Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 1990"

Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils.

Abstract: Aggregation of human platelets induced by a variety of agonists was inhibited by 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dionel (U-73122) (IC50 values 1-5 microM), but not by the close analog 1-[6-[[17 beta-3-methoxyestra- 1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343) in which pyrrolidinedione was substituted for pyrroledione. Inhibition by U-73122 was not mediated by an increase in intracellular cyclic AMP. In contrast, the production of inositol 1,4,5-trisphosphate (IP3) and the subsequent rapid increase in cytosolic Ca++ induced by either thrombin or the thromboxane-mimetic, (5Z,9 alpha, 11 alpha, 13E, 15S) 15-hydroxy-11,9-(epoxymethano)prosta- 5,13,-dien-1-oic acid (U-46619), was inhibited by U-73122 but not by U-73343. Reduction of IP3 levels appeared to reflect an inhibition of IP3 production because the hydrolysis of phosphatidyl[3H]inositol and phosphatidyl[3H]inositol 4,5-bisphosphate catalyzed by a soluble fraction from platelets was inhibited by U-73122 (Ki = 9 and 40 microM, respectively). In addition, U-73122 inhibited thromboxane B2 production induced by collagen but not that supported by exogenously added arachidonic acid, suggesting that U-73122 also inhibited receptor-coupled mobilization of arachidonic acid. After preincubation of platelets with [3H]arachidonic acid, the loss of [3H]phosphatidylinositol and accumulation of [3H]phosphatidic acid induced by thrombin was attenuated by U-73122. U-73122 did not inhibit the activities of phospholipases A2 purified either from porcine pancreas or from the venoms of Crotalus adamanteus and Naja naja. Although U-73122 inhibited neither the conversion of exogenous arachidonic acid to thromboxane B2 nor the binding of the thromboxane receptor antagonist [1S-[1 alpha, 2 beta (5Z), 3 beta, 4 alpha]]-7-[3-[[2- [2-[(phenylamino)-carbonyl]- hydrazino]methyl]-7-oxabicyclo [2.2.1]-hept-2-yl-5-heptenoic acid to platelet membranes, it was an effective inhibitor of arachidonic acid-induced aggregation of platelets. These data are consistent with the observed inhibition by U-73122 of platelet activation by the thromboxane receptor agonist, U-46619, via a mechanism that involves inhibition of a phospholipase C-dependent component(s) of signal transduction. U-73122, but not U-73343, inhibited also N-formyl-methionyl-leucyl-phenylalanine-induced aggregation of human polymorphonuclear neutrophils (PMN) and the associated production of IP3 and diacyglycerol. Diradylglycerol produced in PMN stimulated with N-formyl- methionyl-leucyl-phenylalanine was 74 +/- 7% saponifiable and inhibited by U-73122 (Ki = 2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness.

Abstract: 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C (PLC)-dependent processes in human platelets, was found to be a potent inhibitor of human polymorphonuclear neutrophil (PMN) activation by structurally unrelated receptor-specific agonists. U-73122 caused a time- and concentration-dependent (0.1-1 microM) inhibition of myeloperoxidase and vitamin B12-binding protein release from PMNs exposed to N-formyl-methionyl-leucyl-phenylalanine, recombinant human C5a, leukotriene B4 and platelet-activating factor. Activation of the respiratory burst, as measured by superoxide anion production, in PMNs stimulated with these agonists was also suppressed by U-73122. These data suggested that U-73122 inhibited a component of signal transduction that was common to the mechanisms of action of these stimuli. Production of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol and the rise in the cytosolic free calcium concentration, which are early postreceptor events in PMN activation, were all suppressed in U-73122-treated PMNs stimulated with the agonists. These signal transduction events require activation of PLC. Receptor-coupled activation of PLC in membranes isolated from PMNs was potently inhibited by U-73122. U-73122, however, had no direct effect on PMN protein kinase C activity. 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] -2,5- pyrrolidine-dione (U-73343), a close analog of U-73122 that does not suppress PLC activity, did not inhibit receptor-specific agonist-induced PMN responsiveness. U-73122, therefore, is a novel reagent that is useful in investigating PLC function in receptor-mediated PMN activation.

Cytochrome P 450 isoenzymes, epoxide hydrolase and glutathione transferases in rat and human hepatic and extrahepatic tissues

Abstract: The organ distribution of microsomal cytochrome P 450 isoenzymes (P 450), microsomal epoxide hydrolase (EH) and cytosolic glutathione-S-transferases was investigated by immunoblotting and enzyme measurements in rats and humans. In rats, P 450 IA1 was detected only in the duodenum, and P 450 IA2 and IIC11 were detected only in the liver. The highest concentrations of P 450 IIB1/B2 were found in the lung and in the duodenum; pentoxyresorufin-O-dealkylase activity was closely correlated with the amounts of P 450 IIB1/B2 in the different organs. P 450 IIE1 was present in liver, kidney and lung, whereas EH was found in liver, intestine and kidney. In humans, P 450 IIIA4 was detected in all tissues investigated; the highest concentrations were found in liver and intestine. The P 450 IIIA4 level was closely correlated with that of erythromycin demethylase and pentoxyresorufin-O-dealkylase activities. P 450 IIC8-10, IIE1 and IID6 were expressed in liver and intestine, P 450 9 in liver and kidney and P 450 IA2 in liver. EH was identified only in liver, intestine and kidney. In both species, concentrations and total amounts of P 450 isoenzymes and EH were much lower in all extrahepatic tissues than in the liver. Conversely, glutathione-S-transferase-pi was abundant in human intestine and colon compared to liver. Glutathione-S-transferase-mu polymorphism was confirmed in all tissues investigated. This extensive study showed that the pattern of (iso) enzymes was different in all tissues studied; consequently, xenobiotic metabolism would appear to be very different in each type of tissue.

hCGRP8-37, a calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery.

