Pharmacokinetics of Chlorzoxazone in Rats with Diabetes: Induction of CYP2E1 on 6-Hydroxychlorzoxazone Formation

doi:10.1002/jps.20698

Journal of Pharmaceutical Sciences

Volume 95, Issue 11, November 2006, Pages 2452-2462

https://doi.org/10.1002/jps.20698 Get rights and content

Abstract

Pharmacokinetic parameters of chlorzoxazone (CZX) and its main metabolite, 6-hydroxychlorzoxazone (OH-CZX), were compared after intravenous (20 mg/kg) and oral (50 mg/kg) administration of CZX in rat model of diabetes induced by alloxan (DMIA) or streptozotocin (DMIS), and their respective control rats. In both rat models of diabetes, the expression and mRNA level of CYP2E1 increased, and CZX was metabolized to OH-CZX via CYP2E1 in rats. Hence, it could be expected that formation of OH-CZX increased in both rat models of diabetes. As expected, after intravenous (80.5% and 74.4% increase in rat models of DMIA and DMIS, respectively) and oral (55.6% and 70.5% increase, respectively) administration of CZX, the AUC of OH-CZX was significantly greater than their respective control rats. Since, CZX is an intermediate hepatic extraction ratio drug, the greater AUC values of OH-CZX (the significantly faster CL_NR of CZX) in both rat models of diabetes could be supported by significantly faster CL_int for the formation of OH-CZX (75.9% and 129% increase for rat models of DMIA and DMIS, respectively) and significantly greater free fractions of CZX in plasma (51.9% and 58.9% increase, respectively). Also it was reported that hepatic blood flow rate was faster in male Wister rat model of DMIS.

Section snippets

INTRODUCTION

Chlorzoxazone [5-chloro-2(3H)-benzoxazolone; CZX], once used as a skeletal muscle relaxant for the treatment of painful muscle spasms,¹ primarily undergoes hydroxylation to form 6-hydroxychlorzoxazone (OH-CZX). OH-CZX is then rapidly glucuronidated and excreted in urine.²,³ The formation of OH-CZX is catalyzed mainly by the hepatic microsomal cytochrome P450 (CYP) 2E1 in humans² and rats.4., 5., 6., 7., 8. The use of OH-CZX as a chemical probe has been suggested to assess the activity of CYP2E1

Chemicals

CZX, OH-CZX, reduced form of β-nicotinamide adenine dinucleotide phosphate (NADPH; as a tetrasodium salt), tris(hydroxymethyl)aminomethane (Tris)-buffer, ethylenediamine tetraacetatic acid (EDTA), 3-aminophenyl sulfone (an internal standard of high-performance liquid chromatographic, HPLC, assay for CZX and OH-CZX), β-glucuronidase (Type H-1; from Helixa pomatia), alloxan, and streptozotocin were products from Sigma-Aldrich Corporation (St. Louis, MO). Ketamine hydrochloride was a product from

Measurement of V_max, K_m, and CL_int for the Formation of OH-CZX in Hepatic Microsomes

The V_max, K_m, and CL_int for the formation of OH-CZX in hepatic microsomal fractions of all rats studied are listed in Table 1. In rat models of DMIA and DMIS, the V_max for the formation of OH-CZX was significantly faster (94.0% and 157% increase, respectively) than controls, suggesting that the maximum velocity for the formation of OH-CZX was faster than controls. However, the K_m was not significantly different among three groups of rats, suggesting that the affinity of CZX to the enzyme(s)

DISCUSSION

Induction of diabetes mellitus by injection of alloxan or streptozotocin was evident based on the significantly higher blood glucose level, significantly lager 24-h urine output, and significant decrease in body weight gain than those of their respective controls (Tabs. 2 and 3).

The contribution of CL_R to CL of CZX after the intravenous administration of CZX was almost negligible; the Ae_{0–24 h} was less than 1.69% of the intravenous dose for all rats studied (Tab. 2), indicating that almost all

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Diabetes affects the expression of many isoforms of cytochrome P450. Baek et al. [3] proved increased CYP2E1 activity, which enhanced the concentration of chlorzoxazone metabolite – 6-hydroxy chlorzoxazone (OH-CZX) in rats with experimentally induced diabetes. Due to higher activity of CYP2E1, CYP1A1/2 and CYP3A1/2 isoenzymes in diabetic animals, the clearance of many drugs were faster and AUC values were reduced, when compared with the control group [4].
Alterations in blood glucose levels observed in diabetes, may change the pharmacokinetics of co-administered drugs and in consequence, the efficacy and safety of therapy. Many oncological patients are diabetics and it is important to determine the interaction of anticancer drugs with this chronic disease. Erlotinib is a tyrosine kinase inhibitor (TKI), approved for the treatment of patients with non-small-cell lung cancer and pancreatic cancer in combination with gemcitabine. The aim of the study was to investigate the influence of the diabetes on the pharmacokinetics of erlotinib in rabbits. Additionally, the effect of erlotinib on glucose levels was examined.
The pharmacokinetics of erlotinib was studied in healthy rabbits (n = 6, control group) and type 1 diabetic rabbits (n = 6, diabetic group). Erlotinib was administered in a single oral dose of 25 mg. Plasma concentrations of erlotinib and its metabolite (OSI420) were measured with the validated method.
The plasma concentrations of erlotinib and OSI420 were markedly increased in diabetic rabbits. Statistically significant differences between the groups were revealed for almost all analysed pharmacokinetic parameters for erlotinib and OSI420. The maximum glycaemia drop of 7.7–33.5% was observed in the diabetic animals, but no significant changes in glucose concentration were observed in the control group.
The research proved the significant influence of diabetes on the pharmacokinetics of erlotinib and OSI420. Due to higher exposure to erlotinib, there may be an increased risk of adverse drug reactions in diabetic patients. Therefore, in some cases lower doses of the drug should be considered.
Characterization of novel cytochrome P450 2E1 knockout rat model generated by CRISPR/Cas9
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The adult rats were divided into WT and KO groups and each group contains at least six rats. For intravenous injection treatment, chlorzoxazone was dissolved in normal saline with a minimum amount of 10 mol/L NaOH (3 μL/mL) [27,28], and then injected into WT and KO rats via the tail vein at a dose of 1 mg/kg. Blood samples (approximately 500 μL) were collected from orbital venous plexus at 2, 5, 10, 20, 40, 60, 90, 120, 180 min after chlorzoxazone treatment.
A bacterial CRISPR-associated protein-9 nuclease (CRISPR/Cas9) from Streptococcus pyogenes has generated considerable excitement as a new tool to edit the targeted genome. Cytochrome P450 (CYP) 2E1 not only plays an important role in the xenobiotic metabolism and chemical toxicity, but also is involved in many kinds of diseases, such as alcoholic liver diseases and diabetes. Despite its importance, few animal models are used to predict CYP2E1 properties in physiology, pathology, as well as carcinogen activation. To establish a novel model for investigating the functions of CYP2E1 in vivo, this study has successfully generated the Cyp2e1 knockout (KO) rat model without detectable off-target effects using CRISPR/Cas9 system. The Cyp2e1 KO rats were viable and fertile and did not display any obvious physiological abnormities. The absent expression of CYP2E1 in KO rats also resulted in inactive behaviors in the metabolism of CYP2E1 substrates. The Cyp2e1 KO rats as a novel and available rodent animal model provide a powerful tool for the study of CYP2E1 in the chemical metabolism, toxicity, carcinogenicity, and its core factor in drug–drug interactions.
Glucose dominates the regulation of carboxylesterases induced by lipopolysaccharide or interleukin-6 in primary mouse hepatocytes
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It is shown that diabetes is associated with a significant decrease in hepatic CYP 3A4 without knowing the type of diabetes and diabetes treatment (Dostalek et al., 2011). In contrast, uncontrolled diabetes in experimental animal models results in enhanced expression of several CYP450 isoforms (CYP1A1, 1A2, 1B1, 2B1, 2C12, 2E1, 3A4 and 3A1) and suppressed expression of CYP 2C11, 2C13, 2A2 and 3A2 (Baek et al., 2006; Kim et al., 2005; Lee et al., 2007; Sindhu et al., 2006). An increase in CYP1A2 activity is also observed in patients with type 1 diabetes mellitus (Matzke et al., 2000).
Altered drug disposition has been associated with inflammation and diabetes, leading to the alteration of drug efficacy and toxicity. Carboxylesterases are major hydrolytic enzymes in the liver, catalyzing the hydrolytic biotransformation of numerous therapeutic agents. Therefore, how glucose affects the regulation of carboxylesterases by interleukin-6 (IL-6) and lipopolysaccharide (LPS) were investigated.
Primary mouse hepatocytes were cultured. Protein levels were measured by Western blot or enzyme linked immunosorbent assay (ELISA), while confocal laser scanning microscope and flow cytometry were used to confirm the activation of pregnane X receptor (PXR). Carboxylesterase activity was evaluated by enzymatic and toxicological assays.
