Reductive metabolism of the anticonvulsant agent zonisamide, a 1,2-benzisoxazole derivative.

1. The metabolism of zonisamide in vitro was characterized through aerobic and anaerobic incubations with rat liver subcellular fractions and cultured gastrointestinal microflora. 2. Zonisamide reacted with rat hepatic microsomal cytochrome P-450 and exhibited a Type I binding spectrum. 3. Metabolism of zonisamide in vitro by hepatic subcellular fractions and cultured gastrointestinal flora produced a single metabolite, 2-(sulphamoylacetyl)-phenol (2-SMAP), by reductive cleavage of the 1,2-benzisoxazole ring. 4. The reductive metabolism of zonisamide was primarily mediated by microsomal cytochrome P-450. The soluble fraction enhanced reduction when combined with the microsomal fraction but itself possessed only weak reductive activity. 5. Reduction of zonisamide by the most enzymically active liver fractions required NADPH, was stimulated by FMN and SKF-525A, and was inhibited by CO or air, as well as by n-octylamine. 6. Unlike their involvement in the reduction of numerous nitro, azo, and N-oxide compounds, cultured aerobic and anaerobic intestinal flora were not principally involved in the reduction of zonisamide.     

Preclinical abuse potential assessment of the anticonvulsant zonisamide

Zonisamide (Zonegran^®) is a broad‐spectrum antiepileptic agent that shares some pharmacological properties with other anticonvulsants, including phenytoin, carbamazepine, and valproic acid, but is differentiated from these agents by the ability to significantly block T‐type calcium channels. Zonisamide interacts with the γ‐amino‐butyric acid (GABA) receptor in an allosteric manner, and thus does not modulate GABA receptor effects. However, given the potential of drugs within the latter class for drug abuse in humans, an evaluation of zonisamide for abuse potential is an important component of its potential side‐effect profile. In the present study, zonisamide was tested in animal models of the subjective and reinforcing effects of central nervous system (CNS) depressant drugs, e.g., diazepam discrimination in rats and intravenous self‐administration in rhesus monkeys, respectively. In addition, zonisamide was evaluated for physical dependence liability in a chronic infusion model using rats. Zonisamide did not substitute for diazepam in rats trained to discriminate 2.5‐mg/kg diazepam from vehicle nor was it self‐administered by rhesus monkeys experienced in methohexital‐reinforced responding. Continuous infusion of zonisamide (400 or 600 mg/kg/day) did not prevent the loss of body weight associated with discontinued pentobarbital infusion. These doses of zonisamide did produce some incomplete attenuation of observable signs of pentobarbital withdrawal, likely due to direct sedative or depressant effects of these high doses. These results suggest that zonisamide would not produce diazepam‐like intoxication in humans nor would it likely be subject to abuse when made more widely available. Further, when administered chronically, zonisamide would not be expected to produce physical dependence of the CNS depressant type. Taken together, these results support the prediction that zonisamide would have low abuse liability. Drug Dev. Res. 54:66–74, 2001. © 2001 Wiley‐Liss, Inc.     

Metabolism of the hypolipidemic agent tiadenol in man and in the rat

The metabolism of the hypolipidemic agent 1,10-bis(hydroxyethylthio)decane (tiadenol, Eulip) has been studied in vivo in man and in the rat and in vitro in the rat. Following oral administration, in both species tiadenol was completely absorbed, extensively metabolized by the liver and more than 95% of the dose was eliminated in this form via kidneys within 48 h. Insignificant was the excretion of the unchanged drug in urine (approximately 1%) as well as that of its metabolites in the feces. 8 metabolites were isolated from human or rat urine and their structures were elucidated by means of electron impact, field desorption and positive and negative fast atom bombardment mass spectrometry. Both in man and in the rat the main metabolic pathway was the oxidation of the thioether sulfur, followed by oxidation or conjugation of the primary alcohol group(s). The urinary excretion of S-oxidized metabolites and sulfoxidized carboxylic metabolites accounted for 75% of the dose and that of S-oxidized conjugated metabolites for 20%. Rat in vitro studies showed that hepatic microsomal cytochrome P-450-dependent monooxygenase catalyzes the S-oxidative pathway, which governs the in vivo elimination of the drug in both species. Thus cytochrome P-450 is the key enzyme in the hepatic detoxification of tiadenol.     