Abstract: The C-terminal fragment of human calcitonin gene-related peptide (CGRP), hCGRP8-37, fails to induce any biological activity in a variety of isolated tissues and behavioral assays even though it possesses nanomolar affinity for [125I]hCGRP alpha binding sites in the central nervous system and peripheral membrane preparations. However, hCGRP8-37 displays relatively potent, competitive antagonist properties toward the action of native hCGRP alpha in guinea pig atrial and ileal preparations and on the central nervous system-mediated inhibition of CGRP on food intake. On the contrary, in the rat vas deferens the antagonistic potency of hCGRP8-37 was much weaker and was ineffective against CGRP-induced hyperthermia after i.c.v. injection. Such evidence suggests the existence of at least two classes of CGRP receptors, the first (CGRP1) being sensitive to the antagonist properties of hCGRP8-37, whereas the second (CGRP2) is not. The use of hCGRP8-37 should greatly facilitate the characterization of the physiological roles of CGRP-like peptides, especially those which are mediated via the activation of CGRP1 receptors.

Nonpeptide angiotensin II receptor antagonists. XI. Pharmacology of EXP3174: an active metabolite of DuP 753, an orally active antihypertensive agent.

Abstract: This report describes the pharmacology of (2-n-butyl-4-chloro-1- [(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-5-carboxylic acid (EXP3174). EXP3174 is a major metabolite generated after the oral dosing of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H- tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole, potassium salt in rats. It displaced [3H]angiotensin II (AII) from its specific binding sites in rat adrenal cortical membranes with an IC50 of 3.7 x 10(-8) M. In the isolated rabbit aorta, EXP3174 caused nonparallel shifts to the right of the AII concentration-contractile response curves and reduced the maximal response by 30 to 40% with an apparent pA2 value of 10.09 and a KB value of 10(-10) M. At 10(-6) M, EXP3174 did not alter the contractile responses to norepinephrine and KCl. In the spinal pithed rat, EXP3174 at 0.03 to 0.3 mg/kg i.v. also inhibited the pressor responses to AII and angiotensin III noncompetitively and did not change the pressor responses to vasopressin and norepinephrine. When given i.v. and cumulatively to normotensive rats at 0.003 to 0.3 mg/kg, EXP3174 did not alter blood pressure but inhibited the pressor response to AII. In conscious renal artery-ligated rats, EXP3174 decreased blood pressure with an i.v. ED30 of 0.038 mg/kg and a p.o. ED30 of 0.66 mg/kg. These results demonstrate that EXP3174 is a selective and noncompetitive AII receptor antagonist and lacks agonistic effect. As EXP3174 is a potent antihypertensive agent, it may be responsible for part of the antihypertensive effect of DuP 753 in rats.

Nonpeptide angiotensin II receptor antagonists. VII. Cellular and biochemical pharmacology of DuP 753, an orally active antihypertensive agent.

Abstract: 2-n-Butyl-4-chloro-5-hydroxymethyl-1-[2'-(1H-tetrazole-5-yl)biphenyl-4-y l) methyl]imidazole, potassium salt (DuP 753) is a potent, p.o. active antihypertensive agent exerting its action by specific blockade of angiotensin II receptors. It inhibited the specific binding of labeled angiotensin II to its receptor sites in rat adrenal cortical membranes and in cultured rat smooth muscle cells with IC50 values of 19 and 20 X 10(-9) M, respectively. Functional antagonism was demonstrated by its blockage of angiotensin II (3 X 10(-8) M)-induced 45Ca++ efflux in rat aortic smooth muscle cells with an IC50 of 2 X 10(-8) M. In rabbit aorta, DuP 753 antagonized the contractile response to angiotensin II competitively with a pA2 value of 8.48 but had no effect on the responses induced by norepinephrine or KCl. In both in vitro and in vivo assays, no partial agonistic effect was detected even with concentrations of up to 10(-5) M. In addition, this agent (10(-5) or 10(-4) M) exhibited no direct effect on converting enzyme (rabbit lung) or renin (rat plasma). These data demonstrate that DuP 753, is a potent and highly specific angiotensin II receptor antagonist. This agent may be a useful experimental or therapeutic tool for interference with the renin-angiotensin system in health and diseases.

Role of imidazole receptors in the vasodepressor response to clonidine analogs in the rostral ventrolateral medulla.

Abstract: The rostral ventrolateral medulla is the primary site of action for clonidine, a centrally acting antihypertensive. In the rostral ventrolateral medulla, clonidine binds not only to alpha-2 adrenergic receptors but also to specific imidazole sites. In order to determine whether a putative imidazole receptor mediates the hypotensive action of clonidine, a series of compounds was tested 1) in vitro for binding affinity at imidazole and alpha-2 sites and 2) in vivo for ability to lower arterial pressure and heart rate when microinjected directly into the rostral ventrolateral medulla. Hypotensive potency was correlated with affinity at imidazole sites (r = 0.84), but not with alpha-2 affinity (r = -0.05). The bradycardia elicited by this series of compounds also correlated with affinity at imidazole receptors (r = 0.89), but not with alpha-2 affinity (r = 0.10). Furthermore, the imidazole idazoxan selectively reversed the fall in arterial pressure elicited by clonidine, whereas SKF-86466, an alpha-2 antagonist which is not an imidazole, failed to attenuate clonidine's action. An imidazole receptor in the rostral ventrolateral medulla appears to mediate the central hypotensive actions of clonidine and related centrally acting imidazoles and may participate in the regulation of arterial pressure and heart rate.

Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo: the protective effect of allopurinol.

Abstract: Acetaminophen (500 mg/kg i.p.) induced hepatotoxicity in fasted ICR mice in vivo. Acetaminophen also caused a long-lasting 50% reduction of the hepatic ATP content, an irreversible loss of hepatic xanthine dehydrogenase activity and a transient increase of the xanthine oxidase activity. All effects occurred before parenchymal cell damage, i.e., the release of cellular enzymes. The hepatic content of GSH and GSSG was initially depleted by acetaminophen without affecting the GSSG:GSH ratio (1:200), however, during the recovery phase of the hepatic GSH levels the GSSG content increased faster than GSH, resulting in a GSSG:GSH ratio of 1:18 24 h after acetaminophen administration. The mitochondrial GSSG content increased from 2% in controls to greater than 20% in acetaminophen-treated mice. The extremely elevated tissue GSSG levels were accompanied by a 4-fold increase of the plasma GSSG concentrations but not by an enhanced biliary efflux, although hepatic GSSG formation and biliary excretion were not affected by acetaminophen. Allopurinol protected dose-dependently against acetaminophen-induced cell injury, the loss of ATP and the increase of the GSSG content in the total liver and in the mitochondrial compartment without inhibiting reactive metabolite formation. High, protective as well as low, nonprotective doses of allopurinol almost completely inhibited hepatic xanthine oxidase and dehydrogenase activity, but only high doses prevented the increase of the mitochondrial GSSG content. The data indicate a long-lasting, primarily intracellular oxidant stress during the progression phase of acetaminophen-induced cell necrosis. The protective effect of allopurinol is unlikely to involve the inhibition of reactive oxygen formation by xanthine oxidase but could be the result of its antioxidant property.

