonatal rat cerebellar granule cells, grown in basal minimal Eagle's medium containing 10% (vol/vol) fetal calf serum, was extended to 21-30 days by weekly supplementation with glucose. Addition of 1% fetal calf serum to the culture at 14 days killed 85% of the cells within 1 hr. This lethal effect could be prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists dibenzocyclohepteneimine (MK-801) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP). These findings suggested that the glutamate in the serum caused the dramatic neuronal death through action on the NMDA receptor. Indeed, a 5-min incubation in a Locke physiological salt solution containing 20 microM glutamate and 5 microM glycine killed 55-90% of the cells. This acute toxicity could be prevented by a lyso-GM1 ganglioside with N-acetylated sphingosine. The relatively low glutamate content of the sera analyzed suggests that factors in addition to glycine potentiate serum neurotoxicity. The above noted antagonists of the NMDA receptor also greatly reduced the lethal effect of depolarization by 90 mM KCl or 10 microM veratridine. Therefore, it is likely that the toxicity of the depolarizing agents is mediated by glutamate released from the cells. It is concluded that survival of cerebellar neurons in primary culture may be strongly affected by unsuspected neurotoxic phenomena elicited by brief action of a rather low glutamate concentration.

A possible role of AA2 excitatory amino acid receptors in the expression of stimulant drug effects.

GDEE, an antagonist of the AA2 or quisqualic acid category of excitatory amino acid receptor, decreases behavioral activity and locomotor stimulation induced by cocaine and amphetamine when locally injected into the nucleus accumbens. The present experiment was intended to examine the effects of systemic GDEE and other excitatory amino acid antagonists on stimulant-induced locomotor activity. GDEE markedly attenuated the stimulant effect of amphetamine, and partially blocked the effects of phencyclidine (PCP). Apomorphine-induced cage climbing behavior was partially decreased by lower dosages of GDEE, but was almost completely blocked by the highest dosage tested. Amphetamine-induced stimulation of locomotor activity was not decreased by any of the other excitatory amino acid antagonists that were tested, including MK-801, 2-amino-7-phosphonoheptanoic acid (APH), or CNQX. APH decreased stereotypy only at a high dosage (250 mg/kg), which also produces ataxia. Several other compounds, including L-glutamic acid gamma ethyl ester (GMEE), L-glutamic acid, glycine, and L-glutamine did not block amphetamine-induced stimulation in molar dosages equivalent to the highest dosage of GDEE (8 mmol/kg). It is concluded that the AA2 excitatory amino acid receptor is important in the expression of activating effects of stimulant drugs.

Ketamine and MK-801 prevent degeneration of thalamic neurons induced by focal cortical seizures.

Ketamine and MK-801 act at phencyclidine receptors to block transmitter activity through the N-methyl-D-aspartate (NMDA) subtype of glutamate (GLU) receptor. These agents also block the potent excitotoxic actions of NMDA and are of interest for their potential ability to protect against neurodegenerative processes mediated by the excitotoxic action of endogenous Glu at NMDA receptors. Here we show that degeneration of thalamic neurons caused by persistent seizure activity in the corticothalamic tract (putative glutamergic transmitter pathway) is prevented by systemic administration of ketamine or MK-801, despite the failure of these agents to eliminate persistent electrographic seizure activity recorded from cortex and thalamus.

Behavioral evaluation of the anti-excitotoxic properties of MK-801: comparison with neurochemical measurements.

The ability of MK-801 to protect striatal neurons from the excitotoxic action of quinolinic acid was evaluated by means of apomorphine-induced rotational behavior and by measurement of striatal choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activity, neurochemical markers for cholinergic and GABAergic neurons, respectively. Animals with a unilateral quinolinic acid lesion of the striatum exhibited a vigorous rotational response when challenged with apomorphine (0.5 mg/kg, s.c.) 6 days later and were found to have an 88 90% depletion of striatal ChAT and GAD activity. Treatment with a high dose of MK-801 (10 mg/kg, i.p.) prior to intrastriatal injection of quinolinic acid eliminated the subsequent rotational response to apomorphine and resulted in complete protection of striatal ChAT and GAD activity. Lower doses of MK-801 (1, 3 and 5 mg/kg, i.p.) failed to significantly reduce the rotational response to apomorphine but provided partial, dose-dependent protection of both ChAT and GAD activity. The rotational response to apomorphine correlated with the percent reduction in both ChAT activity (r = 0.57, P less than 0.0005) and GAD activity (r = 0.49, P less than 0.0005). Rotational behavior may thus provide a means to evaluate the functional integrity of the striatum.

Pulsatile release of gonadotropin-releasing hormone from hypothalamic explants is restrained by blockade of N-methyl-D,L-aspartate receptors.

We have shown previously that N-methyl-D,L-aspartate (NMDA) and kainate, two neuroexcitatory amino acids acting through distinct receptors, may induce the release of GnRH from hypothalamic explants. However, that effect could have no physiological significance, since very high concentrations (50 mM) of NMDA and kainate were required. Here, using agents blocking the activation of receptors to neuroexcitatory amino acids, we evaluated their possible physiological involvement in the pulsatile release of GnRH from the hypothalamus of 50-day-old male rats in vitro. In control conditions (10 nM glycine and 1 mM mg2+), the release of GnRH in 7.5-min fractions collected for 2-4 h showed an obvious pulsatile pattern. The mean (+/- 1 SD) interval between pulses, identified by PULSAR program, was 34.3 +/- 11.4 min. The stimulation of GnRH release by NMDA (50 mM) added to the medium for 7.5 min could be blocked reversibly in the presence of MK-801 (100 microM) using medium without glycine or enriched with Mg2+ (2 mM). The endogenous pulses of GnRH secretion were abolished in the presence of MK-801 or using increased Mg2+ concentrations as well as in the absence of glycine. In contrast, pulsatile release of GnRH was not affected in the presence of 6,7-dinitroquinoxaline-2,3-dione (0.1 mM), a selective inhibitor of kainate and quisqualate receptors which suppressed the increase in GnRH release induced by kainate (50 mM) without affecting the response to NMDA. These data indicate that the physiological mechanism of pulsatile GnRH secretion in the hypothalamus may involve endogenous neuroexcitatory factors acting through NMDA-sensitive receptors.