Metabolism of the neuroleptic agent zetidoline in the rat and the dog

The metabolism of zetidoline, a new neuroleptic, in the rat and the dog has been studied. From the urine of rats and dogs given 5 mg/kg of [2-14C] zetidoline orally, unchanged drug and five metabolites were isolated and the structures of four of them assigned by physicochemical analysis. They are: metabolite B, 4'-hydroxy-3'-chlorophenyl zetidoline; metabolite D, zetidoline without the aryl group; metabolite E, the 6'-hydroxy-4'-beta-D-glucuronide of metabolite B, and metabolite F, the 4'-beta-D-glucuronide of metabolite B. The plasma levels of zetidoline and its metabolites after iv administration show that the drug is rapidly excreted and/or metabolized in both animal species. The plasma radioactivity in the dog consists mainly of the pharmacologically active (neuroleptic) metabolite B, whereas in the rat it consists of the more polar metabolites. After oral administration, elimination in both species occurs mostly via the kidneys. In the dog, within a 24-hr period, 6.2 +/- 0.4% of the dose is accounted for as unchanged zetidoline, 7.6 +/- 0.5% as metabolite B, 10.1 +/- 0.7% as the unidentified metabolite C, and 21.4 +/- 1.1% as metabolite F. In the rat, over the same period, zetidoline is present in traces, metabolite B accounts for 6.9 +/- 0.3% of the dose, metabolite D for 6.6 +/- 0.9%, metabolite E for 15.2 +/- 1.4%, and metabolite F for 31.7 +/- 2.2%.     

Effect of immobilization stress on anticonvulsant actions and pharmacokinetics of zonisamide in mice

The effects of immobilization stress on anticonvulsant actions and pharmacokinetics of zonisamide were investigated in mice. Oral administrations of zonisamide (10, 20, and 50 mg/kg) dose-dependently reduced incidence of tonic extension (TE) induced by maximal electroshock seizure (MES). Immobilization stress for 2 h immediately after the administration of zonisamide further enhanced the anticonvulsive actions of it. On the other hand, the serum zonisamide concentrations in stressed group were lower during the first 30 min after the administration compared with that in nonstressed control group. Thereafter, there were no significant differences in the serum concentrations between two groups. The brain zonisamide concentration and the concentration ratio of brain/serum at 2 h after administration of zonisamide (50 mg/kg) were significantly higher in stressed group, rather than that in the nonstressed control group without changing the serum concentration. These results suggest that immobilization stress enhances anticonvulsant actions of zonisamide, and that increases of brain zonisamide concentration by immobilization stress may be related with this phenomenon.     

Metabolism of antiparkinson agent dopazinol by rat liver microsomes

Metabolism of dopazinol (DZ) by liver microsomes from control and phenobarbital- and 3-methylcholanthrene-treated rats has been investigated. Liver microsomes from control and treated rats metabolized DZ to N-despropyl-DZ (39-53% of total metabolites); 8-hydroxy-DZ, a catechol metabolite (32-39%); and 5- or 6-hydroxy-DZ (12-20%). The last metabolite was identified as its dehydration product 5,6-dehydro-DZ. N-Dealkylation was favored only slightly over catechol formation (ratio = 1.2) by liver microsomes from control and phenobarbital-treated rats, whereas with liver microsomes from 3-methylcholanthrene-treated rats, N-dealkylation predominated (ratio = 1.7). Liver microsomes from control rats metabolized DZ at a rate of 0.86 nmol/nmol cytochrome P-450/min. Pretreatment of rats with phenobarbital or 3-methylcholanthrene stimulated rates of metabolism by 2.4- and 3-fold, respectively. Metabolism of DZ was inhibited by SKF 525-A, methimazole, and thiobenzamide. SKF 525-A completely inhibited metabolism of DZ, while methimazole and thiobenzamide, two alternate substrates of the microsomal flavin-containing monooxygenase (MFMO) inhibited N-dealkylation only. These results indicated that while the cytochrome P-450-dependent monooxygenase is the primary enzyme system in DZ oxidation, the MFMO also catalyzes the N-dealkylation reaction. The catechol metabolite was converted to isomeric O-methylated derivatives in approximately 1:1 ratio by purified catechol-O-methyl transferase or 105,000g liver cytosol. The late eluting isomer was 8-methoxy-DZ.     