Functional studies of nonpeptide angiotensin II receptor subtype-specific ligands: DuP 753 (AII-1) and PD123177 (AII-2).

Abstract: DuP 753 and PD123177 are two nonpeptide angiotensin II (AII)-specific ligands, which show high affinities for two respective and distinct subtypes of AII binding sites, i.e., AII-1 and AII-2 sites, respectively, in the rat adrenal gland, brain and uterus. The objective of this study is to identify the functions of these subtype binding sites in the adrenal, sympathetic ganglia, brain and vascular smooth muscle. In conscious rats, DuP 753 at 1, 3 and 10 mg/kg i.v. but not PD123177 at 30 and 100 mg/kg i.v. inhibited the AII-induced aldosterone increase. In the isolated perfused rat adrenal gland, DuP 753 at 10(-6) and 10(-4) M but not PD123177 at 10(-3) M blocked the AII-induced epinephrine secretion. In control and chemically sympathectomized pithed rats, the pressor and tachycardiac responses to AII were blocked by DuP 753 at 10 mg/kg i.v. but not by PD123177 at 100 mg/kg i.v. In conscious rats, DuP 753 at 10 mg/kg s.c. but not PD123177 at 100 mg/kg s.c. inhibited the AII-induced water drinking. In the rabbit aorta, DuP 753 at 10(-6) M but not PD123177 at 10(-4) M inhibited the contractile effect of AII. In conscious renal artery-ligated hypertensive rats, DuP 753 but not PD123177 at 0.1 to 10 mg/kg i.v. lowered blood pressure. In summary, a function of the PD123177-sensitive AII binding site (AII-2) has not yet been identified. However, the DuP 753-sensitive site (AII-1) appears to mediate the AII-induced responses such as adrenal aldosterone and catecholamine secretion, release of catecholamine from sympathetic ganglia, drinking and vasoconstriction.

Pharmacological characterization of cannabinoids in the elevated plus maze.

Abstract: delta 9-Tetrahydrocannabinol (delta 9-THC) induced in both rats and mice an increased aversion to the open arms of the elevated plus maze which was similar to that produced by anxiogenic agents. This effect of delta 9-THC was approximately three times greater in rats than in mice. When the behavioral effects of the cannabinoids were characterized further in the mouse, it was found that delta 9-11-THC, 12 beta-NH2-delta 8-THC, levonantradol and (-)-11-OH-delta 8-THC-DMH produced effects that were similar to those of delta 9-THC. The effect was found to be enantioselective in that (+)-11-OH-delta 8-THC-DMH was inactive even at a dose 200 times greater than an active dose of (-)-11-OH-delta 8-THC-DMH. In contrast to the effects of delta 9-THC, mice treated with cannabidiol and nabilone spent a greater amount of time in the open arm of the maze, an effect similar to that produced by diazepam, the reference anxiolytic agent. In this test situation, 11-nor-delta 8-THC-9-carboxylic acid and abnormal cannabidiol did not alter the behavior of the animals at doses up to 20 and 100 mg/kg, respectively. Pretreatment with either the bidirectional inverse agonist carboline-3-carboxylate or diazepam (at doses that did not modify normal behavior on the elevated plus maze) blocked the effect of delta 9-THC.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonpeptide angiotensin II receptor antagonists. VIII. Characterization of functional antagonism displayed by DuP 753, an orally active antihypertensive agent.

Abstract: In the spinal pithed rat, DuP 753, 2-n-butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl) methyl] imidazole potassium salt, inhibited competitively the pressor response to angiotensin II (AII), whereas saralasin showed a noncompetitive pattern of interaction. It did not alter the pressor responses to vasopressin and norepinephrine as well as the heart rate response to isoproterenol. In the anesthetized rat, DuP 753 did not affect the vasodepressor response to bradykinin. Given p.o. or i.v., DuP 753 did not lower blood pressure in conscious normotensive rats, but it inhibited the pressor response to AII but not to vasopressin. It lowered blood pressure in furosemide-treated normotensive rats. Unlike saralasin, DuP 753 did not cause a transient increase in blood pressure even at 100 mg/kg i.v. DuP 753 at 3.5 micrograms i.c.v. inhibited the pressor response to i.c.v. AII, whereas DuP 753 at 10 mg/kg p.o. did not, suggesting that a single p.o. administration of DuP 753 does not affect brain AII receptors which are accessible by i.c.v. injection. Our study indicates that DuP 753 is a p.o. active, nonpeptide, selective, competitive AII receptor antagonist.

Modulation of A10 dopamine neurons by gamma-aminobutyric acid agonists.

