Chlorzoxazone Alleviates Experimental Autoimmune Encephalomyelitis via Inhibiting IL-6 Secretion of Dendritic Cells.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease with chronic inflammatory demyelination of the CNS. Experimental autoimmune encephalomyelitis (EAE) is an important animal model to study MS, with many pathological phenomena similar to MS. Th17 cells are important regulators of EAE and MS pathogenesis. Most cytokines needed for Th cell development are secreted by APCs, such as dendritic cells (DCs). Consequently, MS could be improved by inhibiting cytokine secretion from DCs. In this study, we reported that chlorzoxazone could ameliorate EAE pathogenesis via inhibiting IL-6 production by DCs. The EAE signs in the chlorzoxazone-treated group of mice were relieved, which was mainly manifested as lower clinical scores, a decrease in the number of immune cells, and a reduction of demyelination in the CNS. Moreover, the proportion of Th17 cells in the spleen and CNS decreased significantly. In vitro experiments showed that chlorzoxazone treatment significantly reduced DC-derived IL-6 production. In the DC-T cell coculture experiment, significantly decreased Th17 differentiation was observed after chlorzoxazone treatment. In addition, mass spectrometric analysis was performed to elucidate the mechanism by which chlorzoxazone affected EAE and DC function. We showed that the effect of chlorzoxazone on inhibiting the secretion of IL-6 by DCs may be mediated via the AMP-activated protein kinase pathway. Overall, our study elucidated the key role of chlorzoxazone in regulating EAE pathogenesis and suggested that it might be used as a new drug for MS patients.

Silymarin Protects against Acute Liver Injury Induced by Acetaminophen by Downregulating the Expression and Activity of the CYP2E1 Enzyme.

Previous studies have shown that silymarin protects against various types of drug-induced liver injury, but whether the protective mechanism of silymarin against acetaminophen-induced liver injury is related to the CYP2E1 enzyme remains unclear. In this study, we investigated the effect of silymarin on the activity and expression of CYP2E1 in vitro and in vivo. The results of in vitro studies showed that silymarin not only inhibited the activity of CYP2E1 in human and rat liver microsomes but also reduced the expression of CYP2E1 in HepG2 cells. In vivo studies showed that silymarin pretreatment significantly reduced the conversion of chlorzoxazone to its metabolite 6-OH-CLX and significantly increased the t1/2, area under the curve (AUC) and mean residence time (MRT) of chlorzoxazone. In addition, silymarin pretreatment significantly inhibited the upregulation of Cyp2e1 expression, reduced the production of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS), and restored the liver glutathione level. The results of our study show that silymarin plays an important protective role in the early stage of acetaminophen-induced acute liver injury by reducing the activity and expression of CYP2E1, reducing the generation of toxic metabolites, and alleviating liver injury.

Enzyme Kinetics and Molecular Docking Studies on Cytochrome 2B6, 2C19, 2E1, and 3A4 Activities by Sauchinone.

Sauchinone, an active lignan isolated from the aerial parts of Saururus chinensis (Saururaceae), exhibits anti-inflammatory, anti-obesity, anti-hyperglycemic, and anti-hepatic steatosis effects. As herb-drug interaction (HDI) through cytochrome P450s (CYPs)-mediated metabolism limits clinical application of herbs and drugs in combination, this study sought to explore the enzyme kinetics of sauchinone towards CYP inhibition in in vitro human liver microsomes (HLMs) and in vivo mice studies and computational molecular docking analysis. In in vitro HLMs, sauchinone reversibly inhibited CYP2B6, 2C19, 2E1, and 3A4 activities in non-competitive modes, showing inhibition constant (Ki) values of 14.3, 16.8, 41.7, and 6.84 µM, respectively. Also, sauchinone time-dependently inhibited CYP2B6, 2E1 and 3A4 activities in vitro HLMs. Molecular docking study showed that sauchinone could be bound to a few key amino acid residues in the active site of CYP2B6, 2C19, 2E1, and 3A4. When sibutramine, clopidogrel, or chlorzoxazone was co-administered with sauchinone to mice, the systemic exposure of each drug was increased compared to that without sauchinone, because sauchinone reduced the metabolic clearance of each drug. In conclusion, when sauchinone was co-treated with drugs metabolized via CYP2B6, 2C19, 2E1, or 3A4, sauchinone-drug interactions occurred because sauchinone inhibited the CYP-mediated metabolic activities.