Metabolism of denopamine, a new cardiotonic agent, in the rat and dog

The metabolism of denopamine, (R)-(-)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol, a new, orally active, selectively inotropic cardiotonic agent, was studied in the rat and dog. Animals were given single oral doses of 5 mg/kg of denopamine labeled with 14C. Denopamine was metabolized in the rat and dog by several pathways including conjugation, side chain oxidation, and ring hydroxylation followed by O-methylation. Rats excreted the drug in the urine almost entirely as unchanged drug and its phenolic O-glucuronide whereas in the dog, the major metabolites were the phenolic O-glucuronide, the alcoholic O-glucuronide, and the phenolic O-sulfate of denopamine and the phenolic O-glucuronide of 3-methoxydenopamine. Demethylation, which has been shown to be a major metabolic pathway in man, and side chain oxidation were minor pathways in the rat and dog. Furthermore, a high degree of stereoselective resistance of the alcoholic O-glucuronide of denopamine to hydrolysis by beta-glucuronidase was observed.     

Pharmacokinetics of twelve anticonvulsant drugs in the rat

The pharmacokinetic data for 12 anticonvulsant drugs was evaluated after administration to laboratory rats. In all cases the half-life and elimination rate constants were significantly different from clinically-determinant values which suggests that pharmacokinetic parameters should be treated as species specific.     

Metabolism of the anti-psoriatic agent 5-methoxypsoralen in humans: comparison with rat and dog

1. Single oral doses of ₁₄C-5-methoxypsoralen (5-MOP) to human subjects (50 mg), rats (1 mg/kg) and dogs (1 mg/kg) were fairly well absorbed but subjected to extensive first-pass metabolism, at least in rat and human. Means of 62, 51 and 40% dose in urine and 31, 38 and 48% dose in faeces, were excreted by humans (during 5 days), rats (3 days) and dogs (1 day), respectively. In dogs, faecal ₁₄C was probably derived, in part, from biliary excreted material.

2. Total ₁₄C in human plasma reached peak concentrations after 2 h (mean 235 ng 5-MOP equivalent/ml) and declined relatively slowly, to about 60% of this value within 24 h. Unchanged 5-MOP was not detected in plasma using h.p.l.c. (< 5 ng/ml).

3. Tissue concentrations of ₁₄C were generally greater in dogs than rats and reached peak levels at 1 h in dogs but at 24 h in rats. Apart from liver and bile, dog tissue ₁₄C concentrations were lower than those in the corresponding plasma, whereas in rat they were lower only until the time of peak concentrations, after which they were generally greater.

4. 5-MOP was extensively metabolized in all three species. The major ₁₄C-components in human and dog urine were glucuronic acid conjugates, mainly of an arylacetic acid and arylalcohols, resulting from initial oxidative metabolism of the furan ring of 5-MOP. In rat, these metabolites were excreted mainly unconjugated. An unusual metabolite was formed by reduction of the lactone moiety of 5-MOP, probably by the gut flora, giving rise to an arylpropionic acid, excreted as a glucuronic acid conjugate in the urine of all three species.

5. Unchanged drug was a very minor component of human and rat plasma, but a major component of dog plasma. In all three species, circulating ₁₄C-metabolites were similar to those in the urine but were present mainly unconjugated. On the basis of these data, the metabolic fate of 5-MOP in humans was more similar to that in dog than to that in rat, although humans appeared to metabolize 5-MOP more rapidly than did dog.     