Abstract: Microinjection of the gamma-aminobutyric acidA agonist, muscimol, into the A10 region of the rat produced a dose-dependent increase in motor activity. This effect was antagonized by intra-A10 administration of the gamma-aminobutyric acidA antagonist, bicuculline, and by peripheral administration of haloperidol, and was associated with an increase in extracellular levels of dopamine metabolites in the nucleus accumbens. Although microinjection of the gamma-aminobutyric acidB agonist, baclofen, into the A10 region did not alter motor activity, it abolished the capacity of intra-A10 injection of mu opioid agonist, Tyr-D-Ala-Gly-MePhe-Gly(ol), or muscimol to increase motor activity. Baclofen also prevented the motor stimulant response to peripheral injection of cocaine or amphetamine, but was ineffective in blocking caffeine-induced behavioral activity. Pretreatment with baclofen prevented the capacity of a mu opioid agonist to elevate dopamine metabolite levels in the nucleus accumbens and prefrontal cortex in postmortem tissue. Baclofen also prevented the elevation of extracellular dopamine content in the nucleus accumbens produced by injection of a mu opioid agonist into the A10 region, as measured in the conscious rat with in vivo dialysis. Finally, when dopamine metabolite levels were elevated in the prefrontal cortex by mild footshock, it was shown that pretreatment with baclofen in the A10 region abolished this response. These data support electrophysiological studies suggesting that activation of gamma-aminobutyric acidB receptors on dopamine perikarya inhibits dopaminergic activity, while activation of gamma-aminobutyric acidA receptors results in an indirect disinhibition of dopaminergic function.

Comparison of in vitro and in vivo models of drug transcytosis through the blood-brain barrier.

Abstract: Drug and solute transport through in vitro and in vivo models of the blood-brain barrier (BBB) were compared to provide a measure of how well the in vitro model predicted BBB permeability found in vivo. The in vitro model employed bovine brain capillary endothelial cells in either primary tissue culture or as a continuous line grown on Transwells and placed in side-by-side diffusion chambers. The in vivo model of BBB transport utilized an internal carotid artery perfusion/capillary depletion method in anesthetized rats. BBB permeability in vivo and in vitro was measured for 15 radiolabeled drugs and for L-[3H]dopa, D-[14C]glucose and [3H]albumin. [3H]- or [14C]sucrose was used in vivo as a blood volume reference. Lipid solubility of each drug was measured based on the 1-octanol/Ringer's partition coefficient. The morphology of the endothelial cell in primary tissue culture was spindle-shaped and the morphology of the endothelial cell in continuous culture was cuboid-shaped. The cuboidal morphology demonstrated a 2-fold greater resistance to solute transport and was used for the majority of the in vitro studies. Drug and solute permeability coefficients (Pe) ranged from 3.9 X 10(-3) to 2.5 X 10(-1) cm/min in vitro and from 1.0 X 10(-5) to 2.1 X 10(-2) cm/min in vivo. The In of the permeability.surface area product in vitro correlated with the In partition coefficient (r = 0.62, P less than .0125) and the In permeability.surface area product in vivo correlated with the In partition coefficient (r = 0.84, P less than .0005).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization of a neuronal receptor for 5-hydroxytryptamine in guinea pig ileum with properties similar to the 5-hydroxytryptamine receptor.

Abstract: Experiments were undertaken to characterize pharmacologically a neuronal receptor to 5-HT in guinea pig ileum. Segments of longitudinal muscle myenteric plexus preparations were treated with phenoxybenzamine and exposed to submaximal electrical field stimulation to evoke the cholinergically mediated "twitch" response. The ability of 5-HT to enhance the submaximal twitch response was investigated. Results using several antagonists (metergoline, spiperone, cyanopindolol, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, ICS 205-930, GR 38032F, MDL 72222 and cocaine) indicate that 5-HT (3 X 10(-10) to 1 x 10(-7) M) agonizes a novel 5-HT receptor site distinct from the 5-HT1, 5-HT2, 5-HT3 and 5-HT1P subtypes as well as the M receptor. The receptor site is located neuronally and is characterized positively by a low affinity for ICS 205-930 (pA2 = 6.5 vs. 5-HT) and by the following order of agonist potency: 5-HT greater than 5-methoxytryptamine greater than BRL 24924 greater than alpha-methyl-5-hydroxytryptamine greater than zacopride = cisapride = 5-carboxamidotryptamine. Agonist-independent pA2 estimates for ICS 205-930 (6.3-6.6) suggest a single site of agonism. 2-Methyl-5-hydroxytryptamine and 5-hydroxyindalpine were inactive at 1 x 10(-5) M either as agonists or antagonists. Thus, the receptor site exhibits a pharmacological profile similar to that characterizing the recently described 5-HT4 [corrected] receptor. Unlike Gaddum's M receptor, which equates with the 5-HT3 [corrected] receptor, the putative 5-HT4 [corrected] receptor site exhibits a higher sensitivity to agonism by 5-HT and is resistant to antagonism by cocaine.

Spontaneous liberation of nitric oxide cannot account for in vitro vascular relaxation by S-nitrosothiols.

Abstract: S-nitrosothiols are potent vasodilators in vivo and in vitro, and have recently been proposed as possible endogenous precursors of endothelium-derived nitric oxide (NO) NO release from S-nitrosothiols has generally been assumed to be spontaneous, but this has not been proven This hypothesis was examined by altering the NO release profiles of two S-nitrosothiols, those of S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-glutathione (GSNO), and observing their relaxation potency on isolated endothelium-denuded rat aortic rings Spontaneous degradation of SNAP and GSNO in tissue bathing medium (monitored by high-performance liquid chromatography) and the associated NO release (assessed by chemiluminescence detection of headspace NO) were enhanced in the presence of 100 microM N-acetylpenicillamine and inhibited in the presence of 100 U/ml superoxide dismutase However, the relaxant effects of SNAP and GSNO were enhanced in the presence of superoxide dismutase, and diminished in the presence of N-acetylpenicillamine In addition, the relaxation potencies of SNAP, GSNO, S-nitrosocystein, S-nitroso-N-acetylcysteine and S-nitroso-coenzyme A were not correlated with spontaneous NO generation These findings therefore argue against spontaneous liberation of NO as a predominant mechanism of S-nitrosothiol action The highly polar SNAP, GSNO, S-nitrosocysteine and S-nitroso-N-acetylcysteine (octanol, pH 74 buffer partition coefficient from less than 025-052) and the bulky and polar S-nitroso-coenzyme A (MW 797) have similar relaxation potencies, indicating that intracellular penetration of intact S-nitrosothiols may not be required for activity NO generation from SNAP was examined in subcellular fractions of bovine coronary arterial smooth muscle cells(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-inflammatory and safety profile of DuP 697, a novel orally effective prostaglandin synthesis inhibitor.