Elevated glucose (11 or 25 mM) significantly increased carboxylesterase expression compared to 5.6 mM glucose. Carboxylesterase expression and activity were inhibited by LPS or IL-6 in 25 mM glucose, but stimulated in 5.6 mM glucose. The altered expression of carboxylesterases was not consistent with the activation of nuclear factor kappa B (NFκB) but repeatedly with the expression and activation of pregnane X receptor (PXR). The altered activation of PXR was further evidenced by the differential subcellular translocation and the expression of its target gene multidrug resistance 1 (MDR1). It implies that PXR, instead of inflammatory signaling, mediates the regulation of carboxylesterases by inflammatory mediators in different glucose concentrations.
The findings contribute to clarify the regulation of carboxylesterases by inflammatory mediators, and indicate that carboxylesterase-involved drug metabolism and drug–drug interactions in diabetes should be reevaluated according to the intensity of inflammatory reactions and hyperglycemia.
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Accumulating evidences have shown that diabetes upregulated the function and expression of CYP3A4, but the mechanism remained unclear. In this study, HepG2 cells were incubated with serum from diabetic rats induced by streptozotocin, and the activity of CYP3A4 was measured by substrate metabolism. Results showed that incubation with diabetic serum significantly induced CYP3A4 activity in HepG2 cells. To identify the specific factors contributing to the regulation, the abnormally altered components in diabetic serum, including glucose, insulin, cholesterol, and free fatty acids were screened. It was found that only fatty acids concentration-dependently up-regulated CYP3A4 activity, and the induction by fatty acids was further confirmed in Fa2N-4 cells. Data from western blotting and QT-PCR showed that induction of CYP3A4 activity was associated with up-regulation of CYP3A4 protein and mRNA levels. In addition, effects of pharmacological inhibitors on fatty acid–induced CYP3A4 activity were studied. The results indicated that the induction of CYP3A4 activity by oleic acid may be partly via AMPK-, PKC-, and NF-κB–dependent pathways, whereas that by palmitic acid was possibly associated with the PKC-dependent pathway. In conclusion, the increased levels of fatty acids may be one of the reasons leading to the elevated function and expression of CYP3A4 under diabetic conditions.
Chlorzoxazone, an SK-type potassium channel activator used in humans, reduces excessive alcohol intake in rats
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Prolonged alcohol exposure can upregulate CYP2E1 activity (19–21), including in the brain (22), which could decrease CZX concentrations in alcohol-drinking rats. However, alcohol can act as a moderate competitive inhibitor of CYP2E1 (23) and increase CZX peak concentrations, although this effect would be more pronounced across time and less evident in the first several hours (24–26). We have not examined CZX metabolism here, in part because repeated blood collection would likely disrupt ongoing alcohol intake.
Alcoholism imposes a tremendous social and economic burden. There are relatively few pharmacological treatments for alcoholism, with only moderate efficacy, and there is considerable interest in identifying additional therapeutic options. Alcohol exposure alters SK-type potassium channel (SK) function in limbic brain regions. Thus, positive SK modulators such as chlorzoxazone (CZX), a US Food and Drug Administration–approved centrally acting myorelaxant, might enhance SK function and decrease neuronal activity, resulting in reduced alcohol intake.
We examined whether CZX reduced alcohol consumption under two-bottle choice (20% alcohol and water) in rats with intermittent access to alcohol (IAA) or continuous access to alcohol (CAA). In addition, we used ex vivo electrophysiology to determine whether SK inhibition and activation can alter firing of nucleus accumbens (NAcb) core medium spiny neurons.
Chlorzoxazone significantly and dose-dependently decreased alcohol but not water intake in IAA rats, with no effects in CAA rats. Chlorzoxazone also reduced alcohol preference in IAA but not CAA rats and reduced the tendency for rapid initial alcohol consumption in IAA rats. Chlorzoxazone reduction of IAA drinking was not explained by locomotor effects. Finally, NAcb core neurons ex vivo showed enhanced firing, reduced SK regulation of firing, and greater CZX inhibition of firing in IAA versus CAA rats.
The potent CZX-induced reduction of excessive IAA alcohol intake, with no effect on the more moderate intake in CAA rats, might reflect the greater CZX reduction in IAA NAcb core firing observed ex vivo. Thus, CZX could represent a novel and immediately accessible pharmacotherapeutic intervention for human alcoholism.

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Research ArticlesPharmacokinetics of Chlorzoxazone in Rats with Diabetes: Induction of CYP2E1 on 6-Hydroxychlorzoxazone Formation

Abstract

Section snippets

INTRODUCTION

Chemicals

Measurement of Vmax, Km, and CLint for the Formation of OH-CZX in Hepatic Microsomes

DISCUSSION

Life Sci

J Phram Sci

J Pharm Sci

Life Sci

Biochem Pharmacol

Biochem Pharmacol

Eur J Pharm Sci

Anal Biochem

J Chromatogr B

Methods Enzymol

Biochem Pharmacol

J Pharm Sci

J Pharm Sci

Eur J Pharm Sci

Physiological disposition and metabolic fate of chlorzoxazone (Paraflex) in man

J Pharmacol Exp Ther

Effect of the acute-phase response on the pharmacokinetics of chlorzoxazone and cytochrome P-450 2E1 in vitro activity in rats

Drug Metab Dispos

Research Articles
Pharmacokinetics of Chlorzoxazone in Rats with Diabetes: Induction of CYP2E1 on 6-Hydroxychlorzoxazone Formation

Measurement of V_max, K_m, and CL_int for the Formation of OH-CZX in Hepatic Microsomes