The effects of non-competitive NMDA receptor antagonists on rats exposed to hyperbaric pressure.

The high pressure neurological syndrome (HPNS) occurs when man or animals are exposed to hyperbaric pressure. Four non-competitive N-methyl-D-aspartate (NMDA) antagonists - MK-801, phencyclidine (PCP), SKF 10,047 and ketamine were tested in rats for effects on the HPNS. All drugs were injected i.p. prior to compression; ketamine was also infused i.v. Control rats received saline. Rats were exposed individually to increasing helium pressure (PO2 0.5 atmospheres absolute ATA). Three endpoints were used to assess HPNS: onset pressures for tremor, myoclonus and convulsions. Neither MK-801 (0.03 and 0.3 mg/kg) nor SKF 10,047 (50 mg/kg) had any effect on the onset pressures for tremor, myoclonus or convulsions, although the type of seizure was modified from the clonic/tonic seizure seen in controls to purely clonic. PCP (5 mg/kg) had no effect on the endpoints, but pressure enhanced the excitation and stereotypy seen at 1 ATA. Ketamine (100 mg/kg i.p.) did not affect tremor or myoclonus; ketamine infused i.v. at pressure only prevented tremor and myoclonus at 'anaesthetizing' concentrations. Our results show that these non-competitive NMDA antagonists had little effect on HPNS, in contrast to competitive NMDA antagonists, such as AP7, which are highly effective. Possible explanations for this lack of effect include (1) interactions with NMDA receptor channels are pressure dependent; (2) other actions of these antagonists override their effects on the NMDA receptor channel.

Antagonism of N-methylaspartate and synaptic responses of neurones in the rat ventrobasal thalamus by ketamine and MK-801.

1. Extracellular single neurone recordings were made in the ventrobasal thalamus of urethane-anaesthetized rats. 2. Iontophoretically and intravenously administered ketamine and MK-801 were found to be selective antagonists of responses of neurones to iontophoretically applied N-methylaspartate. 3. Both antagonists, administered in N-methylaspartate-selective quantities, reduced the synaptic responses of ventrobasal thalamus neurones to a two-second air jet directed at the peripheral receptive field.

[Effect of butaclamol enantiomers on hypothalamic tyrosine hydroxylase in the rat brain]. / Vliianie énantiomerov butaklamola na tirozingidroksilazu gipotalamusa mozga krys.

The authors demonstrate stereospecificity of the action of butaclamol enantiomers on substrate inhibition of hypothalamic tyrosine hydroxylase (TH) and regulation of the tyrosine hydroxylase response by the presynaptic membrane (presynaptic receptors) of rat hypothalamus synaptosomes under membrane activation with dopamine. The effect of (+)-butaclamol on the substrate inhibition of TH was noticeable at a concentration of 10(-8)M, reaching a maximum at 10(-5)M. (-)-Butaclamol administered at the same concentrations did not influence the substrate inhibition of the enzyme. (+)-Butaclamol added to the incubation medium containing hypothalamic synaptosomes concurrently with dopamine (10(-5)M) completely blocked the regulatory action of the latter on TH, with this action mediated via presynaptic receptors. (-)-Butaclamol (10(-5)M) antagonized the action of dopamine under the same conditions. The data obtained indicate high stereo-specificity of butaclamol enantiomers as regards their effect on presynaptic regulation of TH, suggesting that elimination of the substrate inhibition of hypothalamic TH is a stereoselective effect of neuroleptics and can be a prognostically important criterion in the appraisal of compounds with potential neuroleptic activity.

The antiinflammatory profile of (5H-dibenzo[a,d]-cyclohepten-5-ylidene)acetic acid (WY-41,770), an agent possessing weak prostaglandin synthetase inhibitory activity that is devoid of gastric side effects.

Wy-41,770 [(5H-dibenzo[a,d]cyclohepten-5-ylidene)acetic acid], a novel acrylic acid, was compared to indomethacin and aspirin in standard antiinflammatory, analgesic and antipyretic animal models. The acute antiinflammatory, analgesic and antipyretic activity of Wy-41,770 (oral ED50S 50-170 mg/kg) was similar to aspirin; however, it was considerably more potent orally in adjuvant arthritis in the rat (ED50, 16 mg/kg) and urate-induced synovitis in the dog (ED50, 4.5 mg/kg). Wy-41,770 was a weak inhibitor of prostaglandin biosynthesis and did not inhibit either 5- or 15-lipoxygenase. Furthermore, the cellular migration characteristic of carrageenan pleurisy was not affected by Wy-41,770. Unlike a majority of NSAIDs, it produced no gastric irritation in rats after either acute or chronic oral administration over the range 400-800 mg/kg. The major mechanism of action of Wy-41,770 has yet to be identified but does not seem to involve interference of arachidonic acid metabolism.