Effect of piperine on CYP2E1 enzyme activity of chlorzoxazone in healthy volunteers.

1. The purpose of the present study was to investigate the effect of piperine (PIP) on CYP2E1 enzyme activity and pharmacokinetics of chlorzoxazone (CHZ) in healthy volunteers. 2. An open-label, two period, sequential study was conducted in 12 healthy volunteers. A single dose of PIP 20 mg was administered daily for 10 days during treatment phase. A single dose of CHZ 250 mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected at predetermined time intervals after CHZ dosing and analyzed by HPLC. 3. Treatment with PIP significantly enhanced maximum plasma concentration (Cmax) (3.14-4.96 µg/mL), area under the curve (AUC) (10.46-17.78 µg h/mL), half life (T1/2) (1.26-1.82 h) and significantly decreased elimination rate constant (Kel) (0.57-0.41 h - 1), apparent oral clearance (CL/F) (24.76-13.65 L/h) of CHZ when compared to control. In addition, treatment with PIP significantly decreased Cmax (0.22-0.15 µg/mL), AUC (0.94-0.68 µg h/mL), T1/2 (2.54-1.68 h) and significantly increased Kel (0.32-0.43 h - 1) of 6-hydroxychlorzoxazone (6-OHCHZ) as compared to control. Furthermore, treatment with PIP significantly decreased metabolite to parent (6-OHCHZ/CHZ) ratios of Cmax, AUC, T1/2 and significantly increased Kel ratio of 6-OHCHZ/CHZ, which indicate the decreased formation of CHZ to 6-OHCHZ. 4. The results suggest that altered pharmacokinetics of CHZ might be attributed to PIP mediated inhibition of CYP2E1 enzyme, which indicate significant pharmacokinetic interaction present between PIP and CHZ. The inhibition of CYP2E1 by PIP may represent a novel therapeutic benefit for minimizing ethanol induced CYP2E1 enzyme activity and results in reduced hepatotoxicity of ethanol.

Influence of Shenxiong Glucose Injection on the Activities of Six CYP Isozymes and Metabolism of Warfarin in Rats Assessed Using Probe Cocktail and Pharmacokinetic Approaches.

Shenxiong glucose injection (SGI), a traditional Chinese medicine (TCM) preparation, has been widely used for the treatment of various cardiovascular and cerebrovascular diseases for many years. We assessed the potential influences of SGI on the activities of six CYP enzymes (CYP1A2, CYP2C11, CYP2C19, CYP2D4, CYP2E1, and CYP3A2) and on the pharmacokinetics of warfarin in rats. We compared plasma pharmacokinetics of six probe drugs (caffeine/CYP1A2, tolbutamide/CYP2C11, omeprazole/CYP2C19, metoprolol/CYP2D4, chlorzoxazone/CYP2E1, and midazolam/CYP3A2) and of warfarin between control and SGI-pretreated groups, to estimate the effect on the relative activities of the six isozymes and warfarin metabolism. There were no significant differences in the pharmacokinetic parameters of caffeine, omeprazole, metoprolol, chlorzoxazone, and midazolam between the SGI-pretreated and control groups. However, many pharmacokinetic parameters of tolbutamide in SGI-pretreated rats were affected significantly (p < 0.05), and indicated tolbutamide metabolism in the former group was markedly slower. Moreover, SGI reduced the clearance of warfarin. These results suggested SGI showed no effects on the enzyme activities of rat CYP1A2, CYP2C19, CYP2D4, CYP2E1, and CYP3A2, but inhibited the enzyme activity of CYP2C11, and improved the blood concentration of warfarin. This suggests that the dose of warfarin may need be adjusted when co-administrated with SGI.

In vivo analysis of cerebellar Purkinje cell activity in SCA2 transgenic mouse model.

