Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 2004"
TL;DR: Variable activity of these P450s, brought about by genetic polymorphisms and drug interactions, may alter the balance of TAM effects in vivo.
Abstract: We performed comprehensive kinetic, inhibition, and correlation analyses in human liver microsomes and experiments in expressed human cytochromes P450 (P450s) to identify primary and secondary metabolic routes of tamoxifen (TAM) and the P450s catalyzing these reactions at therapeutically relevant concentrations. N-Desmethyl-TAM formation catalyzed by CYP3A4/5 was quantitatively the major primary metabolite of TAM; 4-hydroxy-TAM formation catalyzed by CYP2D6 (and other P450s) represents a minor route. Other minor primary metabolites include alpha -, 3-, and 4'-hydroxyTAM and one unidentified metabolite (M-I) and were primarily catalyzed by CYP3A4, CYP3A5, CYP2B6/2C19, and CYP3A4, respectively. TAM secondary metabolism was examined using N-desmethyl- and 4-hydroxy-TAM as intermediate substrates. N-Desmethyl-TAM was predominantly biotransformed to alpha-hydroxy N-desmethyl-, N-didesmethyl-, and 4-hydroxy N-desmethyl-TAM (endoxifen), whereas 4-hydroxy-TAM was converted to 3,4-dihydroxyTAM and endoxifen. Except for the biotransformation of N-desmethyl-TAM to endoxifen, which was exclusively catalyzed by CYP2D6, all other routes of N-desmethyl- and 4-hydroxy-TAM biotransformation were catalyzed predominantly by the CYP3A subfamily. TAM and its primary metabolites undergo extensive oxidation, principally by CYP3A and CYP2D6 to metabolites that exhibit a range of pharmacological effects. Variable activity of these P450s, brought about by genetic polymorphisms and drug interactions, may alter the balance of TAM effects in vivo.
628 citations
TL;DR: OATP2 is the most important transporter for the hepatic uptake of pitavastatin in humans, according to the observed uptake clearance in human hepatocytes, and about 90% of the total hepatic clearance could be accounted for by OATP 2.
Abstract: Pitavastatin, a novel potent 3-hydroxymethylglutaryl-CoA reductase inhibitor, is selectively distributed to the liver in rats. However, the hepatic uptake mechanism of pitavastatin has not been clarified yet. In the present study, we investigated the contribution of organic anion transporting polypeptide 2 (OATP2/OATP1B1) and OATP8 (OATP1B3) to pitavastatin uptake using transporter-expressing HEK293 cells and human cryopreserved hepatocytes. Uptake studies using OATP2- and OATP8-expressing cells revealed a saturable and Na+-independent uptake, with Km values of 3.0 and 3.3 μM for OATP2 and OATP8, respectively. To determine which transporter is more important for its hepatic uptake, we proposed a methodology for estimating their quantitative contribution to the overall hepatic uptake by comparing the uptake clearance of pitavastatin with that of reference compounds (a selective substrate for OATP2 (estrone-3-sulfate) and OATP8 (cholecystokinin octapeptide) in expression systems and human hepatocytes. The concept of this method is similar to the so-called relative activity factor method often used in estimating the contribution of each cytochrome P450 isoform to the overall metabolism. Applying this method to pitavastatin, the observed uptake clearance in human hepatocytes could be almost completely accounted for by OATP2 and OATP8, and about 90% of the total hepatic clearance could be accounted for by OATP2. This result was also supported by estimating the relative expression level of each transporter in expression systems and hepatocytes by Western blot analysis. These results suggest that OATP2 is the most important transporter for the hepatic uptake of pitavastatin in humans.
427 citations
TL;DR: Considering the possibly concentrated high unbound concentrations of GEM-1-O-glu in the liver and its relatively larger plasma unbound fraction compared with GEM itself, the glucuronide inhibition of the CYP2C8-mediated metabolism of CER appears to be the main mechanism for the clinically relevant drug-drug interaction.
Abstract: A serious pharmacokinetic interaction between cerivastatin (CER) and gemfibrozil (GEM) has been reported. In the present study, we examined the inhibitory effects of GEM and its metabolites, M3 and gemfibrozil 1- O -β-glucuronide (GEM-1- O -glu), on the uptake of CER by human organic anion transporting polypeptide 2 (OATP2)-expressing cells and its metabolism in cytochrome P450 expression systems. Uptake studies showed that GEM and GEM-1- O -glu significantly inhibited the OATP2-mediated uptake of CER with IC50 values of 72 and 24 μM, respectively. They also inhibited the CYP2C8-mediated metabolism of CER with IC50 values of 28 and 4 μM, respectively, whereas M3 had no effects. GEM and GEM-1- O -glu minimally inhibited the CYP3A4-mediated metabolism of CER. The IC50 values of GEM and GEM-1- O -glu for the uptake and the metabolism of CER obtained in the present study were lower than their total, and not unbound, plasma concentrations. However, considering the possibly concentrated high unbound concentrations of GEM-1- O -glu in the liver and its relatively larger plasma unbound fraction compared with GEM itself, the glucuronide inhibition of the CYP2C8-mediated metabolism of CER appears to be the main mechanism for the clinically relevant drug-drug interaction. Previously reported clinical drug interaction studies showing that coadministration of GEM with pravastatin or pitavastatin, both of which are known to be cleared from the plasma by the uptake transporters in the liver, only minimally (less than 2-fold) increased the area under the plasma concentration-time curve of these statins, also supported our present conclusion.
382 citations
TL;DR: The results indicate that the histamine H4 receptor plays a role in the inflammatory process and may have the potential to be useful in treating inflammation in humans.