Abstract: DuP 697 (5-bromo-2[4-fluorophenyl]-3-[4-methylsulfonylphenyl]-thiophene) is a potent inhibitor of paw swelling in nonestablished and established adjuvant arthritis in rats (ED50 = 0.03 and 0.18 mg/kg/day, respectively). DuP 697 had no effect on phenylquinone writhing in rats (ED50 greater than 100 mg/kg), but was analgetic against inflammation-related pain in the Randall-Selitto assay (ED30 = 3.5 mg/kg) and was a very potent antipyretic agent (ED50 = 0.05 mg/kg). The drug was not ulcerogenic in rats at single doses up to 400 mg/kg. DuP 697 (5 mg/kg i.v.) did not alter renal blood flow or the renal vascular response to angiotensin II in furosemide-pretreated, volume-depleted rats. In contrast, indomethacin (5 mg/kg i.v.) decreased renal blood flow and potentiated the renal vascular response to angiotensin II in these animals. DuP 697 was a moderate inhibitor of bull seminal vesicle prostaglandin (PG) synthesis (IC50 = 2.4 X 10(-5) M) and a potent inhibitor of rat brain PG synthesis (IC50 = 4.5 X 10(-6) M) but was ineffective against rat kidney PG synthesis (IC50 7.5 X 10(-5) M). These differential effects of DuP 697 on PG synthesis by various tissues may account for its high potency as an anti-inflammatory and antipyretic agent and its minimal toxicity profile.

Stimulation of a dopamine D1 receptor enhances inositol phosphates formation in rat brain.

Abstract: Accumulation of inositol phosphates was determined in rat brain slices prelabeled with 2-[3H]inositol and incubated with various drugs. In the striatum, micromolar concentrations of dopamine, apomorphine and SKF38393 induced significant accumulations of inositol phosphates in a dose-dependent manner, whereas quinpirole lacked effect. The EC50 values for the accumulation of inositol monophosphate induced by dopamine, apomorphine and SKF38393 were, respectively, 148, 159 and 129 microM. SKF 38393 effect was time-dependent on the accumulation of all three inositol phosphates, with peak effects occurring 64-128 min after drug addition. The action of the dopamine D1 receptor agonist, SKF38393, was blocked by SCH23390 (D1-selective antagonist), but not by sulpiride (D2-selective antagonist), atropine (muscarinic antagonist), prazosin (alpha-1 adrenoceptor antagonist) or methiotepin and methysergide (serotonergic antagonists), indicating that the observed effects of dopaminergic agonists were selectively mediated through the D1 dopamine receptor. On examining the effect of SKF38393 in several brain regions, the highest dopaminergic stimulation of inositol phosphates formation was obtained in the amygdala, followed by the hippocampus and then the striatum and frontal cortex. The finding of an SKF38393-stimulated PI hydrolysis in amygdala, a brain region that is enriched in SCH23390 and SKF38393 binding sites but devoid of dopamine-stimulated adenylate cyclase, suggests that the D1 receptor that is linked to PI metabolism is independent of the D1 receptor which stimulates cyclic AMP formation.

Tolbutamide and mephenytoin hydroxylation by human cytochrome P450s in the CYP2C subfamily.

Abstract: Previous biochemical studies have suggested that tolbutamide and mephenytoin are metabolized by the same cytochrome P450 enzyme. Conversely, clinical studies indicate the involvement of different P450 forms in tolbutamide and mephenytoin metabolism. Our objective was to elucidate further those P450 enzymes responsible for hydroxylation of these two drugs. We studied both tolbutamide and (S)-mephenytoin hydroxylation in microsomes from 38 different normal adult human livers, and found large variability in the rates of metabolism for both reactions (1.75-47.4 nmol/mg/hr for hydroxytolbutamide formation and 0.1-7.2 nmol/mg/hr for 4-hydroxymephenytoin formation). No significant correlation was found between the two activities. However, both reactions shared common inhibitors in vitro, including inhibition by antikidney-liver-microsome autoantibodies (Meier and Meyer, Biochemistry 26: 8466-8474, 1987) and by teniposide. Two human liver cDNAs for P450s of the CYP2C subfamily designated IIC8 and IIC9 (S. Kimura, J. Pastewka, H. V. Gelboin and F. J. Gonzalez, Nucl. Acids Res. 15: 10053-10054, 1987), were functionally expressed in human HepG2 and TK- cells using a vaccinia virus vector. Interestingly, tolbutamide was hydroxylated by both expressed P450s. Only IIC9 catalyzed the 4-hydroxylation of (R)-mephenytoin and neither enzyme metabolized (S)-mephenytoin. We conclude that tolbutamide and (R)-mephenytoin are both metabolized by the same P450 enzyme, IIC9, and that tolbutamide is hydroxylated by an additional highly related enzyme, IIC8, contributing to the lack of correlation of the two hydroxylase activities among human liver microsomes and indicating the absence of a monogenically controlled polymorphism for tolbutamide.

Persistence of neurochemical changes in dopamine systems after repeated cocaine administration.

Abstract: The purpose of this study was to test whether persistent changes consistent with behavioral sensitization occur in dopamine (DA) uptake, release or receptors following repeated cocaine administration. Our neurochemical experiments focused primarily on the striatum; however, quantitative autoradiography was used to measure D-1 and D-2 DA receptors in both cell body and terminal regions of the nigrostriatal and mesolimbic dopaminergic pathways. After receiving eight once-daily injections of cocaine (10 mg/kg, i.p.), rats remained behaviorally sensitized for 1 week. This repeated treatment with cocaine induced two changes consistent with increased dopaminergic transmission. Postsynaptic D-2 DA receptors were selectively increased in nucleus accumbens one day after termination of the repeated cocaine administration; however, these receptors returned to control levels one week after cocaine administration had been terminated. In contrast, amphetamine-stimulated [3H] DA release from striatal slices was increased in rats receiving repeated cocaine injections, but this increase was not apparent until 1 week after the drug administration had been terminated. While neither of these two changes is sufficient to explain cocaine-induced behavioral sensitization, both are consistent with increased dopaminergic responsiveness and may contribute to sensitization.

Metabolism of methylenedioxymethamphetamine: formation of dihydroxymethamphetamine and a quinone identified as its glutathione adduct.

Abstract: The in vitro conversion of (+)-3,4-methylenedioxymethamphetamine and (-)-3,4-methylenedioxymethamphetamine to the corresponding catecholamine, 3,4-dihydroxymethamphetamine (N-methyl-alpha-methyldopamine), by rat liver microsomes was examined. Metabolite formation was monitored after short-term incubations using high-performance liquid chromatography-electrochemical detection to determine concentrations of the catecholamine. The formation of N-methyl-alpha-methyldopamine exhibited enantioselectivity and levels were significantly higher after incubation of the (+)-isomer. The reaction appears to be cytochrome P-450 dependent as it was sensitive to SKF 525A and carbon monoxide. The catecholamine was unstable and was metabolized rapidly to a compound capable of forming an adduct with glutathione (GSH) and other thiol compounds. This second oxidation did not appear to be cytochrome P-450-dependent but required NADPH and microsomal protein. Catecholamine oxidation was inhibited by superoxide dismutase and by reducing agents. The same catecholamine oxidation product, characterized as the GSH adduct, could be generated by a xanthine-xanthine oxidase mixture and by tyrosinase. Mass spectral data showed that it was a 1:1 amine GSH adduct. These results indicate that MDMA is oxidized by cytochrome P-450 to the catechol and the catecholamine oxidized by superoxide to a quinone to which GSH or other thiol functions add. The formation of this quinone and its thiol adducts may account for some of the irreversible actions of this compound on serotonergic neurons.

Nonpeptide angiotensin II receptor antagonists. IX. Antihypertensive activity in rats of DuP 753, an orally active antihypertensive agent.

Abstract: In conscious renal artery-ligated rats, a high renin hypertensive rat model, DuP 753, a p.o. active nonpeptide angiotensin II (AII) receptor antagonist, decreased blood pressure at 0.1 to 3 mg/kg given i.v. or at 0.3 to 10 mg/kg given p.o. with an i.v. ED30 of given i.v. or at 0.3 to 10 mg/kg given p.o. with an i.v. ED30 of 0.78 mg/kg and a p.o. ED30 of 0.59 mg/kg. The antihypertensive efficacy of DuP 753 was similar to that of captopril. Unlike the peptide AII antagonist saralasin, DuP 753 did not cause a transient increase in blood pressure, suggesting absence of agonistic activity. At 3 mg/kg p.o., DuP 753 lowered blood pressure for at least 24 hr and did not change heart rate, suggesting a long duration of antihypertensive effect. At 3 mg/kg i.v., DuP 753 inhibited the pressor response to AII but not to norepinephrine or vasopressin. Pretreatment of renal artery-ligated rats with captopril, saralasin or propranolol, but not with prazosin, hydralazine or indomethacin, abolished or reduced the antihypertensive effect of DuP 753. In the deoxycorticosterone acetate hypertensive rat, a low renin model, DuP 753 did not lower blood pressure. These results suggest that DuP 753 is a p.o. active, antihypertensive agent in renal artery-ligated rats with a similar antihypertensive efficacy as captopril. The antihypertensive effect of DuP 753 is most likely related to the blockade of the vasoconstrictor effect of AII. Unlike saralasin, DuP 753 does not have agonistic activity.

Selective blockade of N-methyl-D-aspartate (NMDA)-induced convulsions by NMDA antagonists and putative glycine antagonists: relationship with phencyclidine-like behavioral effects.

Abstract: Antagonism of N-methyl-D-aspartate (NMDA)-induced convulsions by a variety of drugs was compared with their ability to produce phencyclidine (PCP)-like behavioral effects (locomotion and falling) in mice. Convulsions produced by i.c.v. administration of NMDA were antagonized, at doses that did not block kainate- and quisqualate-induced convulsions, by competitive NMDA antagonists (e.g., CPP and CGS 19755), noncompetitive antagonists (e.g., PCP and MK-801) and also by some putative glycine antagonists (7-chlorokynurenic acid and HA-966). Only the competitive and the noncompetitive NMDA antagonists produced locomotion and falling, and their potencies to do so correlated (r = 0.92) with their relative potencies to antagonize NMDA-induced convulsions. However, the PCP-like behavioral effects produced by the competitive antagonists were of a lesser magnitude than those of the noncompetitive antagonists, and occurred at doses higher than those needed to block NMDA-induced convulsions. The putative glycine antagonists 7-chlorokynurenic acid and HA-966 selectively blocked NMDA-induced convulsions, without producing PCP-like behavioral effects. The extent to which compounds produce PCP-like behavioral effects might depend in part on the specific component of the NMDA receptor complex with which they interact: i.e., the NMDA receptor, the NMDA receptor-associated ion channel or the glycine-sensitive modulatory site.

Antidepressant binding sites in brain: autoradiographic comparison of [3H]paroxetine and [3H]imipramine localization and relationship to serotonin transporter.

Abstract: Binding of two different antidepressant drugs, [3H]paroxetine and [3H]imipramine in 30 rat brain regions was visualized, compared and quantified by means of autoradiography and densitometry. Specific binding of [3H]paroxetine to coronal sections of diencephalon represented 85% of total binding and was saturable and of high affinity (KD, 0.36 +/- 0.07 nM) with a maximum number of binding sites of 276 +/- 41 fmol/mg protein. The autoradiograms showed a heterogenous distribution of [3H]paroxetine in brain with selective accumulation of label in brain regions known to contain serotonergic terminals, axons and cell bodies (amygdaloid and raphe nuclei, superior colliculus, substantia nigra and medial forebrain bundle). Binding was displaced selectively with other serotonin uptake inhibitors (clomipramine and fluoxetine) and almost abolished by lesioning the serotonergic neurons with p-chloroamphetamine. The desipramine-sensitive [3H]imipramine binding was more diffuse with relatively high density in cerebral cortex and hippocampus and was only decreased partially in animals treated with p-chloroamphetamine. The results indicate that [3H]paroxetine, but not [3H]imipramine, is a ligand of choice to selectively label serotonergic structures in brain.

Identification of the subcellular site for nitroglycerin metabolism to nitric oxide in bovine coronary smooth muscle cells.

Abstract: The vasodilating action of nitroglycerin (NTG) is thought to be mediated by its metabolic activation to nitric oxide (NO) in the vascular smooth muscle cell, but the site at which this process occurs has not been defined. To determine which cellular component is primarily responsible for this metabolic activation, subcellular fractions of bovine vascular smooth muscle cells were prepared and incubated with NTG along with cofactors. Time-dependent headspace NO concentrations generated in these preparations were determined directly by chemiluminescence detection. A mathematical model was developed to relate headspace NO with the NO-generating activity in each incubation, correcting for the concurrent processes of NO partitioning between the headspace and the incubation medium, and NO degradation in these two phases. The estimated NO-generating activities from different subcellular fractions were well correlated with the activities of two enzyme markers of the plasma membrane (K(+)-activated ouabain sensitive p-nitrophenyl phosphatase and 5'-nucleotidase), but not with those of the mitochondria, endoplasmic reticulum or the cytosol. These results indicate that the enzyme(s) responsible for the metabolic activation of NTG, and possibly other organic nitrate vasodilators, are associated with the plasma membrane in bovine coronary smooth muscle cells.

Chemically induced hypoglycemia and anoxia: relationship to glutamate receptor-mediated toxicity in retina.

Abstract: Chemically induced hypoglycemia and anoxia were evaluated in embryonic day 13 chicken retina to determine if excitotoxicity was a consequence of these conditions and if this was preceded by the net release of glutamate or aspartate. Retina incubated with iodoacetate (IOA), to inhibit glycolysis, or potassium cyanide (KCN), to inhibit electron transport, produced histological lesions similar to those found with N-methyl-D-aspartate (NMDA) or kainate. An increase in gamma-aminobutyric acid release, which has been used previously as a marker of excitatory amino acid-induced acute excitotoxicity, was also found to occur with IOA or KCN treatment. The NMDA antagonists 2-amino-5-phosphonovalerate and MK-801 [(+)-11-dihydro-5H-dibenzo[a,d]cyclohepten,5,10-imine maleate] protected retina from IOA- or KCN-induced lesioning and prevented the increase in gamma-aminobutyric acid release. The non-NMDA glutamate antagonist, 6-nitro,7-cyano-quinoxaline,2,3-dion, had little effect suggesting that the damage was mediated predominantly by the NMDA receptor. Extracellular glutamate and aspartate concentrations remained low (less than 0.2 microM) throughout incubation. Thus, the data furnish no evidence that an increase in released glutamate or aspartate is responsible for the activation of the NMDA receptor. Lactate production, ATP and phosphocreatine concentrations were also measured. ATP and phosphocreatine, but not lactate, levels were correlated with the induction of an acute excitotoxic lesion. The depletion of high energy phosphates and the first appearance of acute excitotoxicity were temporally distinct. Possible mechanisms linking metabolic inhibition and NMDA receptor-mediated acute excitotoxicity are discussed.

Serotonin release contributes to the locomotor stimulant effects of 3,4-methylenedioxymethamphetamine in rats.

Abstract: Methylenedioxymethamphetamine (MDMA) is a phenylethylamine with novel mood-altering properties in humans. MDMA shares the dopamine-releasing properties of amphetamine but has been found to be a more potent releaser of serotonin (5-HT). The present study undertook to determine the relative roles of dopamine and 5-HT release in MDMA-induced locomotor hyperactivity. S-(+)MDMA produced dose-dependent increases of rat locomotion. Investigatory behaviors such as holepokes and rearings were suppressed by (+)MDMA. Pretreatment with the selective 5-HT uptake inhibitors fluoxetine, sertraline and zimelidine inhibited (+)MDMA-induced locomotor hyperactivity but failed to antagonize the reduction of holepokes and rearings. Because 5-HT uptake inhibitors have been found previously to block the MDMA-induced release of 5-HT in vitro, and because fluoxetine was found to have no effect on (+)amphetamine-induced hyperactivity, the present results suggest that (+) MDMA-induced locomotor hyperactivity is dependent on release of endogenous 5-HT. Additionally, prior depletion of central 5-HT with p-chlorophenylalanine partially antagonized the (+)MDMA-induced hyperactivity, although catecholamine synthesis inhibition with alpha-methyl-p-tyrosine did not block the effects of (+)MDMA. Taken together, these studies suggest that (+)MDMA increases locomotor activity via mechanisms that are dependent on the release of central 5-HT and that are qualitatively different from the mechanism of action of (+)amphetamine.

Effects of the selective adenosine A2 receptor agonist CGS 21680 on in vitro electrophysiology, cAMP formation and dopamine release in rat hippocampus and striatum.

Abstract: Evaluation of adenosine A2 receptor function in the mammalian CNS has been impeded by the lack of highly selective A2 receptor agonists. The present investigations describe the actions of a recently introduced A2 selective adenosine agonist, CGS 21680 (2-[p-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne), on various functional neural responses known to be affected by adenosine. In hippocampal slices, CGS 21680 appeared to be a weak agonist on pre- and postsynaptic measures of electrophysiological activity (putative A1 receptor mediated events) and was ineffective at stimulating the formation of cAMP (a putative A2b mediated response). 5'-N-ethycarboxamidoadenosine (NECA), which is known to act at both A2a and A2b receptors, increased hippocampal cAMP levels 4-fold. In striatal slices, CGS 21680 potently stimulated the formation of cAMP with an EC50 of 110 nM but was ineffective at inhibiting electrically stimulated dopamine release. In contrast, adenosine and cyclohexyladenosine both inhibited the stimulus-evoked overflow of dopamine. These results agree with previous receptor binding studies suggesting that CGS 21680 is a relatively selective agonist at the high affinity adenosine A2a receptor in striatum, with little intrinsic activity at the low affinity A2b site in hippocampus.

Pharmacological characterization of the discriminative stimulus effects of cocaine in rhesus monkeys.