Cerebellar Purkinje cells (PCs) are primarily affected in many spinocerebellar ataxias (SCA). In this study we investigated functional activity of PCs in transgenic mouse model of SCA2, a polyglutamine neurodegenerative hereditary disorder. In our studies we used extracellular single-unit recording method to compare spontaneous activity of PCs in age-matched wild-type mice and SCA2-58Q transgenic mice. We discovered that the fraction of PCs with bursting and an irregular pattern of spontaneous activity dramatically increases in aged SCA2-58Q mice compared with wild-type littermates. Small-conductance calcium-activated potassium (SK) channels play an important role in determining firing rate of PCs. Indeed, we demonstrated that intraperitoneal (IP) injection of SK channel inhibitor NS8593 induces an irregular pattern of PC activity in wild-type mice. Furthermore, we demonstrated that IP injection of SK channel-positive modulator chlorzoxazone (CHZ) decreases spontaneous firing rate of cerebellar PCs. Finally, we have shown that IP injections with CHZ normalize firing activity of cerebellar PCs from aging SCA2-58Q mice. We propose that alterations in PC firing patterns is one of potential causes of ataxic symptoms in SCA2 and in other SCAs and that positive modulators of SK channels can be used to normalize activity of PCs and alleviate ataxic phenotype in patients with SCA.

Effects of eleutheroside B and eleutheroside E on activity of cytochrome P450 in rat liver microsomes.

BACKGROUND: Chemicals of herbal products may cause unexpected toxicity or adverse effect by the potential for alteration of the activity of CYP450 when co-administered with other drugs. Eleutherococcus senticosus (ES), has been widely used as a traditional herbal medicine and popular herbal dietary supplements, and often co-administered with many other drugs. The main bioactive constituents of ES were considered to be eleutherosides including eleutheroside B (EB) and eleutheroside E (EE). This study was to investigate the effects of EB and EE on CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in rat liver microsomes in vitro. METHOD: Probe drugs of tolbutamide (TB), dextromethorphan (DM), chlorzoxazone (CLZ) and testosterone (TS) as well as eleutherosides of different concentrations were added to incubation systems of rat liver microsomes in vitro. After incubation, validated HPLC methods were used to quantify relevant metabolites. RESULTS: The results suggested that EB and EE exhibited weak inhibition against the activity of CYP2C9 and CYP2E1, but no effects on CYP2D6 and CYP3A4 activity. The IC50 values for EB and EE were calculated to be 193.20 µM and 188.36 µM for CYP2E1, 595.66 µM and 261.82 µM for CYP2C9, respectively. Kinetic analysis showed that inhibitions of CYP2E1 by EB and EE were best fit to mixed-type with Ki value of 183.95 µM and 171.63 µM, respectively. CONCLUSIONS: These results indicate that EB and EE may inhibit the metabolism of drugs metabolized via CYP2C9 and CYP2E1, and have the potential to increase the toxicity of the drugs.

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata.

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17-21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (ß4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell firing associated with genetic ataxias.

Gut microbiota and host Cyp450s co-contribute to pharmacokinetic variability in mice with non-alcoholic steatohepatitis: Effects vary from drug to drug.

INTRODUCTION: Pharmacokinetic variability in disease state is common in clinical practice, but its underlying mechanism remains unclear. Recently, gut microbiota has been considered to be pharmacokinetically equivalent to the host liver. Although some studies have explored the roles of gut microbiota and host Cyp450s in drug pharmacokinetics, few have explored their effects on pharmacokinetic variability, especially in disease states. OBJECTIVES: In this study, we aim to investigate the effects of gut microbiota and host Cyp450s on pharmacokinetic variability in mice with non-alcoholic steatohepatitis (NASH), and to elucidate the contribution of gut microbiota and host Cyp450s to pharmacokinetic variability in this setting. METHODS: The pharmacokinetic variability of mice with NASH was explored under intragastric and intravenous administrations of a cocktail mixture of omeprazole, phenacetin, midazolam, tolbutamide, chlorzoxazone, and metoprolol, after which the results were compared with those obtained from the control group. Thereafter, the pharmacokinetic variabilities of all drugs and their relations to the changes in gut microbiota and host Cyp450s were compared and analyzed. RESULTS: The exposures of all drugs, except metoprolol, significantly increased in the NASH group under intragastric administration. However, no significant increase in the exposure of all drugs, except tolbutamide, was observed in the NASH group under intravenous administration. The pharmacokinetic variabilities of phenacetin, midazolam, omeprazole, and chlorzoxazone were mainly associated with decreased elimination activity in the gut microbiota. By contrast, the pharmacokinetic variability of tolbutamide was mainly related to the change in the host Cyp2c65. Notably, gut microbiota and host Cyp450s exerted minimal effects on the pharmacokinetic variability of metoprolol. CONCLUSION: Gut microbiota and host Cyp450s co-contribute to the pharmacokinetic variability in mice with NASH, and the degree of contribution varies from drug to drug. The present findings provide new insights into the explanation of pharmacokinetic variability in disease states.

