Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 2003"
TL;DR: Recent advances on the study of the role of microglia based on findings from animal and cell culture models in the pathogenesis of neurodegenerative diseases, with particular emphasis on Parkinson's disease are summarized.
Abstract: Evidence from postmortem analysis implicates the involvement of microglia in the neurodegenerative process of several degenerative neurological diseases, including Alzheimer's disease and Parkinson's disease. It remains to be determined, however, whether microglial activation plays a role in the initiation stage of disease progression or occurs merely as a response to neuronal death. Activated microglia secrete a variety of proinflammatory and neurotoxic factors that are believed to induce and/or exacerbate neurodegeneration. In this article, we summarize recent advances on the study of the role of microglia based on findings from animal and cell culture models in the pathogenesis of neurodegenerative diseases, with particular emphasis on Parkinson's disease. In addition, we also discuss novel approaches to potential therapeutic strategies.
1,069 citations
TL;DR: Cholinergic abnormalities may also contribute to noncognitive behavioral abnormalities as well as the deposition of toxic neuritic plaques in AD and cholinergic-based strategies will likely remain valid as one approach to rational drug development for the treatment of AD other forms of dementia.
Abstract: The cholinergic hypothesis was initially presented over 20 years ago and suggests that a dysfunction of acetylcholine containing neurons in the brain contributes substantially to the cognitive decline observed in those with advanced age and Alzheimer's disease (AD). This premise has since served as the basis for the majority of treatment strategies and drug development approaches for AD to date. Recent studies of the brains of patients who had mild cognitive impairment or early stage AD in which choline acetyltransferase and/or acetylcholinesterase activity was unaffected (or even up-regulated) have, however, led some to challenge the validity of the hypothesis as well as the rationale for using cholinomimetics to treat the disorder, particularly in the earlier stages. These challenges, primarily based on assays of post mortem enzyme activity, should be taken in perspective and evaluated within the wide range of cholinergic abnormalities known to exist in both aging and AD. The results of both post mortem and antemortem studies in aged humans and AD patients, as well as animal experiments suggest that a host of cholinergic abnormalities including alterations in choline transport, acetylcholine release, nicotinic and muscarinic receptor expression, neurotrophin support, and perhaps axonal transport may all contribute to cognitive abnormalities in aging and AD. Cholinergic abnormalities may also contribute to noncognitive behavioral abnormalities as well as the deposition of toxic neuritic plaques in AD. Therefore, cholinergic-based strategies will likely remain valid as one approach to rational drug development for the treatment of AD other forms of dementia.
1,008 citations
TL;DR: It is demonstrated that inhibition of microglial activation attenuated the development of behavioral hypersensitivity in a rat model of neuropathic pain but had no effect on the treatment of existing mechanical allodynia and hyperalgesia.
Abstract: Microglia, the intrinsic macrophages of the central nervous system, have previously been shown to be activated in the spinal cord in several rat mononeuropathy models. Activation of microglia and subsequent release of proinflammatory cytokines are known to play a role in inducing a behavioral hypersensitive state (hyperalgesia and allodynia) in these animals. The present study was undertaken to determine whether minocycline, an inhibitor of microglial activation, could attenuate both the development and existing mechanical allodynia and hyperalgesia in an L5 spinal nerve transection model of neuropathic pain. In a preventive paradigm (to study the effect on the development of hypersensitive behaviors), minocycline (10, 20, or 40 mg/kg intraperitoneally) was administered daily, beginning 1 h before nerve transection. This regimen produced a decrease in mechanical hyperalgesia and allodynia, with a maximum inhibitory effect observed at the dose of 20 and 40 mg/kg. The attenuation of the development of hyperalgesia and allodynia by minocycline was associated with an inhibitory action on microglial activation and suppression of proinflammatory cytokines at the L5 lumbar spinal cord of the nerveinjured animals. The effect of minocycline on existing allodynia was examined after its intraperitoneal administration initiated on day 5 post-L5 nerve transection. Although the postinjury administration of minocycline significantly inhibited microglial activation in neuropathic rats, it failed to attenuate existing hyperalgesia and allodynia. These data demonstrate that inhibition of microglial activation attenuated the development of behavioral hypersensitivity in a rat model of neuropathic pain but had no effect on the treatment of existing mechanical allodynia and hyperalgesia.
766 citations
TL;DR: Efavirenz systemic exposure is likely to be subject to interindividual variability in CYP2B6 activity and to drug interactions involving this isoform, and may be a valuable phenotyping tool to study the role of CYP1B6 in human drug metabolism.
Abstract: We used human liver microsomes (HLMs) and recombinant cytochromes P450 (P450s) to identify the routes of efavirenz metabolism and the P450s involved. In HLMs, efavirenz undergoes primary oxidative hydroxylation to 8-hydroxyefavirenz (major) and 7-hydroxyefavirenz (minor) and secondary metabolism to 8,14-dihydroxyefavirenz. The formation of 8-hydroxyefavirenz in two HLMs showed sigmoidal kinetics (average apparent Km, 20.2 micro M; Vmax, 140 pmol/min/mg protein; and Hill coefficient, 1.5), whereas that of 7-hydroxyefavirenz formation was characterized by hyperbolic kinetics (Km, 40.1 micro M and Vmax, 20.5 pmol/min/mg protein). In a panel of 10 P450s, CYP2B6 formed 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz from efavirenz (10 micro M) at the highest rate. The Km value for the formation of 8-hydroxyefavirenz in CYP2B6 derived from hyperbolic Eq. 12.4 micro M) was close to that obtained in HLMs (Km, 20.2 micro M). None of the P450s tested showed activity toward 7-hydroxylation of efavirenz. When 8-hydroxyefavirenz (2.5 micro M) was used as a substrate, 8,14-dihydroxyefavirenz was formed by CYP2B6 at the highest rate, and its kinetics showed substrate inhibition (Ksi, approximately 94 micro M in HLMs and approximately 234 micro M in CYP2B6). In a panel of 11 HLMs, 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz formation rates from efavirenz (10 micro M) correlated significantly with the activity of CYP2B6 and CYP3A. N,N',N"-Triethylenethiophosphoramide (thioTEPA; 50 micro M) inhibited the formation rates of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz from efavirenz (10 micro M) by > or = 60% in HLMs) and CYP2B6, with Ki values < 4 micro M. In conclusion, CYP2B6 is the principal catalyst of efavirenz sequential hydroxylation. Efavirenz systemic exposure is likely to be subject to interindividual variability in CYP2B6 activity and to drug interactions involving this isoform. Efavirenz may be a valuable phenotyping tool to study the role of CYP2B6 in human drug metabolism.
590 citations
TL;DR: In contrast to melatonin, agomelatine behaves as an antagonist at 5- HT2B and 5-HT2C receptors: blockade of the latter reinforces frontocortical adrenergic and dopaminergic transmission.
Abstract: Agomelatine (S20098) displayed p K i values of 6.4 and 6.2 at native (porcine) and cloned, human (h)5-hydroxytryptamine (5-HT)2C receptors, respectively. It also interacted with h5-HT2B receptors (6.6), whereas it showed low affinity at native (rat)/cloned, human 5-HT2A (<5.0/5.3) and 5-HT1A (<5.0/5.2) receptors, and negligible (<5.0) affinity for other 5-HT receptors. In antibody capture/scintillation proximity assays, agomelatine concentration dependently and competitively abolished h5-HT2C receptor-mediated activation of Gq/11 and Gi3 (p A 2 values of 6.0 and 6.1). As measured by [3H]phosphatidylinositol depletion, agomelatine abolished activation of phospholipase C by h5-HT2C (p K B value of 6.1) and h5-HT2B (p K B value of 6.6) receptors. In vivo, it dose dependently blocked induction of penile erections by the 5-HT2C agonists ( S )-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (Ro60,0175) and 1-methyl-2-(5,8,8-trimethyl-8 H -3-aza-cyclopenta[ a ]inden-3-yl) ethylamine (Ro60,0332). Furthermore, agomelatine dose dependently enhanced dialysis levels of dopamine in frontal cortex of freely moving rats, whereas they were unaffected in nucleus accumbens and striatum. Although the electrical activity of ventrotegmental dopaminergic neurons was unaffected agomelatine, it abolished their inhibition by Ro60,0175. Extracellular levels of noradrenaline in frontal cortex were also dose dependently enhanced by agomelatine in parallel with an acceleration in the firing rate of adrenergic cell bodies in the locus coeruleus. These increases in noradrenaline and dopamine levels were unaffected by the selective melatonin antagonist N -[2-(5-ethyl-benzo[ b ]thien-3-yl)ethyl] acetamide (S22153) and likely flect blockade of 5-HT2C receptors inhibitory to frontocortical dopaminergic and adrenergic pathways. Correspondingly, distinction to agomelatine, melatonin showed negligible activity 5-HT2C receptors and failed to modify the activity of adrenergic and dopaminergic pathways. In conclusion, in contrast to melatonin, agomelatine behaves as an antagonist at 5-HT2B and 5-HT2C receptors: blockade of the latter reinforces frontocortical adrenergic and dopaminergic transmission.
494 citations
TL;DR: In this paper, it was shown that histamine induces chemotaxis of mouse mast cells, without affecting mast cell degranulation, and that activation of H4 receptors by histamine resulted in calcium mobilization from intracellular calcium stores.
Abstract: The diverse physiological functions of histamine are mediated through distinct histamine receptors. Mast cells are major producers of histamine, yet effects of histamine on mast cells are currently unclear. The present study shows that histamine induces chemotaxis of mouse mast cells, without affecting mast cell degranulation. Mast cell chemotaxis toward histamine could be blocked by the dual H3/H4 receptor antagonist thioperamide, but not by H1 or H2 receptor antagonists. This chemotactic response is mediated by the H4 receptor, because chemotaxis toward histamine was absent in mast cells derived from H4 receptor-deficient mice but was detected in H3 receptor-deficient mast cells. In addition, Northern blot analysis showed the expression of H4 but not H3 receptors on mast cells. Activation of H4 receptors by histamine resulted in calcium mobilization from intracellular calcium stores. Both G alpha i/o proteins and phospholipase C (PLC) are involved in histamine-induced calcium mobilization and chemotaxis in mast cells, because these responses were completely inhibited by pertussis toxin and PLC inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5 (10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122). In summary, histamine was shown to mediate signaling and chemotaxis of mast cells via the H4 receptor. This mechanism might be responsible for mast cell accumulation in allergic tissues.
488 citations
TL;DR: The study provides a rationale as well as clinically comparable dosing regimens for animal studies and raises questions about the inferences drawn from previous studies that have used doses unrepresentative of the clinical situation.
Abstract: What is the appropriate dose of an antipsychotic in an animal model? The literature reveals no standard rationale across studies. This study was designed to use in vivo dopamine D2 receptor occupancy as a cross-species principle for deriving clinically comparable doses for animal models. The relationship between dose, plasma levels, and in vivo dopamine D2 receptor occupancy was established in rats for a range of doses administered as a single dose or multiple doses (daily injections or osmotic minipump infusions) for five of the most commonly used antipsychotics. As a single dose, haloperidol (0.04–0.08 mg/kg), clozapine (5–15 mg/kg), olanzapine (1–2 mg/kg), risperidone (0.5–1 mg/kg), and quetiapine (10–25 mg/kg) reached clinically comparable occupancies. However, when these “optimal” single doses were administered as multiple doses, either by injection or by a mini-pump, it led to no or inappropriately low trough (24-h) occupancies. This discrepancy arises because the half-life of antipsychotics in rodents is 4 to 6 times faster than in humans. Only when doses 5 times higher than the optimal single dose were administered by pump were clinically comparable occupancies obtained (e.g., haloperidol, 0.25 mg/kg/day; olanzapine, 7.5 mg/kg/day). This could not be achieved for clozapine or quetiapine due to solubility and administration constraints. The study provides a rationale as well as clinically comparable dosing regimens for animal studies and raises questions about the inferences drawn from previous studies that have used doses unrepresentative of the clinical situation.