Novel potent anticonvulsant agent containing a tetrahydroisoquinoline skeleton

In our studies on the development of new anticonvulsants, we planned the synthesis of N-substituted 1,2,3,4-tetrahydroisoquinolines to explore the structure-activity relationships. All derivatives were evaluated against audiogenic seizures in DBA/2 mice, and the 1-(4'-bromophenyl)-6,7-dimethoxy-2-(piperidin-1-ylacetyl) derivative (26) showed the highest activity with a potency comparable to that of talampanel, the only noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist in clinical trials as an anticonvulsant agent. Electrophysiological experiments indicated that 26 acts as noncompetitive AMPA receptor modulator.     

Metabolism of the anti-psoriatic agent 5-methoxypsoralen in humans: comparison with rat and dog.

1. Single oral doses of 14C-5-methoxypsoralen (5-MOP) to human subjects (50 mg), rats (1 mg/kg) and dogs (1 mg/kg) were fairly well absorbed but subjected to extensive first-pass metabolism, at least in rat and human. Means of 62, 51 and 40% dose in urine and 31, 38 and 48% dose in faeces, were excreted by humans (during 5 days), rats (3 days) and dogs (1 day), respectively. In dogs, faecal 14C was probably derived, in part, from biliary excreted material. 2. Total 14C in human plasma reached peak concentrations after 2 h (mean 235 ng 5-MOP equivalent/ml) and declined relatively slowly, to about 60% of this value within 24 h. Unchanged 5-MOP was not detected in plasma using h.p.l.c. (< 5 ng/ml). 3. Tissue concentrations of 14C were generally greater in dogs than rats and reached peak levels at 1 h in dogs but at 24 h in rats. Apart from liver and bile, dog tissue 14C concentrations were lower than those in the corresponding plasma, whereas in rat they were lower only until the time of peak concentrations, after which they were generally greater. 4. 5-MOP was extensively metabolized in all three species. The major 14C-components in human and dog urine were glucuronic acid conjugates, mainly of an arylacetic acid and arylalcohols, resulting from initial oxidative metabolism of the furan ring of 5-MOP. In rat, these metabolites were excreted mainly unconjugated. An unusual metabolite was formed by reduction of the lactone moiety of 5-MOP, probably by the gut flora, giving rise to an arylpropionic acid, excreted as a glucuronic acid conjugate in the urine of all three species. 5. Unchanged drug was a very minor component of human and rat plasma, but a major component of dog plasma. In all three species, circulating 14C-metabolites were similar to those in the urine but were present mainly unconjugated. On the basis of these data, the metabolic fate of 5-MOP in humans was more similar to that in dog than to that in rat, although humans appeared to metabolize 5-MOP more rapidly than did dog.     

Metabolism of tetrachlorophenols in the rat

The three isomers of tetrachlorophenol were administrated intraperitoneally to rats and the urinary excretion products studied. Tetrachloro-p-hydroquinone was identified as a major metabolite of 2,3,5,6-tetrachlorophenol, constituing about 35% of the dose given. Trichloro-p-hydroquinone was identified as a minor metabolite of both 2,3,4,5- and 2,3,4,6-tetrachlorophenol. 2,3,5,6-tetrachlorophenol was eliminated within 24 h, 2,3,4,6-tetrachlorophenol within 48 h while only 60% of the given dose of 2,3,4,5-tetrachlorophenol could be recovered within 72 h.The acute toxicity of the tetrachlorophenols and tetrachloro-p-hydroquinone was studied in mice upon oral and intraperitoneal administration. 2,3,5,6-tetrachlorophenol (LD₅₀ p.o. 109 mg · kg^–1) was the most toxic compound followed y 2,3,4,6- and 2,3,4,5-tetrachlorophenol (LD₅₀ p.o. 131 and 400 mg · kg^–1, respectively). Tetrachloro-p-hydroquinone proved to have low oral toxicity (LD₅₀ p.o. 500 mg · kg^–1) but was more toxic than the tetrachlorophenols when administered intraperitoneally. The oral LD₅₀ for pentachlorophenol, under identical experimental conditions, was found to be 74 mg · kg^–1.

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Zusammenfassung Die drei Isomeren von Tetrachlorphenol wurden an Ratten intraperitoneal verabreicht und die Ausscheidungsprodukte in Harn untersucht. Tetrachlor-p-hydrochinon erwies sich als Hauptmetabolit von 2,3,5,6-Tetrachlorphenol, entsprechend etwa 35% der zugeführten Menge. Bei sowohl 2,3,4,5-als auch 2,3,4,6-Tetrachlorphenol wurde Trichlor-p-hydrochinon als Metabolit identifiziert, jedoch in geringer Menge. Das 2,3,5,6-Tetrachlorphenol wurde innerhalb von 24 Std und das 2,3,4,6-Tetrachlorphenol innerhalb von 48 Std eliminiert, während bei 2,3,4,5-Tetrachlorphenol innerhalb von 72 Std nur 60% der gegebenen Menge erfaßt werden konnte.Die akute Toxizität von Tetrachlorphenolen und Tetrachlor-p-hydrochinon wurden an Mäusen oral sowie intraperitoneal studiert. 2,3,5,6-Tetrachlorphenol erwies sich als die toxischste der Substanzen (LD₅₀ p.o. 109 mg · kg^–1), gefolgt von 2,3,4,6- und 2,3,4,5-Tetrachlorphenol (LD₅₀ p.o. 131 mg · kg^–1 und 400 mg · kg^–1). Tetrachlor-p-hydrochinon zeigte nur geringe Toxizität, wenn oral verabreicht (LD₅₀ 500 mg · kg^–1), war aber toxischer als die Tetrachlorphenole, wenn intraperitoneal gegeben.

     

Metabolism of aminoglutethimide in the rat

The metabolism of aminoglutethimide was studied in the rat by use of the 14C-labeled compound. Following oral doses of 5 and 50 mg/kg, the drug was almost completely eliminated within 48 hr into urine and feces, mostly in the form of metabolites. In bile duct-cannulated rats, biliary excretion of radioactivity amounted to about 52% within 24 hr of an orally administered 50 mg/kg dose, with the remainder of the dose being eliminated into urine. The major urinary metabolites resulted from acetylation of the aniline moiety, hydroxylation of the glutarimide ring at positions 3 and 4, and oxidative elimination of the ethyl sidechain. The polar metabolites are accounted for by aromatic hydroxylation with subsequent sulfate conjugation and by a glutarimide ring-opened compound. In addition, a gamma-butyrolactone derivative was also identified.     

Cannabidivarin is anticonvulsant in mouse and rat

Background and Purpose

Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and several exert notable anticonvulsant effects. For the first time, we investigated the anticonvulsant profile of the phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models.

Experimental Approach

The effect of CBDV (1–100 μM) on epileptiform local field potentials (LFPs) induced in rat hippocampal brain slices by 4-aminopyridine (4-AP) application or Mg²⁺-free conditions was assessed by in vitro multi-electrode array recordings. Additionally, the anticonvulsant profile of CBDV (50–200 mg·kg⁻¹) in vivo was investigated in four rodent seizure models: maximal electroshock (mES) and audiogenic seizures in mice, and pentylenetetrazole (PTZ) and pilocarpine-induced seizures in rats. The effects of CBDV in combination with commonly used antiepileptic drugs on rat seizures were investigated. Finally, the motor side effect profile of CBDV was investigated using static beam and grip strength assays.

Key Results

CBDV significantly attenuated status epilepticus-like epileptiform LFPs induced by 4-AP and Mg²⁺-free conditions. CBDV had significant anticonvulsant effects on the mES (≥100 mg·kg⁻¹), audiogenic (≥50 mg·kg⁻¹) and PTZ-induced seizures (≥100 mg·kg⁻¹). CBDV (200 mg·kg⁻¹) alone had no effect against pilocarpine-induced seizures, but significantly attenuated these seizures when administered with valproate or phenobarbital at this dose. CBDV had no effect on motor function.

Conclusions and Implications

These results indicate that CBDV is an effective anticonvulsant in a broad range of seizure models. Also it did not significantly affect normal motor function and, therefore, merits further investigation as a novel anti-epileptic in chronic epilepsy models.

Linked Articles

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8