KCa channels as therapeutic targets in episodic ataxia type-2.

Episodic ataxia type-2 (EA2) is an inherited movement disorder caused by mutations in the gene encoding the Ca(v)2.1alpha1 subunit of the P/Q-type voltage-gated calcium channel that result in an overall reduction in the P/Q-type calcium current. A consequence of these mutations is loss of precision of pacemaking in cerebellar Purkinje cells. This diminished precision reduces the information encoded by Purkinje cells and is thought to contribute to symptoms associated with this disorder. The loss of the precision of pacemaking in EA2 is the consequence of reduced activation of calcium-dependent potassium channels (K(Ca)) by the smaller calcium current and in vitro can be pharmacologically restored by K(Ca) activators. We used a well established mouse model of EA2, the tottering (tg/tg) mouse, to examine the potential therapeutic utility of one such Food and Drug Administration (FDA)-approved compound, chlorzoxazone (CHZ). Compared with wild-type Purkinje cells, we found the firing rate of tg/tg Purkinje cells in acutely prepared cerebellar slices to be very irregular. Bath application of CHZ successfully restored the precision of pacemaking in a dose-dependent manner. Oral administration of CHZ to tg/tg mice improved their baseline motor performance and reduced the severity, frequency, and duration of episodes of dyskinesia without producing any adverse effects. We propose the use of CHZ, which is currently FDA approved as a muscle relaxant, as a safe and novel treatment of EA2.

The therapeutic mode of action of 4-aminopyridine in cerebellar ataxia.

Episodic ataxia type 2 (EA2) is a hereditary cerebellar ataxia associated with mutations in the P/Q-type voltage-gated calcium (Ca(2+)) channels. Therapeutic approaches for treatment of EA2 are very limited. Presently, the potassium (K(+)) channel blocker 4-aminopyridine (4-AP) constitutes the most promising treatment, although its mechanism of action is not understood. Here we show that, in contrast to what is commonly believed, therapeutic concentrations of 4-AP do not increase the inhibitory drive of cerebellar Purkinje cells. Instead, 4-AP restores the severely diminished precision of pacemaking in Purkinje cells of EA2 mutant mice by prolonging the action potential and increasing the action potential afterhyperpolarization. Consistent with this mode of action, the therapeutic efficacy of 4-AP was comparable, and not additive, to chlorzoxazone, an activator of Ca(2+)-dependent K(+) channels that also restores the precision of Purkinje cell pacemaking. The likely target of 4-AP at the concentrations used are the K(v)1 family of K(+) channels, possibly the K(v)1.5 subtype. Because at higher concentrations 4-AP blocks a large array of K(+) channels and is a proconvulsant, use of selective K(v)1 channel blockers is likely to be a safer substitute for treatment of cerebellar ataxia.

In vivo analysis of the spontaneous firing of cerebellar Purkinje cells in awake transgenic mice that model spinocerebellar ataxia type 2.

Cerebellar Purkinje cells (PCs) fire spontaneously in a tonic mode, although the precision of this pacemaking activity is disturbed in many abnormal conditions involving cerebellar atrophy, such as many spinocerebellar ataxias (SCAs). In our previous studies we used the single-unit extracellular recording method to analyze spontaneous PC firing in vivo in the anesthetized SCA2-58Q transgenic mice. We realized that PCs from aging SCA2-58Q mice fire much less regularly compared to PCs from their wild type (WT) littermates and this abnormal activity can be reversed with an intraperitoneal (i. p.) injection of SK channel-positive modulator chlorzoxazone (CHZ). Here we used the same single-unit extracellular recording method to analyze the spontaneous firing in vivo in awake SCA2-58Q transgenic mice. For this purpose, we used the Mobile HomeCage (Neurotar, Finland) floating platform to immobilize the experimental animal's head during the recording sessions. We discovered that generally PCs from awake animals fired much more frequently and much less regularly than previously observed PCs from anesthetized animals. In vivo recordings from awake SCA2/WT mice revealed that complex spikes, which are generated by PCs in reply to the excitation coming by climbing fibers, as well as simple spikes, were much less frequent in SCA2 mice compared to their WT littermates. To test the effect of the SK channel positive modulation on the PCs firing activity in awake SCA2 mice and also the effect on their motor coordination, we started the CHZ trial in these mice. We discovered that the long-term i. p. injections of CHZ did not affect the spike generation in SCA2-58Q mice, however, they did recover the precision of this spontaneous pacemaking activity. Furthermore, we also showed that treatment with CHZ alleviated the age-dependent motor impairment in SCA2-58Q mice. We propose that the lack of precision in PC spike generation might be a key cause for the progression of ataxic symptoms in different SCAs and that the activation of calcium-activated potassium channels, including SK channels, can be used as a potential way to treat SCAs on the physiological level of the disease.