Abstract: Histamine mediates its physiological function through binding to four known histamine receptors. Here, we describe the first selective antagonist of the histamine H4 receptor, the newest member of the histamine receptor family, and provide evidence that such antagonists have anti-inflammatory activity in vivo. 1-[(5-chloro-1 H -indol-2-yl)carbonyl]-4-methylpiperazine (JNJ 7777120) has a K i of 4.5 nM versus the human receptor and a p A 2 of 8.1. It is equipotent against the human, mouse, and rat receptors. It exhibits at least 1000-fold selectivity over H1, H2, or H3 receptors and has no cross-reactivity against 50 other targets. This compound has an oral bioavailability of ∼30% in rats and 100% in dogs, with a half-life of ∼3 h in both species. JNJ 7777120 blocks histamine-induced chemotaxis and calcium influx in mouse bone marrow-derived mast cells. In addition, it can block the histamine-induced migration of tracheal mast cells from the connective tissue toward the epithelium in mice. JNJ 7777120 significantly blocks neutrophil infiltration in a mouse zymosan-induced peritonitis model. This model is reported to be mast cell-dependent, which suggests that the compound effect may be mediated by mast cells. These results indicate that the histamine H4 receptor plays a role in the inflammatory process. Selective H4 receptor antagonists like JNJ 7777120 may have the potential to be useful in treating inflammation in humans.
378 citations
TL;DR: A critical role for superoxide is unraveled in the nociceptive signaling cascade both peripherally and centrally and the discovery of this pathway opens a new therapeutic strategy for the development of novel nonnarcotic antihyperalgesic agents.
Abstract: Novel classes of pain-relieving molecules are needed to fill the void between nonsteroidal anti-inflammatory agents and narcotics. Our studies have identified superoxide as a novel mediator of hyperalgesia (clinically defined as an augmented sensitivity to painful stimuli) and have exposed potential pathways through which this radical modulates the hyperalgesic response. The role of superoxide in pain was elucidated using a superoxide dismutase mimetic, M40403 [a manganese(II) complex with a bis(cyclo-hexylpyridine-substituted) macrocyclic ligand]. Intraplantar injection of carrageenan in rats led to time-dependent development of peripheral inflammation [measured parameters of inflammation included paw edema, cytokine release in the paw exudates, nitrotyrosine formation (a marker of peroxynitrite formation and oxidative stress), and poly-ADP-ribose-polymerase activation (the nuclear enzyme activated by superoxide/peroxynitrite)] and hyperalgesia. M40403 blocked all measured parameters of inflammation and hyperalgesia. Furthermore, when given therapeutically (2 h after the induction of hyperalgesia) either by intravenous or intrathecal administration, M40403 but not its inactive congener M40404 inhibited hyperalgesia with a rapid onset of action. Our results also show that, at the level of the spinal cord and time of peak hyperalgesia, endogenous manganese superoxide dismutase was nitrated and subsequently deactivated, losing its capacity to remove superoxide. The antihyperalgesic effects of M40403 were not reversed by naloxone excluding the potential involvement of an opiate pathway. Collectively, these studies have unraveled a critical role for superoxide in the nociceptive signaling cascade both peripherally and centrally. The discovery of this pathway opens a new therapeutic strategy for the development of novel nonnarcotic antihyperalgesic agents.
372 citations
TL;DR: The recent identification of polymorphic genetic variants of human OCT1 and OCT2 that severely affect transport activity suggests that some of the interpatient differences in response and sensitivity to cationic drugs may be caused by variable activity of these transporters.
Abstract: For the elimination of environmental toxins and metabolic waste products, the body is equipped with a range of broad-specificity transporters that are generally present in the liver, kidney, and intestine. The polyspecific organic cation transporters OCT1, 2, and 3 (SLC22A1-3) mediate the facilitated transport of a variety of structurally diverse organic cations, including many drugs, toxins, and endogenous compounds. OCT1 and OCT2 are found in the basolateral membrane of hepatocytes, enterocytes, and renal proximal tubular cells. OCT3 has a more widespread tissue distribution and is considered to be the major component of the extraneuronal monoamine transport system (or uptake-2), which is responsible for the peripheral elimination of monoamine neurotransmitters. Studies with knockout mouse models have directly demonstrated that these transporters can have a major impact on the pharmacological behavior of various substrate organic cations. The recent identification of polymorphic genetic variants of human OCT1 and OCT2 that severely affect transport activity thus suggests that some of the interpatient differences in response and sensitivity to cationic drugs may be caused by variable activity of these transporters.
365 citations
TL;DR: It is suggested that affective regulation is exerted by CRF/urocortin systems within the brain based upon the sensitivity of local brain sites to CRf/uroCortin ligand administration and the appearance of hypothalamo-pituitary-adrenocortical activation following stressor exposure.
Abstract: Organisms exposed to challenging stimuli that alter the status quo inside or outside of the body are required for survival purposes to generate appropriate coping responses that counteract departures from homeostasis. Identification of an executive control mechanism within the brain capable of coordinating the multitude of endocrine, physiological, and functional coping responses has high utility for understanding the response of the organism to stressor exposure under normal or pathological conditions. The corticotropin-releasing factor (CRF)/urocortin family of neuropeptides and receptors constitutes an affective regulatory system due to the integral role it plays in controlling neural substrates of arousal, emotionality, and aversive processes. In particular, available evidence from pharmacological intervention in multiple species and phenotyping of mutant mice shows that CRF/urocortin systems mediate motor and psychic activation, stimulus avoidance, and threat recognition responses to aversive stimulus exposure. It is suggested that affective regulation is exerted by CRF/urocortin systems within the brain based upon the sensitivity of local brain sites to CRF/urocortin ligand administration and the appearance of hypothalamo-pituitary-adrenocortical activation following stressor exposure. Moreover, these same stress neuropeptides may constitute a mechanism for learning to avoid noxious stimuli by facilitating the formation of so-called emotional memories. A conceptual framework is provided for extrapolation of animal model findings to humans and for viewing CRF/urocortin activation as a continuum measure linking normal and pathological states.
353 citations
TL;DR: The present findings provide compelling evidence that cinacalcet HCl is a potent and stereoselective activator of the parathyroid CaR and, as such, might be beneficial in the treatment of hyperparathyroidism.
Abstract: Calcimimetic compounds, which activate the parathyroid cell Ca(2+) receptor (CaR) and inhibit parathyroid hormone (PTH) secretion, are under experimental study as a treatment for hyperparathyroidism. This report describes the salient pharmacodynamic properties, using several test systems, of a new calcimimetic compound, cinacalcet HCl. Cinacalcet HCl increased the concentration of cytoplasmic Ca(2+) ([Ca(2+)](i)) in human embryonic kidney 293 cells expressing the human parathyroid CaR. Cinacalcet HCl (EC(50) = 51 nM) in the presence of 0.5 mM extracellular Ca(2+) elicited increases in [Ca(2+)](i) in a dose- and calcium-dependent manner. Similarly, in the presence of 0.5 mM extracellular Ca(2+), cinacalcet HCl (IC(50) = 28 nM) produced a concentration-dependent decrease in PTH secretion from cultured bovine parathyroid cells. Using rat medullary thyroid carcinoma 6-23 cells expressing the CaR, cinacalcet HCl (EC(50) = 34 nM) produced a concentration-dependent increase in calcitonin secretion. In vivo studies in rats demonstrated cinacalcet HCl is orally bioavailable and displays approximately linear pharmacokinetics over the dose range of 1 to 36 mg/kg. Furthermore, this compound suppressed serum PTH and blood-ionized Ca(2+) levels and increased serum calcitonin levels in a dose-dependent manner. Cinacalcet was about 30-fold more potent at lowering serum levels of PTH than it was at increasing serum calcitonin levels. The S-enantiomer of cinacalcet (S-AMG 073) was at least 75-fold less active in these assay systems. The present findings provide compelling evidence that cinacalcet HCl is a potent and stereoselective activator of the parathyroid CaR and, as such, might be beneficial in the treatment of hyperparathyroidism.
353 citations
TL;DR: Three lines of evidence link S1P3 receptor activity with acute toxicity and cardiovascular regulation: compound potency on S 1P3 correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus brady Cardia and hypertension was consistent with affinity for S1 p1 relative to S1p3; and toxicity, brady cardia, and hypertension were absent in S1 P3-/- mice.
Abstract: Sphingosine 1-phosphate (S1P) is a bioactive lysolipid with pleiotropic functions mediated through a family of G protein-coupled receptors, S1P(1,2,3,4,5). Physiological effects of S1P receptor agonists include regulation of cardiovascular function and immunosuppression via redistribution of lymphocytes from blood to secondary lymphoid organs. The phosphorylated metabolite of the immunosuppressant agent FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol) and other phosphonate analogs with differential receptor selectivity were investigated. No significant species differences in compound potency or rank order of activity on receptors cloned from human, murine, and rat sources were observed. All synthetic analogs were high-affinity agonists on S1P(1), with IC(50) values for ligand binding between 0.3 and 14 nM. The correlation between S1P(1) receptor activation and the ED(50) for lymphocyte reduction was highly significant (p < 0.001) and lower for the other receptors. In contrast to S1P(1)-mediated effects on lymphocyte recirculation, three lines of evidence link S1P(3) receptor activity with acute toxicity and cardiovascular regulation: compound potency on S1P(3) correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus bradycardia and hypertension was consistent with affinity for S1P(1) relative to S1P(3); and toxicity, bradycardia, and hypertension were absent in S1P(3)(-/-) mice. Blood pressure effects of agonists in anesthetized rats were complex, whereas hypertension was the predominant effect in conscious rats and mice. Immunolocalization of S1P(3) in rodent heart revealed abundant expression on myocytes and perivascular smooth muscle cells consistent with regulation of bradycardia and hypertension, whereas S1P(1) expression was restricted to the vascular endothelium.
347 citations
TL;DR: The pH-sensitive activity of human organic anion transporting polypeptide OATP-B, which is expressed at the apical membrane of human small intestinal epithelial cells, was functionally characterized and clarification of factors, such as pH, that affect the OATp-B activity is essential for an understanding of the physiological and pharmacological relevance of the transporter in the small intestine.
Abstract: The pH-sensitive activity of human organic anion transporting polypeptide OATP-B, which is expressed at the apical membrane of human small intestinal epithelial cells, was functionally characterized. When initial uptake of estrone-3-sulfate, a typical substrate of OATP, was studied kinetically, we observed an increase in V(max) with decrease of pH from 7.4 to 5.0, whereas the change in K(m) was negligible. OATP-B-mediated uptake of estrone-3-sulfate was independent of sodium, chloride, bicarbonate, or glutathione, whereas the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone exhibited a pH-dependent inhibitory effect, suggesting that a proton gradient is a driving force for OATP-B. When OATP-B was expressed in human embryonic kidney 293 cells, uptake activities for anionic compounds showed various kinds of pH sensitivity. Dehydroepiandrosterone-sulfate, estrone-3-sulfate, and fexofenadine were transported by OATP-B at both neutral and acidic pH, whereas estradiol-17beta-glucuronide, acetic acid, and lactic acid were not transported at all. Transport of taurocholic acid and pravastatin by OATP-B was observed only at acidic pH, demonstrating a pH-sensitive substrate specificity of OATP-B. Because the physiological pH close to the surface of intestinal epithelial cells is acidic, the roles of OATP-B in the small intestine might be different from those in other tissues, such as liver basolateral membrane. Although the driving force for OATP-B has not been fully established, the clarification of factors, such as pH, that affect the OATP-B-activity is essential for an understanding of the physiological and pharmacological relevance of the transporter in the small intestine.
342 citations
TL;DR: A class of α-keto-heterocycles are identified that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation.
Abstract: Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of α-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.
TL;DR: It is reported here that methamphetamine activates microglia in a dose-related manner and along a time course that is coincident with dopamine nerve ending damage, suggesting that microglial activation represents an early step in methamphetamine-induced neurotoxicity.
Abstract: Methamphetamine intoxication causes long-lasting damage to dopamine nerve endings in the striatum. The mechanisms underlying this neurotoxicity are not known but oxidative stress has been implicated. Microglia are the major antigen-presenting cells in brain and when activated, they secrete an array of factors that cause neuronal damage. Surprisingly, very little work has been directed at the study of microglial activation as part of the methamphetamine neurotoxic cascade. We report here that methamphetamine activates microglia in a dose-related manner and along a time course that is coincident with dopamine nerve ending damage. Prevention of methamphetamine toxicity by maintaining treated mice at low ambient temperature prevents drug-induced microglial activation. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which damages dopamine nerve endings and cell bodies, causes extensive microglial activation in striatum as well as in the substantia nigra. In contrast, methamphetamine causes neither microglial activation in the substantia nigra nor dopamine cell body damage. Dopamine transporter antagonists (cocaine, WIN 35,428 [(-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate], and nomifensine), selective D1 (SKF 82958 [(+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide]), D2 (quinpirole), or mixed D1/D2 receptor agonists (apomorphine) do not mimic the effect of methamphetamine on microglia. Hyperthermia, a prominent and dangerous clinical response to methamphetamine intoxication, was also ruled out as the cause of microglial activation. Together, these data suggest that microglial activation represents an early step in methamphetamine-induced neurotoxicity. Other neurochemical effects resulting from methamphetamine-induced overflow of DA into the synapse, but which are not neurotoxic, do not play a role in this response.
TL;DR: Duloxetine significantly attenuated late phase paw-licking behavior in a dose-dependent manner in the formalin model of persistent pain and was more potent than venlafaxine, milnacipran, and amitriptyline in this paradigm and was minimally efficacious in the tail-flick model of acute nociceptive pain.
Abstract: 5-Hydroxytryptamine (serotonin) (5-HT) and norepinephrine (NE) are implicated in modulating descending inhibitory pain pathways in the central nervous system. Duloxetine is a selective and potent dual 5-HT and NE reuptake inhibitor (SNRI). The ability of duloxetine to antagonize 5-HT depletion in para-chloramphetamine-treated rats was comparable with that of paroxetine, a selective serotonin reuptake inhibitor (SSRI), whereas its ability to antagonize NE depletion in alpha-methyl-m-tyrosine-treated rats was similar to norepinephrine reuptake inhibitors (NRIs), thionisoxetine or desipramine. In this paradigm, duloxetine was also more potent than other SNRIs, including venlafaxine or milnacipran and amitriptyline. Low doses of the SSRI paroxetine or the NRI thionisoxetine alone did not have an effect on late phase paw-licking pain behavior in the formalin model of persistent pain; however, when combined, significantly attenuated this pain behavior. Duloxetine (3-15 mg/kg intraperitoneal) significantly attenuated late phase paw-licking behavior in a dose-dependent manner in the formalin model and was more potent than venlafaxine, milnacipran, and amitriptyline. These effects of duloxetine were evident at doses that did not cause neurologic deficits in the rotorod test. Duloxetine (5-30 mg/kg oral) was also more potent and efficacious than venlafaxine and milnacipran in reversing mechanical allodynia behavior in the L5/L6 spinal nerve ligation model of neuropathic pain. Duloxetine (3-30 mg/kg oral) was minimally efficacious in the tail-flick model of acute nociceptive pain. These data suggest that inhibition of both 5-HT and NE uptake may account for attenuation of persistent pain mechanisms. Thus, duloxetine may have utility in treatment of human persistent and neuropathic pain states.
TL;DR: The ontogeny of CYP2C9 and -2C19 were dissimilar among both fetal and 0- to 5-months postnatal samples, implying different developmental regulatory mechanisms.
Abstract: The CYP2C subfamily is responsible for metabolizing many important drugs and accounts for about 20% of the cytochrome P450 in adult liver. To determine developmental expression patterns, liver microsomal CYP2C9 and -2C19 were measured ( n = 237; ages, 8 weeks gestation-18 years) by Western blotting and with diclofenac or mephenytoin, respectively, as probe substrates. CYP2C9-specific content and catalytic activity were consistent with expression at 1 to 2% of mature values (i.e., specific content, 18.3 pmol/mg protein and n = 79; specific activity, 549.5 pmol/mg/min and n = 72) during the first trimester, with progressive increases during the second and third trimesters to levels approximately 30% of mature values. From birth to 5 months, CYP2C9 protein values varied 35-fold and were significantly higher than those observed during the late fetal period, with 51% of samples exhibiting values commensurate with mature levels. Less variable CYP2C9 protein and activity values were observed between 5 months and 18 years. CYP2C19 protein and catalytic activities that were 12 to 15% of mature values (i.e., specific content, 14.6 pmol/mg and n = 20; specific activity, 18.5 pmol/mg/min and n = 19) were observed as early as 8 weeks of gestation and were similar throughout the prenatal period. CYP2C19 expression did not change at birth, increased linearly over the first 5 postnatal months, and varied 21-fold from 5 months to 10 years. Adult CYP2C19 protein and activity values were observed in samples older than 10 years. The ontogeny of CYP2C9 and -2C19 were dissimilar among both fetal and 0- to 5-months postnatal samples, implying different developmental regulatory mechanisms.
TL;DR: Inhibition of CYP2B6 was time- and concentration-dependent, and as shown by dialysis experiments, it was irreversible and dependent on NADPH, suggesting a mechanism-based mode of action, and the possibility of drug interactions between thienopyridine derivates and drug substrates of CYp2B 6 and CYP1A2 is suggested.
Abstract: The thienopyridine derivatives ticlopidine and clopidogrel are inhibitors of ADP-induced platelet aggregation. Pharmacological activity of these prodrugs depends on cytochrome P450 (P450)-dependent oxidation to the active antithrombotic agent. In this study, we investigated the interaction potential of clopidogrel and ticlopidine by using human liver microsomes and recombinantly expressed P450 isoforms. Both clopidogrel and ticlopidine inhibited CYP2B6 with highest potency and CYP2C19 with lower potency. Clopidogrel also inhibited CYP2C9, and ticlopidine also inhibited CYP1A2, with lower potency. Inhibition of CYP2B6 was time- and concentration-dependent, and as shown by dialysis experiments, it was irreversible and dependent on NADPH, suggesting a mechanism-based mode of action. Inactivation was of nonpseudo-firstorder type with maximal rates of inactivation (K(inact)) for clopidogrel and ticlopidine in microsomes (recombinant CYP2B6) of 0.35 (1.5 min(-1)) and 0.5 min(-1) (0.8 min(-1)), respectively, and half-maximal inactivator concentrations (KI) were 0.5 microM (1.1 microM) for clopidogrel and 0.2 microM (0.8 microM) for ticlopidine. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. A chemical mechanism is discussed based on the known metabolic activation of clopidogrel and on the finding that hemoprotein integrity of recombinant CYP2B6 was not affected by irreversible inhibition. These results suggest the possibility of drug interactions between thienopyridine derivates and drug substrates of CYP2B6 and CYP2C19.