474 citations
TL;DR: Findings are consistent with the hypothesis that CREB-mediated induction of dynorphin in the NAc “triggers” immobility behavior in the FST and raise the possibility that κ-antagonists may have efficacy as antidepressants, but lack stimulant or reward-related effects.
Abstract: We showed previously that cAMP response element-binding protein (CREB) within the nucleus accumbens (NAc) of rats regulates immobility in the forced swim test (FST), an assay used to study depression. Because CREB regulates expression of dynorphin (which acts at kappa-opioid receptors) in NAc neurons, these findings raised the possibility that kappa-receptors mediate immobility behaviors in the FST. Here, we report that i.c.v. administration of the kappa-antagonist nor-binaltorphimine dose dependently decreased immobility in the FST, suggesting that it has antidepressant-like effects. Implicating a specific effect at kappa-receptors, similar antidepressant-like effects were seen after treatment with either of two novel, structurally dissimilar kappa-antagonists: 5'-guanidinonaltrindole, which was effective after i.c.v. but not systemic treatment, and 5'-acetamidinoethylnaltrindole (ANTI), which was potent and effective after systemic treatment. The behavioral effects of the kappa-antagonists resembled those of tricyclic antidepressants (desipramine) and selective serotonin reuptake inhibitors (fluoxetine and citalopram). Conversely, systemic administration of the kappa-agonist [5alpha,7alpha,8beta]-N-methyl-N-[7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec8-yl]-benzenacetamide (U-69593) dose dependently increased immobility in the FST, consistent with prodepressant-like effects. The effects of the kappa-ligands in the FST were not correlated with nonspecific effects on locomotor activity. Furthermore, the most potent and effective kappa-antagonist (ANTI) did not affect the rewarding impact of lateral hypothalamic brain stimulation at a dose with strong antidepressant-like effects. These findings are consistent with the hypothesis that CREB-mediated induction of dynorphin in the NAc "triggers" immobility behavior in the FST. Furthermore, they raise the possibility that kappa-antagonists may have efficacy as antidepressants, but lack stimulant or reward-related effects.
466 citations
TL;DR: Accumbens DA may be important for enabling rats to overcome behavioral constraints, such as work-related response costs, and may be critical for the behavioral organization and conditioning processes that enable animals to engage in vigorous responses, or to emit large numbers of responses in ratio schedules in the absence of primary reinforcement.
Abstract: For several decades, it has been suggested that dopamine (DA), especially in nucleus accumbens, mediates the primary reinforcing characteristics of natural stimuli such as food, as well as drugs of abuse. Yet, several fundamental aspects of primary food reinforcement, motivation, and appetite are left intact after interference with accumbens DA transmission. Recent studies have shown that accumbens DA is involved in responsiveness to conditioned stimuli and activational aspects of motivation. In concurrent choice tasks, accumbens DA depletions cause animals to reallocate their choice behavior in the direction of instrumental behaviors that involve less effort. Also, an emerging body of evidence has demonstrated that the effects of accumbens DA depletions on instrumental food-seeking behavior can vary greatly depending upon the task. For example, some schedules of reinforcement are insensitive to the effects of DA depletions, whereas others are highly sensitive (e.g., large fixed ratios). Accumbens DA depletions slow the rate of operant responding, blunt the rate-facilitating effects of moderate-sized ratios, and enhance the rate-suppressing effects of very large ratios (i.e., produce ratio strain). Accumbens DA may be important for enabling rats to overcome behavioral constraints, such as work-related response costs, and may be critical for the behavioral organization and conditioning processes that enable animals to engage in vigorous responses, such as barrier climbing, or to emit large numbers of responses in ratio schedules in the absence of primary reinforcement. The involvement of accumbens DA in activational aspects of motivation has implications for energy-related disorders in psychiatry, as well as aspects of drug-seeking behavior.
423 citations
TL;DR: OATP-B plays a role in the absorption of anionic compounds across the apical membrane of human intestinal epithelial cells, although it cannot be decisively concluded that pH-dependent absorption of pravastatin is determined by OATPB alone.
Abstract: Some organic anions are absorbed from the gastrointestinal tract through carrier-mediated transport mechanism(s), which may include proton-coupled transport, anion exchange transport, and others. However, the molecular identity of the organic anion transporters localized at the apical membrane of human intestinal epithelial cells has not been clearly demonstrated. In the present study, we focused on human organic anion transporting polypeptide OATP-B and examined its subcellular localization and functionality in the small intestine. Localization of OATP-B was determined by immunohistochemical analysis. Transport properties of estrone-3-sulfate and the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin by OATP-B-transfected human embryonic kidney 293 cells were measured. OATP-B was immunohistochemically localized at the apical membrane of intestinal epithelial cells in humans. Uptake of [3H]estrone-3-sulfate and [14C]pravastatin by OATP-B at pH 5.5 was higher than that at pH 7.4. [3H]Estrone-3-sulfate transport was decreased by pravastatin, aromatic anion compounds, and the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by small anionic compounds, such as lactic acid and acetic acid. The inhibitory effect of pravastatin on the uptake of [3H]estrone-3-sulfate was concentration-dependent, and the IC50 value was 5.5 mM. The results suggested that OATP-B mediates absorption of anionic compounds and its activity may be optimum at the acidic surface microclimate pH of the small intestine. Accordingly, OATP-B plays a role in the absorption of anionic compounds across the apical membrane of human intestinal epithelial cells, although it cannot be decisively concluded that pH-dependent absorption of pravastatin is determined by OATP-B alone.
408 citations
TL;DR: The 1459C>T SNP, which resulted in the Arg487Cys substitution, was associated with the lowest level of CYP2B6 activity in livers of females, and several common SNPs that are associated with polymorphic CYP 2B6 expression were found.
Abstract: CYP2B6 metabolizes many drugs, and its expression varies greatly. CYP2B6 genotype-phenotype associations were determined using human livers that were biochemically phenotyped for CYP2B6 (mRNA, protein, and CYP2B6 activity), and genotyped for CYP2B6 coding and 5'-flanking regions. CYP2B6 expression differed significantly between sexes. Females had higher amounts of CYP2B6 mRNA (3.9-fold, P G substitution that resulted in an Lys139Glu change. Many CYP2B6 splice variants (SV) were observed, and the most common variant lacked exons 4 to 6. A nonsynonymous SNP in exon 4 (15631G>T), which disrupted an exonic splicing enhancer, and a SNP 15582C>T in an intron-3 branch site were correlated with this SV. The extent to which CYP2B6 variation was a predictor of CYP2B6 activity varied according to sex and ethnicity. The 1459C>T SNP, which resulted in the Arg487Cys substitution, was associated with the lowest level of CYP2B6 activity in livers of females. The intron-3 15582C>T SNP (in significant linkage disequilibrium with a SNP in a putative hepatic nuclear factor 4 (HNF4) binding site) was correlated with lower CYP2B6 expression in females. In conclusion, we found several common SNPs that are associated with polymorphic CYP2B6 expression.
TL;DR: Human plasma contains several catechols, including the catecholamines norepinephrine, epinephrine, and dopamine, their precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), and their deaminated metabolites, dihydroxyphensylglycol, the main neuronal metabolite of nore Pinephrine,
Abstract: Human plasma contains several catechols, including the catecholamines norepinephrine, epinephrine, and dopamine, their precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), and their deaminated metabolites, dihydroxyphenylglycol, the main neuronal metabolite of norepinephrine, and dihydroxyphenylacetic acid, a deaminated metabolite of dopamine. Products of metabolism of catechols include 3-methoxytyrosine (from L-DOPA), homovanillic acid and dopamine sulfate (from dopamine), normetanephrine, vanillylmandelic acid, and methoxyhydroxyphenylglycol (from norepinephrine), and metanephrine (from epinephrine). Plasma levels of catechols and their metabolites have related but distinct sources and therefore reflect different functions of catecholamine systems. This article provides an update about plasma levels of catechols and their metabolites and the relevance of those levels to some issues in human health and disease.
TL;DR: The data show that VR1 antagonists have antihyperalgesic activity in animal models of chronic inflammatory and neuropathic pain, and illustrate species differences in the in vivo pharmacology of VR1 that correlate with differences in pharmacology previously seen in vitro.
Abstract: Vanilloid receptor type 1 (VR1) (TRPV1) is a ligand-gated ion channel expressed on sensory nerves that responds to noxious heat, protons, and chemical stimuli such as capsaicin. Herein, we have examined the activity of the VR1 antagonist capsazepine in models of inflammatory and neuropathic pain in the rat, mouse, and guinea pig. In naive animals, subcutaneous administration of capsazepine (10-100 mg/kg s.c.) did not affect withdrawal thresholds to noxious thermal or mechanical stimuli. However, pretreatment with capsazepine prevented the development of mechanical hyperalgesia induced by intraplantar injection of capsaicin, with a similar potency in all three species. Capsazepine (up to 100 mg/kg s.c.) did not affect mechanical hyperalgesia in the Freund's complete adjuvant (FCA)-inflamed hind paw of the rat or mouse. Strikingly, capsazepine (3-30 mg/kg s.c.) produced up to 44% reversal of FCA-induced mechanical hyperalgesia in the guinea pig. Capsazepine also produced significant reversal of carageenan-induced thermal hyperalgesia in the guinea pig at 30 mg/kg s.c., but was ineffective in the rat. Similarly, in the partial sciatic nerve ligation model of neuropathic pain, capsazepine was surprisingly effective in the guinea pig, producing up to 80% reversal of mechanical hyperalgesia (1-30 mg/kg s.c.) but had no effect in the rat or mouse. These data show that VR1 antagonists have antihyperalgesic activity in animal models of chronic inflammatory and neuropathic pain, and illustrate species differences in the in vivo pharmacology of VR1 that correlate with differences in pharmacology previously seen in vitro.
TL;DR: A role for the endogenous cannabinoid system in modulating neuroexcitation is defined and plasticity of the CB1 receptor occurs with epilepsy, and data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration.
Abstract: Several lines of evidence suggest that cannabinoid compounds are anticonvulsant. However, the anticonvulsant potential of cannabinoids and, moreover, the role of the endogenous cannabinoid system in regulating seizure activity has not been tested in an in vivo model of epilepsy that is characterized by spontaneous, recurrent seizures. Here, using the rat pilocarpine model of epilepsy, we show that the marijuana extract Delta9-tetrahydrocannabinol (10 mg/kg) as well as the cannabimimetic, 4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one [R(+)WIN55,212 (5 mg/kg)], completely abolished spontaneous epileptic seizures. Conversely, application of the cannabinoid CB1 receptor (CB1) antagonist, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A), significantly increased both seizure duration and frequency. In some animals, CB1 receptor antagonism resulted in seizure durations that were protracted to a level consistent with the clinical condition status epilepticus. Furthermore, we determined that during an short-term pilocarpine-induced seizure, levels of the endogenous CB1 ligand 2-arachidonylglycerol increased significantly within the hippocampal brain region. These data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of the CB1 receptor. Furthermore, Western blot and immunohistochemical analyses revealed that CB1 receptor protein expression was significantly increased throughout the CA regions of epileptic hippocampi. By demonstrating a role for the endogenous cannabinoid system in regulating seizure activity, these studies define a role for the endogenous cannabinoid system in modulating neuroexcitation and suggest that plasticity of the CB1 receptor occurs with epilepsy.
TL;DR: Using purified recombinant HDAC proteins, several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8 are identified and displayed distinct properties in inducing histone acetylation and reporter gene expression.
Abstract: Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC50 value of approximately 0.1-0.3 microM), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC50 value of approximately 0.3 microM) versus HDAC3 (IC50 value of approximately 8 microM) and had no inhibitory activity toward HDAC8 (IC50 value >100 microM). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.
TL;DR: The objective of this study was to characterize the developmental expression of hepatic CYP3A forms from early gestation to 18 years of age using up to 212 fetal and pediatric liver samples and based on immunoquantitation, CYP 3A5 protein expression was found to be highly variable, generally independent of age, and more frequently observed for African-American individuals.
Abstract: The human cytochrome P4503A forms show expression patterns subject to developmental influence. CYP3A7 and CYP3A4 are generally classified as the major fetal and adult liver forms, respectively. However, characterization of CYP3A4, -3A5, and -3A7 developmental expression has historically been confounded by the lack of CYP3A isoform-specific antibodies or marker enzyme activities. Therefore, the objective of this study was to characterize the developmental expression of hepatic CYP3A forms from early gestation to 18 years of age using up to 212 fetal and pediatric liver samples. Based on immunoquantitation, CYP3A5 protein expression was found to be highly variable, generally independent of age, and more frequently observed for African-American individuals. For differentiation of CYP3A4 and -3A7 levels, dehydroepiandrosterone metabolite patterns for expressed CYP3A forms were characterized and used for simultaneous quantitation of protein levels within liver microsome samples. The major metabolite formed by CYP3A4, 7beta-hydroxy-dehydroepiandrosterone, was identified based on cochromatography and mass spectra matching with the authentic standard. Kinetic analysis showed a 34-fold greater intrinsic clearance of 7beta-hydroxy-dehydroepiandrosterone by CYP3A4 versus -3A7, whereas CYP3A7 showed the highest 16alpha-hydroxy-dehydroepiandrosterone intrinsic clearance. Metabolite profiles for the expressed enzymes were fit to a multiple response model and CYP3A4 and -3A7 levels in fetal and pediatric liver microsome samples were calculated. Fetal liver microsomes showed extremely high CYP3A7 levels (311-158 pmol/mg protein) and significant expression through 6 months postnatal age. Low CYP3A4 expression was noted for fetal liver (< or =10 pmol/mg), with mean levels increasing with postnatal age.
TL;DR: These studies support the previous proposal that the therapeutic action of galantamine is mainly produced by its sensitizing action on nAChRs rather than by general cholinergic enhancement due to cholinesterase inhibition.
Abstract: Galantamine (Reminyl), an approved treatment for Alzheimer9s disease (AD), is a potent allosteric potentiating ligand (APL) of human α3β4, α4β2, and α6β4 nicotinic receptors (nAChRs), and of the chicken/mouse chimeric α7/5-hydroxytryptamine3 receptor, as was shown by whole-cell patch-clamp studies of human embryonic kidney-293 cells stably expressing a single nAChR subtype. Galantamine potentiates agonist responses of the four nAChR subtypes studied in the same window of concentrations (i.e., 0.1–1 μM), which correlates with the cerebrospinal fluid concentration of the drug at the recommended daily dosage of 16 to 24 mg. At concentrations >10 μM, galantamine acts as an nAChR inhibitor. The other presently approved AD drugs, donepezil and rivastigmine, are devoid of the nicotinic APL action; at micromolar concentrations they also block nAChR activity. Using five CHO-SRE-Luci cell lines, each of them expressing a different human muscarinic receptor, and a reporter gene assay, we show that galantamine does not alter the activity of M1–M5 receptors, thereby confirming that galantamine modulates selectively the activity of nAChRs. These studies support our previous proposal that the therapeutic action of galantamine is mainly produced by its sensitizing action on nAChRs rather than by general cholinergic enhancement due to cholinesterase inhibition. Galantamine9s APL action directly addresses the nicotinic deficit in AD.
TL;DR: The DDI between CER and CsA is mainly due to the inhibition of transporter (at least partly OATP2)-mediated uptake in the liver, suggesting that the mechanism involved in the clinically relevant drug-drug interaction (DDI) between cerivastatin (CER) and cyclosporin A (CsA) involves the inhibition.
Abstract: The mechanism involved in the clinically relevant drug-drug interaction (DDI) between cerivastatin (CER) and cyclosporin A (CsA) has not yet been clarified. In the present study, we examined the possible roles of transporter-mediated hepatic uptake in this DDI. The uptake of [(14)C]CER into human hepatocytes prepared from three different donors was examined. Kinetic analyses revealed K(m) values for the uptake of [(14)C]CER within the range of 3 to 18 microM, suggesting that more than 70% of the total uptake at therapeutic CER concentrations was accounted for by a saturable process, i.e., transporter-mediated uptake. This uptake was inhibited by CsA with K(i) values of 0.3 to 0.7 microM. The uptake of [(14)C]CER was also examined in human organic anion transporting polypeptide-2 (OATP2)-expressing Madin-Darby canine kidney cells (MDCKII). Saturable OATP2-mediated uptake of [(14)C]CER was observed and was also inhibited by CsA, with a K(i) value of 0.2 microM. These results suggest that the DDI between CER and CsA involves the inhibition of transporter-mediated uptake of CER and, at least in part, its OATP2-mediated uptake. The effect of CsA on the in vitro metabolism of [(14)C]CER was also examined. The metabolism of [(14)C]CER was inhibited by CsA with an IC(50) value of more than 30 microM. From these results, we conclude that the DDI between CER and CsA is mainly due to the inhibition of transporter (at least partly OATP2)-mediated uptake in the liver.
TL;DR: This is the first demonstration of an uptake transporter such as OATP-C, in modulating PXR function, and sheds important new insight into the understanding of the molecular determinants of P XR-mediated inductive processes.
Abstract: Rifampin, a member of the rifamycin class of antibiotics, is well known for its ability to induce drug-metabolizing enzymes and transporters, through activation of the pregnane X receptor. Available data suggest rifampin entry into hepatocytes may be transporter-mediated. Accordingly, it is therefore plausible that modulation of the achievable intracellular concentration of rifampin by drug uptake transporters would influence the degree of induction. In this study, we expressed an array of known hepatic uptake transporters to show the key hepatic rifampin uptake transporters are liver-specific members of the organic anion transporting polypeptide family (OATP). Indeed, both OATP-C and OATP8 seemed capable of mediating rifampin uptake into HeLa cells. OATP-C, however, seemed to have far greater affinity and capacity for rifampin transport. In addition, several allelic variants of OATP-C known to be present among European and African Americans were found to have markedly decreased rifampin transport activity. In cell-based, transactivation assays, OATP-C expression was associated with increased cellular rifampin retention as well as potentiation of PXR reporter gene activity. This is the first demonstration of an uptake transporter such as OATP-C, in modulating PXR function, and sheds important new insight into our understanding of the molecular determinants of PXR-mediated inductive processes.
TL;DR: A potential for significant flavonoid-drug interactions with P-gp substrates is indicated, at least in part, a direct interaction.
Abstract: Flavonoids are constituents of fruits, vegetables, and plant-derived beverages, as well as components in herbal-containing dietary supplements. The objective of this investigation was to characterize the effect of flavonoids on P-glycoprotein (P-gp)-mediated cellular efflux and to determine the molecular mechanism(s) of the flavonoid-drug interaction. Studies were conducted in the sensitive and multidrug resistant human breast cancer cell lines MCF-7 and MDA435/LCC6 and examined the effects of the flavonoids biochanin A, morin, phloretin, and silymarin on daunomycin (DNM) accumulation and doxorubicin cytotoxicity. The potential mechanism(s) involved in the interaction was evaluated by determining flavonoid effects on 1) P-gp ATPase activity, 2) [ 3 H]azidopine photoaffinity labeling of P-gp, and 3) cellular P-gp levels. The flavonoids increased [ 3 H]DNM accumulation in P-gp positive cells, but not P-gp negative cells, and these effects were both flavonoid concentration- and P-gp expression level-dependent. Biochanin A and silymarin potentiated doxorubicin cytotoxicity in P-gp positive cells. Biochanin A and phloretin stimulated, whereas morin and silymarin inhibited P-gp ATPase activity, confirming that these flavonoids interact with P-gp. Morin and silymarin significantly inhibited [ 3 H]azidopine photoaffinity labeling of P-gp, suggesting a direct interaction with P-gp substrate binding. A 24-h preincubation with all flavonoids, followed by flavonoid removal, did not alter cellular P-gp level in P-gp positive cells. In conclusion, biochanin A, morin, phloretin, and silymarin all inhibited P-gp-mediated cellular efflux and the mechanism of the interaction involved, at least in part, a direct interaction. The findings of this study indicate a potential for significant flavonoid-drug interactions with P-gp substrates.
TL;DR: Results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.
Abstract: Alpha-D-glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-alpha and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 microM. E5564 blocked LPS-mediated activation of nuclear factor-kappaB in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to (6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-beta-O-phosphono-alpha-D-glucopyranose tetrasodium salt (E5531), another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose- and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.
TL;DR: The results suggested that the protective anti-inflammatory effect of treatment with FTY720 was, to a large extent, due to the inhibition of encephalitogenic T-cell responses and/or their migration into the central nervous system and may be a potential candidate for use in treating patients with MS.
Abstract: Experimental autoimmune encephalomyelitis (EAE) is a T-cell-dependent autoimmune disease that reproduces the inflammatory demyelinating pathology of multiple sclerosis (MS). We investigated the efficacy and mechanism of immunosuppression against EAE by administering 2-amino-[2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride (FTY720) in Lewis rats immunized with myelin basic protein together with complete Freund's adjuvant. FTY720 treatment almost completely protected the rats against disease. The protection by FTY720 was associated with a dramatic reduction in the number of lymphocytes staining for T-cell receptors in the spinal cord as examined by immunohistochemistry. The mRNA expression of Th1 cytokines interleukin (IL)-2, IL-6, and interferon-gamma in the spinal cord was also reduced dramatically as assessed by reverse-transcription polymerase chain reaction. Furthermore, lymphocytes isolated from the spleen of FTY720-treated rats were transferred into naive recipient rats against EAE manifestation by reducing both disease incidence and clinical score. These results suggested that the protective anti-inflammatory effect of treatment with FTY720 was, to a large extent, due to the inhibition of encephalitogenic T-cell responses and/or their migration into the central nervous system and may be a potential candidate for use in treating patients with MS.
TL;DR: Drug administration increased urinary normetanephrine excretion, but, contrary to the typical MAO-inhibitor effect profile, deaminated monoamine metabolite levels were not decreased, suggesting a predominantly peripheral (gastrointestinal and liver) site of action for harmine.
Abstract: The effects of the South American psychotropic beverage ayahuasca on subjective and cardiovascular variables and urine monoamine metabolite excretion were evaluated, together with the drug's pharmacokinetic profile, in a double-blind placebo-controlled clinical trial. This pharmacologically complex tea, commonly obtained from Banisteriopsis caapi and Psychotria viridis, combines N,N-dimethyltryptamine (DMT), an orally labile psychedelic agent showing 5-hydroxytryptamine2A agonist activity, with monoamine oxidase (MAO)-inhibiting beta-carboline alkaloids (harmine, harmaline, and tetrahydroharmine). Eighteen volunteers with prior experience in the use of psychedelics received single oral doses of encapsulated freeze-dried ayahuasca (0.6 and 0.85 mg of DMT/kg of body weight) and placebo. Ayahuasca produced significant subjective effects, peaking between 1.5 and 2 h, involving perceptual modifications and increases in ratings of positive mood and activation. Diastolic blood pressure showed a significant increase at the high dose (9 mm Hg at 75 min), whereas systolic blood pressure and heart rate were moderately and nonsignificantly increased. Cmax values for DMT after the low and high ayahuasca doses were 12.14 ng/ml and 17.44 ng/ml, respectively. Tmax (median) was observed at 1.5 h after both doses. The Tmax for DMT coincided with the peak of subjective effects. Drug administration increased urinary normetanephrine excretion, but, contrary to the typical MAO-inhibitor effect profile, deaminated monoamine metabolite levels were not decreased. This and the negligible harmine plasma levels found suggest a predominantly peripheral (gastrointestinal and liver) site of action for harmine. MAO inhibition at this level would suffice to prevent first-pass metabolism of DMT and allow its access to systemic circulation and the central nervous system.
TL;DR: The fact that some of the compounds tested exert Pgp inhibitor effects at similar concentrations as quinidine suggests that pharmacokinetic drug-drug interactions between the newer antidepressants and Pgp substrates should now be thoroughly studied in vivo.
Abstract: Pharmacokinetic drug-drug interactions often occur at the level of P-glycoprotein (Pgp). To study possible interactions caused by the newer antidepressants we investigated citalopram, fluoxetine, fluvoxamine, paroxetine, reboxetine, sertraline, and venlafaxine and their major metabolites desmethylcitalopram, norfluoxetine, paroxetine-metabolite (paroxetine-M), desmethylsertraline, N-desmethylvenlafaxine, and O-desmethylvenlafaxine for their ability to inhibit Pgp. Pgp inhibition was studied by a fluorometric assay using calcein-acetoxymethylester as Pgp substrate and two different cell systems: L-MDR1 cells (model for human Pgp) and primary porcine brain capillary endothelial cells (pBCECs, model for the blood-brain barrier). Both cell systems proved to be suitable for the evaluation of Pgp inhibitory potency of drugs. All antidepressants tested except O-desmethylvenlafaxine showed Pgp inhibitory activity with sertraline, desmethylsertraline, and paroxetine being the most potent, comparable with the well known Pgp inhibitor quinidine. In L-MDR1 cells fluoxetine, norfluoxetine, fluvoxamine, reboxetine, and paroxetine-M revealed intermediate Pgp inhibition and citalopram, desmethylcitalopram, venlafaxine, and N-desmethylvenlafaxine were only weak inhibitors. The ranking order was similar in pBCECs. The fact that some of the compounds tested exert Pgp inhibitor effects at similar concentrations as quinidine suggests that pharmacokinetic drug-drug interactions between the newer antidepressants and Pgp substrates should now be thoroughly studied in vivo.
TL;DR: The results suggest that VR1 expression in keratinocytes may have a role in the inflammation that occurs secondary to epidermal damage or insult, and thus may function as a sensor for noxious cutaneous stimulation.
Abstract: During dermal injury and the associated trauma a number of compounds are released that can mediate the inflammatory response. Determining the cellular mechanisms that initiate the inflammatory responses to acute keratinocyte damage is important for understanding the regulation of epidermal inflammation. The recently cloned vanilloid receptor-1 (VR1) is a polymodal receptor, responding to thermal, pH, or vanilloids such as capsaicin stimulation. Although VR1 has been localized only on sensory neurons and within the central nervous system, recent evidence suggests a functional VR1 is expressed in human skin and epidermal cells. Using reverse transcription-polymerase chain reaction and immunoblotting we report that human keratinocytes and the human keratinocyte cell line HaCaT express VR1. Consistent with neuronal VR1, activation of epidermal VR1 by capsaicin induced a calcium influx. Treating HaCaT cells with capsaicin resulted in a dose-dependent expression of cyclooxygenase-2 (COX-2), whereas pretreatment with the VR1 receptor antagonist capsazepine abolished the capsaicin-stimulated increase in COX-2 expression. Furthermore, the capsaicin-induced expression of COX-2 was dependent on extracellular calcium. Activation of the epidermal VR1 by capsaicin also resulted in an increased release of interleukin-8 and prostaglandin E2, and the stimulated release was attenuated by capsazepine. The finding that VR1 is expressed by keratinocytes is of great importance because it expands the putative role of VR1 beyond that of pain perception. Our results suggest that VR1 expression in keratinocytes may have a role in the inflammation that occurs secondary to epidermal damage or insult, and thus may function as a sensor for noxious cutaneous stimulation.
TL;DR: The effects of BCTC on acute, inflammatory, and neuropathic pain in rats suggest a role for VR1 in persistent and chronic pain arising from inflammation or nerve injury.
Abstract: The vanilloid receptor 1 (VR1) is a cation channel expressed predominantly by nociceptive sensory neurons and is activated by a wide array of pain-producing stimuli, including capsaicin, noxious heat, and low pH. Although the behavioral effects of injected capsaicin and the VR1 antagonist capsazepine have indicated a potential role for VR1 in the generation and maintenance of persistent pain states, species differences in the molecular pharmacology of VR1 and a limited number of selective ligands have made VR1 difficult to study in vivo. N- (4-Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropryazine-1(2 H )-carbox-amide (BCTC) is a recently described inhibitor of capsaicin- and acid-mediated currents at rat VR1. Here, we report the effects of BCTC on acute, inflammatory, and neuropathic pain in rats. Administration of BCTC (30 mg/kg p.o.) significantly reduced both mechanical and thermal hyperalgesia induced by intraplantar injection of 30 μg of capsaicin. In rats with Freund9s complete adjuvantinduced inflammation, BCTC significantly reduced the accompanying thermal and mechanical hyperalgesia (3 mg/kg and 10 mg/kg p.o., respectively). BCTC also reduced mechanical hyperalgesia and tactile allodynia 2 weeks after partial sciatic nerve injury (10 and 30 mg/kg p.o.). BCTC did not affect motor performance on the rotarod after administration of doses up to 50 mg/kg p.o. These data suggest a role for VR1 in persistent and chronic pain arising from inflammation or nerve injury.
TL;DR: It is indicated that STI-571 is a substrate of P-glycoprotein, and that the inhibition of P -glycop protein affects the transport of STI -571 across MDCKII monolayers.
Abstract: The adequate distribution of STI-571 (Gleevec) to the central nervous system (CNS) is critical for its effective use in CNS tumors. P-glycoprotein-mediated efflux in the blood-brain barrier may play a role in the CNS delivery of this drug. Whether STI-571 is a substrate of P-glycoprotein was determined by examining the directional flux of [14C]STI-571 in parental and MDR1-transfected Madin-Darby canine kidney (MDCK) II epithelial cell monolayers. The basolateral-to-apical flux of STI-571 was 39-fold greater than the apical-to-basolateral flux in the MDR1-transfected cells and 8-fold greater in the parental cell monolayers. This difference in directional flux was significantly reduced by a specific P-glycoprotein inhibitor (2 R )-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979). The role of P-glycoprotein in the CNS distribution of STI-571 was examined in vivo, using wild-type and mdr1a/b (−/−) knockout mice that were orally administered 25 mg/kg [14C]STI-571. In the wild-type mice, the brain-to-plasma STI-571 concentration ratio at all time points was low (1–3%); however, there was an 11-fold greater brain partitioning of STI-571 at 1 h postdose in the mdr1a/b (−/−) mice compared with the wild-type mice. When 12.5 mg/kg STI-571 was given intravenously, the brain-to-plasma ratio of STI-571 in the mdr1a/b (−/−) mice was approximately 7-fold greater than that of wild-type mice up to 120 min postdose. These data indicate that STI-571 is a substrate of P-glycoprotein, and that the inhibition of P-glycoprotein affects the transport of STI-571 across MDCKII monolayers. Moreover, P-glycoprotein plays an important role in limiting the distribution of STI-571 to the CNS.
TL;DR: Pluronic block copolymers with intermediate lipophilic properties showed the strongest fluidization effect on the cell membranes along with the most efficient reduction of intracellular ATP synthesis in BBMEC monolayers.
Abstract: Pluronic block copolymer P85 was shown to inhibit the P-glycoprotein (Pgp) drug efflux system and to increase the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). However, there is an entire series of Pluronics varying in lengths of propylene oxide and ethylene oxide and overall lipophilicity. This study identifies those structural characteristics of Pluronics required for maximal impact on drug efflux transporter activity in bovine brain microvessel endothelial cells (BBMECs). Using a wide range of block copolymers, differing in hydrophilic-lipophilic balance (HLB), this study shows that lipophilic Pluronics with intermediate length of propylene oxide block (from 30 to 60 units) and HLB <20 are the most effective at inhibiting Pgp efflux in BBMECs. The methods used included 1) cellular accumulation studies with the Pgp substrate rhodamine 123 in BBMECs to assess Pgp activity; 2) luciferin/luciferase ATP assay to evaluate changes in cellular ATP; 3) 1,6-diphenyl-1,3,5-hexatriene membrane microviscosity studies to determine alterations in membrane fluidity; and 4) Pgp ATPase assays using human Pgp-expressing membranes. Pluronics with intermediate lipophilic properties showed the strongest fluidization effect on the cell membranes along with the most efficient reduction of intracellular ATP synthesis in BBMEC monolayers. The relationship between the structure of Pluronic block copolymers and their biological response-modifying effects in BBMECs are useful for determining formulations with maximal efficacy for increasing BBB permeability.
TL;DR: In vitro pharmacology of a high potency, selective VR1 antagonist that, unlike capsazepine, has potent blocking effects on low pH-induced activation of rat VR1 is described, making it a more suitable candidate for testing the role played by VR1 in rat models of human disease.
Abstract: Vanilloids such as capsaicin have algesic properties and seem to mediate their effects via activation of the vanilloid receptor 1 (VR1), a ligand-gated ion channel highly expressed on primary nociceptors. Although blockade of capsaicin-induced VR1 activation has been demonstrated in vitro and in vivo with the antagonist capsazepine, efficacy in rat models of chronic pain has not been observed with this compound. Here, we describe the in vitro pharmacology of a highly potent VR1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC). Similar to capsazepine, this compound inhibits capsaicin-induced activation of rat VR1 with an IC50 value of 35 nM. Interestingly however, BCTC also potently inhibits acid-induced activation of rat VR1 (IC50 value of 6.0 nM), whereas capsazepine is inactive. Similarly, in the rat skin-nerve preparation both BCTC and capsazepine block capsaicin-induced activation, whereas the response to acidification is inhibited by BCTC, but not by capsazepine. Specificity for VR1 was demonstrated against 63 other receptor, enzyme, transporter, and ion channel targets. BCTC was orally bioavailable in the rat, demonstrating a plasma half-life of approximately 1 h and significant penetration into the central nervous system. Thus, BCTC is a high potency, selective VR1 antagonist that, unlike capsazepine, has potent blocking effects on low pH-induced activation of rat VR1. These properties make it a more suitable candidate than capsazepine for testing the role played by VR1 in rat models of human disease.
TL;DR: It was demonstrated that Syk may play a very critical role in the pathogenesis of allergic reactions and was identified as a potent and selective inhibitor of Syk kinase.
Abstract: Spleen tyrosine kinase (Syk) tyrosine kinase plays essential roles in receptors for Fc portion of immunoglobulins and B cell receptor complex signaling in various inflammatory cells; therefore, inhibitors of Syk kinase may show potential as antiasthmatic/allergic therapeutics. We identified 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY 61-3606), a potent (Ki = 7.5 nM) and selective inhibitor of Syk kinase. BAY 61-3606 inhibited not only degranulation (IC50 values between 5 and 46 nM) but also lipid mediator and cytokine synthesis in mast cells. BAY 61-3606 was highly efficacious in basophils obtained from healthy human subjects (IC50 = 10 nM) and seems to be at least as potent in basophils obtained from atopic (high serum IgE) subjects (IC50 = 8.1 nM). B cell receptor activation and receptors for Fc portion of IgG signaling in eosinophils and monocytes were also potently suppressed by BAY 61-3606. Oral administration of BAY 61-3606 to rats significantly suppressed antigen-induced passive cutaneous anaphylactic reaction, bronchoconstriction, and bronchial edema at 3 mg/kg. Furthermore, BAY 61-3606 attenuated antigen-induced airway inflammation in rats. Based on these anti-inflammatory effects of BAY 61-3606 both in vitro and in vivo, it was demonstrated that Syk may play a very critical role in the pathogenesis of allergic reactions.