Chlorzoxazone, a small molecule drug, augments immunosuppressive capacity of mesenchymal stem cells via modulation of FOXO3 phosphorylation.

Nowadays, immune diseases are a large burden in healthcare. Mesenchymal stem cells (MSCs) have prominent ability in immunomodulation and have been applicated on treating many immune-related diseases. However, the clinical outcomes can be disparate and sometimes completely counterproductive beyond explanation of cell heterogeneity. The theory of immunomodulation plasticity in MSCs has then emerged to explain that MSCs can be induced into proinflammatory MSC1 or anti-inflammatory MSC2 responding to different immune environment. It would be safer and more efficient if we could induce MSCs into a certain immune phenotype, in most cases MSC2, prior to medical treatment. In this study, we screened and identified a classical FDA-approved drug, chlorzoxazone (CZ). Unlike traditional method induced by IFN-γ, CZ can induce MSC into MSC2 phenotype and enhance the immunosuppressive capacity without elevation of immunogenicity of MSCs. CZ-treated MSCs can better inhibit T cells activation and proliferation, promote expression of IDO and other immune mediators in vitro, and alleviate inflammatory infiltration and tissue damage in acute kidney injury rat model more effectively. Moreover, we discovered that CZ modulates phosphorylation of transcriptional factor forkhead box O3 (FOXO3) independent of classical AKT or ERK signaling pathways, to promote expression of downstream immune-related genes, therefore contributing to augmentation of MSCs immunosuppressive capacity. Our study established a novel and effective approach to induce MSC2, which is ready for clinical application.

Chlorzoxazone exhibits neuroprotection against Alzheimer's disease by attenuating neuroinflammation and neurodegeneration in vitro and in vivo.

Alzheimer's disease (AD), a complex and an age-related brain disease, is induced by the accumulation of amyloid beta (Aß) and neuroinflammation. Chlorzoxazone (CZ) is a classical FDA-approved drug, and shows anti-inflammatory effects. However, up until now, its regulatory role in AD has not been investigated. Therefore, in this study we attempted to explore if CZ could be an effective therapeutic strategy for AD treatment. At first, the in vitro study was performed to mimic AD using Aß. We found that Aß caused p65 nuclear translocation in both primary microglial cells and astrocytes, which were, however, restrained by CZ treatments. Meanwhile, CZ incubation markedly decreased the expression of pro-inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß). Aß deposition was also markedly reduced in glial cells treated with CZ. Importantly, we found that glial activation and its-related pro-inflammation induced by Aß led to obvious neurodegeneration and neuroinflammation, which were effectively attenuated by CZ pre-treatment in the isolated primary cortical neurons. Then, the in vivo study was performed using APP/PS1 mice with AD. Behavior tests showed that CZ administration effectively improved cognitive deficits in AD mice. Neuron death in hippocampus of AD mice was also inhibited by CZ. Aß accumulation in brain was markedly decreased in CZ-treated AD mice. We finally found that hippocampal glial activation in AD mice was obviously blocked by CZ supplementation, along with remarkable decreases in TNF-α, IL-1ß and p65 nuclear translocation. Together, these findings above demonstrated that CZ could inhibit glial activation and inflammatory response, contributing to the suppression of neurodegeneration and neuroinflammation. Therefore, CZ may be an effective therapeutic strategy for AD treatment.

Computer-aided identification of recognized drugs as Pseudomonas aeruginosa quorum-sensing inhibitors.