TL;DR: Evaluating the in vitro antiproliferative ability of cannabidiol (CBD), a nonpsychoactive cannabinoid compound, on U87 and U373 human glioma cell lines suggests a possible application of CBD as an antineoplastic agent.
Abstract: Recently, cannabinoids (CBs) have been shown to possess antitumor properties. Because the psychoactivity of cannabinoid compounds limits their medicinal usage, we undertook the present study to evaluate the in vitro antiproliferative ability of cannabidiol (CBD), a nonpsychoactive cannabinoid compound, on U87 and U373 human glioma cell lines. The addition of CBD to the culture medium led to a dramatic drop of mitochondrial oxidative metabolism [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide test] and viability in glioma cells, in a concentration-dependent manner that was already evident 24 h after CBD exposure, with an apparent IC(50) of 25 microM. The antiproliferative effect of CBD was partially prevented by the CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; SR2) and alpha-tocopherol. By contrast, the CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716; SR1), capsazepine (vanilloid receptor antagonist), the inhibitors of ceramide generation, or pertussis toxin did not counteract CBD effects. We also show, for the first time, that the antiproliferative effect of CBD was correlated to induction of apoptosis, as determined by cytofluorimetric analysis and single-strand DNA staining, which was not reverted by cannabinoid antagonists. Finally, CBD, administered s.c. to nude mice at the dose of 0.5 mg/mouse, significantly inhibited the growth of subcutaneously implanted U87 human glioma cells. In conclusion, the nonpsychoactive CBD was able to produce a significant antitumor activity both in vitro and in vivo, thus suggesting a possible application of CBD as an antineoplastic agent.
TL;DR: This in vivo study of Sulforaphane provides the first clue as to the plasma concentrations of SUL, in vivo mitogen-activated protein kinase activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes.
Abstract: Sulforaphane (SUL) is one member of the isothiocyanate class of cancer chemopreventive compounds that has been shown to be effective in blocking initiation and progression of carcinogenesis. Previously, many studies have shown that SUL can potently induce phase II detoxifying enzymes, which contributes to its chemopreventive functions. In this study, we used 4967 oligonucleotides microarray to assess the genes that are modulated by SUL in in vivo rat livers, as well as time course of expression of these genes. The pharmacokinetics of SUL was assessed after oral dose of 50 micromol of SUL. The plasma concentration occurred at 1 h and peaked around 20 microM at 4 h after dosing and declined with a half-life of about 2.2 h. Analysis of the gene expression data found various clusters of genes that are important in cellular defense mechanisms and cell cycle regulation. The most robust cluster of genes is the metallothionein-like genes (MT-1/2 and MT-1a), which are increased up to 10-fold by 2 to 4 h after SUL dosing. The second cluster of genes is the glutathione S-transferase-A3-like genes, which include aflatoxin B1 aldehyde reductase and aldehyde oxidase. These genes are increased slightly by 4 h and peaked at 12 h. Real-time polymerase chain reaction was performed to authenticate the mRNA expression of some of these genes. In summary, this in vivo study of SUL provides the first clue as to the plasma concentrations of SUL, in vivo mitogen-activated protein kinase activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes. The results from this study may yield better insights for its chemopreventive functions.
TL;DR: The data obtained here suggest that positive modulation of GABAB receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines.
Abstract: The role of GABAB receptors in various behavioral processes has been largely defined using the prototypical GABAB receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABAB receptors in these disorders is largely unclear. We recently identified GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) as a selective allosteric positive modulator at GABAB receptors. The aim of the present study was to broadly characterize the effects of GS39783 in well-validated rodent models for motor activity, cognition, and anxiety. The following tests were included: locomotor activity in rats and mice, rotarod and traction tests (including determinations of core temperature) in mice, passive avoidance in mice and rats, elevated plus maze in rats, elevated zero maze in mice and rats, stress-induced hyperthermia in mice, and pentobarbital- and ethanol-induced sleep in mice. Unlike baclofen and/or the benzodiazepine chlordiazepoxide, GS39783 had no effect in any of the tests for locomotion, cognition, temperature, or narcosis. Most interestingly, GS39783 had anxiolytic-like effects in all the tests used. Overall, the data obtained here suggest that positive modulation of GABAB receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines.
TL;DR: This is the first report to demonstrate that the nuclear receptor PXR mediates induction of CYP2C9 with rifampicin, phenobarbital, and hyperforin, and the cis-element essential for this response is identified.
Abstract: Human CYP2C9 is important in the metabolism of numerous clinically used drugs such as the anticoagulant warfarin, the anticonvulsant phenytoin, antidiabetic drugs such as tolbutamide and glipizide, the hypertensive agent losartan, and numerous nonsteroidal anti-inflammatory drugs. Several studies have reported that certain drugs such as rifampicin and phenobarbital induce CYP2C9, but the molecular basis for this induction remains unknown. In the present study, we demonstrate that the human pregnane X receptor (hPXR) mediates induction of CYP2C9 by the prototype drugs rifampicin, hyperforin (found in St. John's Wart), and phenobarbital. Deletion and mutagenesis studies with luciferase reporter constructs showed that a functional PXR-responsive element located -1839/-1824 base pairs upstream from the translation start site was the primary binding site mediating the rifampicin induction of CYP2C9. This site was previously described as a constitutive androstane receptor-responsive element (CAR-RE). Mutational analysis of 3- and 12-kilobase CYP2C9 promoter fragments indicated that this proximal binding site was essential for rifampicin inducibility, although a cooperative effect could be attributed to a second CAR-RE located at -2899/-2883. In summary, we have demonstrated rifampicin induction of CYP2C9 promoter constructs that is consistent with the magnitude of induction of CYP2C9 protein and mRNA reported in vivo and in primary human hepatocytes, and we have identified the cis-element essential for this response. This is the first report to demonstrate that the nuclear receptor PXR mediates induction of CYP2C9 with rifampicin, phenobarbital, and hyperforin.
TL;DR: The results suggest that BCRP may play an important role in drug-drug interactions involving coadministration of the HPIs with drugs that are substrates of the transporter.
Abstract: Breast cancer resistance protein (BCRP) is a recently discovered ATP-binding cassette drug transporter. Hence, the full spectrum of therapeutic agents that interact with BCRP remains to be elucidated. Because human immunodeficiency virus protease inhibitors (HPIs) are well known P-glycoprotein (P-gp) substrates, and there is an overlap in substrate specificity between P-gp and BCRP, this study was performed to investigate whether HPIs are substrates and/or inhibitors of BCRP. First, the effect of HPIs on BCRP efflux activity in human embryonic kidney (HEK) cells stably expressing wild-type BCRP (482R) and its two mutants (482T and 482G) was studied by measuring intracellular mitoxantrone fluorescence using flow cytometry. We found that ritonavir, saquinavir, and nelfinavir were effective inhibitors of wild-type BCRP (482R) with IC50 values of 19.5 +/- 0.8 microM, 19.5 +/- 7.6 microM, and 12.5 +/- 4.1 microM, respectively. Ritonavir, saquinavir, and nelfinavir inhibited 482T and 482G with IC50 values that were approximately 2 times greater than that for 482R. Indinavir and amprenavir had no significant inhibition on BCRP activity. Direct efflux of radiolabeled HPIs in HEK cells was measured to determine whether the HPIs are substrates of BCRP. None of the HPIs were found to be transported by BCRP. Together, ritonavir, saquinavir, nelfinavir, indinavir, and amprenavir are not substrates for BCRP. However, ritonavir, saquinavir, and nelfinavir are effective inhibitors of the transporter. These results suggest that BCRP may play an important role in drug-drug interactions involving coadministration of the HPIs with drugs that are substrates of the transporter.
TL;DR: The data do not support the hypothesis that mentholated cigarette smoking results in a greater absorption of tobacco smoke toxins, but find the finding of impaired metabolism of nicotine while mentholation of cigarettes suggests that ment Holedouble cigarette smoking enhances systemic nicotine exposure.
Abstract: Smoking mentholated cigarettes has been suggested to convey a greater cancer risk compared with smoking nonmentholated cigarettes. Two of the possible mechanisms by which mentholated cigarette smoking could increase risk are by increasing systemic exposure to tobacco smoke toxins and by affecting the metabolism of nicotine or tobacco smoke carcinogens. To examine these possibilities, we performed a crossover study in 14 healthy smokers, one-half of whom were African-Americans and one-half whites. Subjects were randomly assigned to smoke mentholated or nonmentholated cigarettes for 1 week, then to cross over to the other type of cigarettes for another week. Subjects were confined to a Clinical Research Center for 3 days of each week, during which time blood levels of nicotine and carbon monoxide were measured throughout the day and an intravenous infusion of deuterium-labeled nicotine and cotinine was administered to determine the rate and pathways of nicotine metabolism. The systemic intake of nicotine and carbon monoxide was, on average, not affected by mentholation of cigarettes. Mentholated cigarette smoking did significantly inhibit the metabolism of nicotine (clearance: 1289 versus 1431 ml/min, two sided, p = 0.02). Inhibition of nicotine metabolism occurred both by slower oxidative metabolism to cotinine and by slower glucuronide conjugation. Our data do not support the hypothesis that mentholated cigarette smoking results in a greater absorption of tobacco smoke toxins. Our finding of impaired metabolism of nicotine while mentholated cigarette smoking suggests that mentholated cigarette smoking enhances systemic nicotine exposure.
TL;DR: Salvinorin A was found to be a full agonist, being significantly more efficacious than (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for κ-opioid receptors).
Abstract: The diterpene salvinorin A from Salvia divinorum has recently been reported to be a high-affinity and selective kappa-opioid receptor agonist (Roth et al., 2002). Salvinorin A and selected derivatives were found to be potent and efficacious agonists in several measures of agonist activity using cloned human kappa-opioid receptors expressed in human embryonic kidney-293 cells. Thus, salvinorin A, salvinorinyl-2-propionate, and salvinorinyl-2-heptanoate were found to be either full (salvinorin A) or partial (2-propionate, 2-heptanoate) agonists for inhibition of forskolin-stimulated cAMP production. Additional studies of agonist potency and efficacy of salvinorin A, performed by cotransfecting either the chimeric G proteins Gaq-i5 or the universal G protein Ga16 and quantification of agonist-evoked intracellular calcium mobilization, affirmed that salvinorin A was a potent and effective kappa-opioid agonist. Results from structure-function studies suggested that the nature of the substituent at the 2-position of salvinorin A was critical for kappa-opioid receptor binding and activation. Because issues of receptor reserve complicate estimates of agonist efficacy and potency, we also examined the agonist actions of salvinorin A by measuring potassium conductance through G protein-gated K(+) channels coexpressed in Xenopus oocytes, a system in which receptor reserve is minimal. Salvinorin A was found to be a full agonist, being significantly more efficacious than (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50488) or (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593) (two standard kappa-opioid agonists) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for kappa-opioid receptors). Salvinorin A thus represents the first known naturally occurring non-nitrogenous full agonist at kappa-opioid receptors.
TL;DR: HSC activation as assessed by α-smooth muscle actin mRNA expression and immunohistochemistry was markedly reduced in both 3- and 10-day BDL animals, suggesting the pan-caspase inhibitor IDN-6556 is a promising agent for cholestatic liver injury.
Abstract: Liver injury is characterized by hepatocyte apoptosis and collagen-producing activated hepatic stellate cells (HSC). Hepatocyte apoptosis promotes liver injury and fibrosis, whereas activated HSC apoptosis limits hepatic fibrosis. Pharmacological inhibition of liver cell apoptosis may potentially attenuate liver injury and fibrosis by blocking hepatocyte apoptosis or promote fibrosis by permitting accumulation of activated HSCs. To ascertain the net effect of inhibiting liver cell apoptosis on liver injury, inflammation, and hepatic fibrogenesis, we examined the effect of a pancaspase inhibition IDN-6556 on these parameters in the bile duct ligated (BDL) mouse. Hepatocyte apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and immunofluorescence for active caspases 3/7, and liver injury by histopathology and serum alanine aminotransferase (ALT) determinations. Real-time polymerase chain reaction was used to measure mRNA transcripts for markers of hepatic inflammation, HSC activation, and fibrosis. Immunohistochemistry for alpha-smooth muscle actin was performed to identify HSC activation. Collagen deposition was quantitated by Sirius red staining and digital imaging techniques. Hepatocyte apoptosis and liver injury (bile infarcts and serum ALT values) were reduced in IDN-6556-treated versus saline-treated 3-day BDL mice. Markers for liver inflammation [chemokine (C-X-C) ligand 1 and macrophage inflammatory protein-2 chemokine expression] and hepatic fibrogenesis (transforming growth factor-beta and collagen I expression) were also attenuated. Consistent with these data, HSC activation as assessed by alpha-smooth muscle actin mRNA expression and immunohistochemistry was markedly reduced in both 3- and 10-day BDL animals. Collectively, these data suggest hepatocyte apoptosis initiates cascades culminating in liver injury and fibrosis. The pan-caspase inhibitor IDN-6556 is a promising agent for cholestatic liver injury.
TL;DR: EGCG at micromolar concentrations was highly effective in inhibiting the IL-1β-induced glycosaminoglycan release from human cartilage explants in vitro and EGCG showed a differential, dose-dependent inhibitory effect on the expression and activity of MMPs and the activities of transcription factors NF-κB and AP-1.
Abstract: Interleukin-1β (IL-1β)-induced inflammatory response in arthritic joints include the enhanced expression and activity of matrix metalloproteinases (MMPs), and their matrix degrading activity contribute to the irreversible loss of cartilage and may also be associated with sustained chronic inflammation. We have earlier shown that green tea ( Camellia sinensis ) polyphenol epigallocatechin-3-gallate (EGCG) was non-toxic to human chondrocytes [Singh R, Ahmed S, Islam N, Goldberg VM, and Haqqi TM (2002) Arthritis Rheum 46: 2079–2086] and inhibits the expression of inflammatory mediators in arthritic joints [Haqqi TM, Anthony DD, Gupta S, Ahmed N, Lee MS, Kumar GK, and Mukhtar H (1999) Proc Natl Acad Sci USA 96: 4524–4529]. Here we show that EGCG at micromolar concentrations was highly effective in inhibiting the IL-1β-induced glycosaminoglycan (GAG) release from human cartilage explants in vitro. EGCG also inhibited the IL-1β-induced mRNA and protein expression of MMP-1 and MMP-13 in human chondrocytes. Importantly, EGCG showed a differential, dose-dependent inhibitory effect on the expression and activity of MMP-13 and MMP-1. A similar differential dose-dependent inhibition of transcription factors NF-κB and AP-1 by EGCG was also noted. These results for the first time demonstrate a differential dose-dependent effect of EGCG on the expression and activity of MMPs and on the activities of transcription factors NF-κB and AP-1 and provide insights into the molecular basis of the reported anti-inflammatory effects of EGCG. These results also suggest that EGCG or compounds derived from it may be therapeutically effective inhibitors of IL-1β-induced production of matrix-degrading enzymes in arthritis.
TL;DR: It is suggested that there is an efficient MPAG transport mediated by Mrp2 on the bile canalicular membrane of rat hepatocytes and that the therapeutic range of CsA potentially interferes with Mrp1 and tacrolimus does not inhibit the transporter.
Abstract: The onset of diarrhea after the administration of mycophenolate mofetil (MMF) is possibly associated with the biliary excretion of its metabolite, mycophenolic acid glucuronide (MPAG). This study was undertaken to clarify the mechanism underlying the biliary excretion of MPAG. Intravenously administered mycophenolic acid (MPA, 5 mg/kg) rapidly disappeared from plasma and was efficiently excreted as MPAG in the bile of Wistar (26% of dose) and Sprague-Dawley rats (21% of dose) over 1 h. On the other hand, in spite of the rapid disappearance of MPA from plasma, the biliary excretion of MPAG was very limited in Eisai hyperbilirubinemic rats (EHBRs), which display mutations in multidrug resistance-associated protein 2 (Mrp2)/canalicular multispecific organic anion transporter, and constituted only 0.5% of dose. Instead, high levels of MPA were noted in the plasma of EHBRs. Intravenous administration of CsA (5 mg/kg) to Wistar rats significantly lowered the biliary excretion of MPAG. However, intravenously administered tacrolimus (0.1 mg/kg) failed to produce such effect. In conclusion, it is suggested that there is an efficient MPAG transport mediated by Mrp2 on the bile canalicular membrane of rat hepatocytes and that the therapeutic range of CsA potentially interferes with Mrp2. However, the therapeutic range of tacrolimus does not inhibit the transporter. Thus, it should be noted that MMF coadministered with tacrolimus instead of CsA might increase the occurrence of diarrhea related to the biliary excretion of MPAG in transplant recipients.
TL;DR: Although ranolazine alone prolonged APD, it reduced APD and ventricular arrhythmias caused by agents that increased late INa and decreased IK, and attenuated the synergistic effect of MAPD90 increase caused by combinations of ATX-II and blockers of IK.
Abstract: Prolongation of the QT interval of the ECG is associated with increased risk of torsades de pointes ventricular tachycardia. Ranolazine, a novel antianginal agent, is reported to decrease the delayed rectifier potassium current, I(Kr), and to increase action potential duration (APD) and the QT interval. However, ranolazine is also reported to reduce late sodium current (late I(Na)), a depolarizing current that contributes to prolongation of the plateau of the ventricular action potential. We hypothesized that ranolazine would decrease APD and the occurrence of arrhythmias when late I(Na) is increased. Therefore, we measured the effects of ranolazine alone and in the presence of anemone toxin (ATX)-II, whose action mimics the sodium channelopathy associated with long-QT3 syndrome, on epicardial monophasic action potentials and ECGs recorded from guinea pig isolated hearts. Ranolazine (0.1-50 microM) prolonged monophasic APD at 90% repolarization (MAPD(90)) by up to 22% but did not cause either early afterdepolarizations (EADs) or ventricular tachycardia (VT). ATX-II (1-20 nM) markedly increased APD and caused EADs and VT. Ranolazine (5-30 microM) significantly attenuated increases in MAPD(90) and reduced episodes of EADs and VT produced by ATX-II. Ranolazine also attenuated the synergistic effect of MAPD(90) increase caused by combinations of ATX-II and blockers of I(K) [E-4031; 1-[2-(6-methyl-2-pyridyl)ethyl]-4-methylsulfonylaminobenzoyl)piperidine]. Thus, although ranolazine alone prolonged APD, it reduced APD and ventricular arrhythmias caused by agents that increased late I(Na) and decreased I(K).
TL;DR: CES1A1 is the major enzyme responsible for the first-pass, stereoselective metabolism of methylphenidate, and both enantiomers ofethylphenidate can be fit into the three-dimensional model of CES 1A1 to form productive complexes in the active site.
Abstract: Methylphenidate is an important stimulant prescribed to treat attention-deficit hyperactivity disorder. It has two chiral centers, but most current commercial formulations consist of the racemic mixture of the threo pair of methylphenidate isomers ( d- , l-threo- methylphenidate). The d -isomer is the pharmacologically active component. Numerous studies reported that oral administration of the methylphenidate racemate undergoes first-pass, stereoselective clearance in humans with l- methylphenidate being eliminated faster than d- methylphenidate. Accordingly, the kinetics of hydrolysis of individual enantiomers by purified native and recombinant human liver carboxylesterases CES1A1 and CES2 and a colon isoenzyme CES3 were examined with a liquid chromatography/mass spectrometry assay. The expression of CES1A1, CES2, and CES3 in Sf9 cells and the methods for purification of the three isoenzymes are reported. CES1A1 has a high catalytic efficiency for both d - and l -enantiomers of methylphenidate. No catalytic activity was detected with CES2 and CES3 for either enantiomer. The catalytic efficiency of CES1A1 for l- methylphenidate ( k cat/ K m = 7.7 mM–1 min–1) is greater than that of d- methylphenidate ( k cat/ K m = 1.3–2.1 mM–1 min–1). Hence, the catalytic efficiency of CES1A1 for methylphenidate enantiomers agrees with stereoselective clearance of methylphenidate reported in human subjects. Both enantiomers of methylphenidate can be fit into the three-dimensional model of CES1A1 to form productive complexes in the active site. We conclude that CES1A1 is the major enzyme responsible for the first-pass, stereoselective metabolism of methylphenidate.
TL;DR: It is concluded that carbachol-induced contraction of human urinary bladder via M3 receptors largely depends on Ca2+ entry through nifedipine-sensitive channels and activation of a rho kinase, whereas phospholipase D and store-operated Ca2-operated channels contribute only in a minor way.
Abstract: The present study was designed to reexamine the muscarinic acetylcholine receptor subtype mediating carbachol-induced contraction of human urinary bladder and to investigate the underlying signal transduction. Based upon the nonselective tolterodine, the highly M(2)-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M(3)-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M(3) receptors. The phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122) (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. The phospholipase D inhibitor butan-1-ol relative to its negative control butan-2-ol (0.3% each) caused small but detectable inhibition of carbachol-induced bladder contraction. The Ca(2+) entry blocker nifedipine (10-100 nM) strongly inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SK&F 96,365) (1-10 microM), an inhibitor of store-operated Ca(2+) channels, caused little inhibition. The protein kinase C inhibitor bisindolylmaleimide I (1-10 microM) did not significantly affected carbachol-induced bladder contraction. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 microM), an inhibitor of rho-associated kinases, concentration dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of human urinary bladder via M(3) receptors largely depends on Ca(2+) entry through nifedipine-sensitive channels and activation of a rho kinase, whereas phospholipase D and store-operated Ca(2+) channels contribute only in a minor way. Surprisingly, phospholipase C or protein kinase C do not seem to be involved to a relevant extent.
TL;DR: It is indicated that memantine improves hippocampus-based spatial learning in a transgenic mouse model of AD without producing nonspecific effects on locomotion/exploratory activity.
Abstract: Memantine, a low- to moderate-affinity uncompetitive N- methyl-d-aspartate receptor antagonist, has been shown to improve learning and memory in several pharmacological models of Alzheimer9s disease (AD). In the present study, the effect of memantine on locomotor activity, social behavior, and spatial learning was assessed in a transgenic mouse model of AD. Eight-month-old male C57BL/6J mice carrying mutated human APP and PS1 genes (APP/PS1) and their nontransgenic (NT) litter mates were administered a therapeutic dose of memantine (30 mg/kg/day p.o.) for 2 to 3 weeks. At this age, APP/PS1 mice show elevated levels of β-amyloid peptides in several brain regions. APP/PS1 mice exhibited less exploratory rearing and increased aggressive behavior compared with NT mice. In the water maze test for spatial learning, APP/PS1 mice had longer escape latencies to both hidden and visible platforms, but they did not differ from NT mice in their swimming speed. Memantine significantly improved the acquisition of the water maze in APP/PS1 mice without affecting swimming speed. Memantine did not affect either locomotor activity or aggressive behavior in either genotype. These data indicate that memantine improves hippocampus-based spatial learning in a transgenic mouse model of AD without producing nonspecific effects on locomotion/exploratory activity.
TL;DR: It is concluded that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L- type Ca2+ channels, thus rendering it more selective to T-type Ca 2+ channels.
Abstract: Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.