Abstract: Rhesus monkeys (n = 6), trained in a two-lever, food-reinforced paradigm to discriminate cocaine (0.2 or 0.4 mg/kg, i.m.) from saline, received injections of cocaine (0.025-0.40 mg/kg, i.v. or i.m.) or various direct and indirect acting agonists (i.v.). Administration of cocaine resulted in a dose-related increase in the percentage of responses that occurred on the drug-appropriate lever. The indirect dopamine agonists GBR 12909 (0.2-1.6 mg/kg), mazindol (0.025-0.4 mg/kg), nomifensine (0.025-0.2 mg/kg) and bupropion (0.1-1.6 mg/kg) each produced dose-related increases in cocaine-appropriate responding, with complete substitution for cocaine achieved at the highest doses of each drug. In contrast, the norepinephrine re-uptake blockers tomoxetine (0.8-6.4 mg/kg) and nisoxetine (0.4-1.6 mg/kg), the serotonin re-uptake blocker fluoxetine (1.6-12.8 mg/kg), the D1 agonist SKF 38393 (3.2-12.8 mg/kg) and the D2 agonist quinpirole (0.05-0.2 mg/kg) failed to engender cocaine-appropriate responding. Administration of the D1 antagonist SCH 23390 (0.05-0.2 mg/kg, i.m.) 20 min before cocaine resulted in a 4- to 8-fold parallel shift to the right in the cocaine dose-response function. Similarly, the D2 antagonist haloperidol (0.003-0.012 mg/kg, i.m.) produced at least a 2-fold shift to the right in the cocaine dose-response function. The results indicate that blockade of dopamine re-uptake is sufficient to mimic the cocaine discriminative stimulus and suggest that stimulation of either D1 or D2 receptors is necessary but not sufficient for the expression of the discriminative stimulus effects of cocaine.

Autoradiographic characterization of (+-)-1-(2,5-dimethoxy-4-[125I] iodophenyl)-2-aminopropane ([125I]DOI) binding to 5-HT2 and 5-HT1c receptors in rat brain.

Abstract: The 5-HT2 (serotonin) receptor has traditionally been labeled with antagonist radioligands such as [3H]ketanserin and [3H]spiperone, which label both agonist high-affinity (guanyl nucleotide-sensitive) and agonist low-affinity (guanyl nucleotide-insensitive) states of this receptor. The hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) is an agonist which labels the high-affinity guanyl nucleotide-sensitive state of brain 5-HT2 receptors selectively. In the present study, conditions for autoradiographic visualization of (+/-)-[125I]DOI-labeled 5-HT2 receptors were optimized and binding to slide-mounted sections was characterized with respect to pharmacology, guanyl nucleotide sensitivity and anatomical distribution. In slide-mounted rat brain sections (+/-)-[125I]DOI binding was saturable, of high affinity (KD approximately 4 nM) and displayed a pharmacologic profile typical of 5-HT2 receptors. Consistent with coupling of 5-HT2 receptors in the high-affinity state to a guanyl nucleotide regulatory protein, [125I]DOI binding was inhibited by guanyl nucleotides but not by adenosine triphosphate. Patterns of autoradiographic distribution of [125I]DOI binding to 5-HT2 receptors were similar to those seen with [3H]ketanserin- and [125I]-lysergic acid diethylamide-labeled 5-HT2 receptors. However, the density of 5-HT2 receptors labeled by the agonist [125I]DOI was markedly lower (30-50%) than that labeled by the antagonist [3H]ketanserin. High densities of [125I]DOI labeling were present in olfactory bulb, anterior regions of cerebral cortex (layer IV), claustrum, caudate putamen, globus pallidus, ventral pallidum, islands of Calleja, mammillary nuclei and inferior olive. Binding in hippocampus, thalamus and hypothalamus was generally sparse. Of note, choroid plexus, a site rich in 5-HT1c receptors had a high density of [125I]DOI binding sites but [3H]ketanserin binding in this region was low. Studies in which [125I]DOI binding to 5-HT2 receptors was blocked with spiperone revealed persisting robust [125I]DOI binding in choroid plexus, which was guanyl nucleotide-sensitive and displayed a pharmacologic profile consistent with its binding to 5-HT1c receptors. These studies suggest that [125I]DOI may be useful as a radiolabel for visualizing the agonist high-affinity state of 5-HT2 receptors and for visualizing 5-HT1c receptors.

Zolpidem and triazolam in humans: behavioral and subjective effects and abuse liability.

Abstract: Zolpidem, which is currently marketed in Europe as a hypnotic, is a short-duration imidazopyridine whose actions are mediated at the gamma-aminobutyric acid benzodiazepine receptor complex. However, zolpidem produces a variety of biochemical differences from classic benzodiazepine agonists including showing selectivity for the central BZ1 (omega 1) receptor subtype as well as showing a different pattern of distribution of binding sites. This study compared zolpidem to the benzodiazepine hypnotic triazolam in 15 healthy male volunteers with histories of sedative drug abuse. Placebo, zolpidem (15, 30 and 45 mg) and triazolam (0.25, 0.5 and 0.75 mg) were administered p.o. in a mixed sequence in a double-blind, cross-over design. The onset time with zolpidem was faster than with triazolam, with peak effects of both drugs occurring at 1 to 2 hr after administration. Both zolpidem and triazolam produced dose-related decrements in performance on various performance tasks including circular lights, reaction time, balance, number recall and the digit symbol substitution test. Both drugs also produced similar dose-related changes on various observer ratings including overall strength of drug effect. Triazolam, but not zolpidem, increased subject- and observer-rated sleepiness and produced greater impairment on a picture memory task. Zolpidem, but not triazolam, produced increases in subject ratings of various somatic symptoms (e.g., dizzy, anxious and queasy) and there were 9 days on which subjects vomited after zolpidem, but none after triazolam. Although the highest dose of both drugs was identified by subjects as being active, the highest dose of triazolam was identified as being barbiturate, benzodiazepine or alcohol, almost twice as often as the highest dose of zolpidem. Overall, this study shows that although zolpidem produces many effects in common with triazolam, it also has a unique profile of effects distinguishable from classic benzodiazepine agonists. The mechanism(s) underlying these differences is unclear, but may be related to the atypical biochemical profile of zolpidem.