Attenuation of Pseudomonas aeruginosa virulence by the use of small-molecule quorum-sensing inhibitors (referred to as the antipathogenic drug principle) is likely to play a role in future treatment strategies for chronic infections. In this study, structure-based virtual screening was used in a search for putative quorum-sensing inhibitors from a database comprising approved drugs and natural compounds. The database was built from compounds which showed structural similarities to previously reported quorum-sensing inhibitors, the ligand of the P. aeruginosa quorum-sensing receptor LasR, and a quorum-sensing receptor agonist. Six top-ranking compounds, all recognized drugs, were identified and tested for quorum-sensing-inhibitory activity. Three compounds, salicylic acid, nifuroxazide, and chlorzoxazone, showed significant inhibition of quorum-sensing-regulated gene expression and related phenotypes in a dose-dependent manner. These results suggest that the identified compounds have the potential to be used as antipathogenic drugs. Furthermore, the results indicate that structure-based virtual screening is an efficient tool in the search for novel compounds to combat bacterial infections.

Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI) carboxylic acids as mutual prodrugs: design, synthesis, pharmacological investigations and docking studies.

The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxazone ester prodrugs (6-8) of some acidic NSAIDs (1-3) were designed, synthesized and evaluated as mutual prodrugs with the aim of improving the therapeutic potency and retard the adverse effects of gastrointestinal origin. The structure of the synthesized mutual ester prodrugs (6-8) were confirmed by IR, (1)H NMR, mass spectroscopy (MS) and their purity was ascertained by TLC and elemental analyses. In vitro chemical stability revealed that the synthesized ester prodrugs (6-8) are chemically stable in hydrochloric acid buffer pH 1.2 as a non-enzymatic simulated gastric fluid (SGF) and in phosphate buffer pH 7.4 as non-enzymatic simulated intestinal fluid (SIF). In 80% human plasma, the mutual prodrugs were found to be susceptible to enzymatic hydrolysis at relatively faster rate (t(1/2) approximately 37 and 34 min for prodrugs 6 and 7, respectively). Mutual ester prodrugs (6-8) were evaluated for their anti-inflammatory and muscle relaxation activities. Scanning electromicrographs of the stomach showed that the ester prodrugs induced very little irritancy in the gastric mucosa of rats after oral administration for 4days. In addition, docking of the mutual ester prodrugs (6-8) into COX-2 active site was conducted in order to predict the affinity and orientation of these prodrugs at the enzyme active site.

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase-null mice.

Previous studies have demonstrated that the NADH-dependent cytochrome b(5) electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH-cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase-null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH-supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase-null mice, whereas NADH-supported activity was about twofold higher than that found in normal (wild-type) liver microsomes. This greater activity in oxidoreductase-null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4-fold higher level of cytochrome b(5). Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase-null mice was about 5% of that in hepatocytes from wild-type mice and matched the results obtained with wild-type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b(5) electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions.

Altered cytochrome 2E1 and 3A P450-dependent drug metabolism in advanced ovarian cancer correlates to tumour-associated inflammation.

BACKGROUND AND PURPOSE: Previous work has focussed on changes in drug metabolism caused by altered activity of CYP3A in the presence of inflammation and, in particular, inflammation associated with malignancy. However, drug metabolism involves a number of other P450s, and therefore, we assessed the effect of cancer-related inflammation on multiple CYP enzymes using a validated drug cocktail. EXPERIMENTAL APPROACH: Patients with advanced stage ovarian cancer and healthy volunteers were recruited. Participants received caffeine, chlorzoxazone, dextromethorphan, and omeprazole as in vivo probes for CYP1A2, CYP2E1, CYP2D6, CYP3A, and CYP2C19. Blood was collected for serum C-reactive protein and cytokine analysis. KEY RESULTS: CYP2E1 activity was markedly up-regulated in cancer (6-hydroxychlorzoxazone/chlorzoxazone ratio of 1.30 vs. 2.75), while CYP3A phenotypic activity was repressed in cancer (omeprazole sulfone/omeprazole ratio of 0.23 vs. 0.49). Increased activity of CYP2E1 was associated with raised serum levels of IL-6, IL-8, and TNF-α. Repression of CYP3A correlated with raised levels of serum C-reactive protein, IL-6, IL-8, and TNF-α. CONCLUSIONS AND IMPLICATIONS: CYP enzyme activity is differentially affected by the presence of tumour-associated inflammation, affecting particularly CYP2E1- and CYP3A-mediated drug metabolism, and may have profound implications for drug development and prescribing in oncological settings.

Modulation of hepatic and renal metabolism and toxicity of trichloroethylene and perchloroethylene by alterations in status of cytochrome P450 and glutathione.

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver.