Cases of Kava Impairment in Iowa Drivers.

Kava is an Oceanic plant in which the root is consumed as a beverage and is becoming increasingly popular. The effects of kava consumption may include sedation, euphoria, and impairment of motor coordination. This article demonstrates kava impairment through four cases of self-reported kava use supported with Drug Recognition Expert (DRE) evaluations of each subject. Subject's urines screened negative for common drugs of abuse by immunoassay analysis. Urine from cases 3 and 4 were analyzed by liquid chromatography-tandem mass spectrometry, and gas chromatography-mass spectrometry, which yielded the presence of kavalactones. Subjects exhibited poor driving behavior and signs of intoxication. Indicators of impairment from multiple drug categories, central nervous system (CNS) depressants, CNS stimulants, and cannabis were observed, which may be consistent with the presence of multiple kavalactones and their diverse array of mechanisms of action. The consumption of kava can hinder one's ability to operate a vehicle safely.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: urinary organic acid profiles and expanded spectrum of mutations.

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (HMCS2) deficiency results in episodes of hypoglycemia and increases in fatty acid metabolites. Metabolite abnormalities described to date in HMCS2 deficiency are nonspecific and overlap with other inborn errors of metabolism, making the biochemical diagnosis of HMCS2 deficiency difficult. Urinary organic acid profiles from periods of metabolic decompensation were studied in detail in HMCS2-deficient patients from four families. An additional six unrelated patients were identified from clinical presentation and/or qualitative identification of abnormal organic acids. The diagnosis was confirmed by sequencing and deletion/duplication analysis of the HMGCS2 gene. Seven related novel organic acids were identified in urine profiles. Five of them (3,5-dihydroxyhexanoic 1,5 lactone; trans-5-hydroxyhex-2-enoate; 4-hydroxy-6-methyl-2-pyrone; 5-hydroxy-3-ketohexanoate; 3,5-dihydroxyhexanoate) were identified by comparison with synthesized or commercial authentic compounds. We provisionally identified trans-3-hydroxyhex-4-enoate and 3-hydroxy-5-ketohexanoate by their mass spectral characteristics. These metabolites were found in samples taken during periods of decompensation and normalized when patients recovered. When cutoffs of adipic >200 and 4-hydroxy-6-methyl-2-pyrone >20 µmol/mmol creatinine were applied, all eight samples taken from five HMCS2-deficient patients during episodes of decompensation were flagged with a positive predictive value of 80% (95% confidence interval 35-100%). Some ketotic patients had increased 4-hydroxy-6-methyl-2-pyrone. Molecular studies identified a total of 12 novel mutations, including a large deletion of HMGCS2 exon 1 in two families, highlighting the need to perform quantitative gene analyses. There are now 26 known HMGCS2 mutations, which are reviewed in the text. 4-Hydroxy-6-methyl-2-pyrone and related metabolites are markers for HMCS2 deficiency. Detection of these metabolites will streamline the biochemical diagnosis of this disorder.

AFN-1252 in vitro absorption studies and pharmacokinetics following microdosing in healthy subjects.

OBJECTIVES: To investigate the absorption, distribution, metabolism and excretion of AFN-1252, a novel inhibitor of the essential FabI enzyme in Staphylococcus spp., in vitro and following microdosing in healthy adult male subjects following intravenous and oral administration. METHODS: Three ADME studies, comprising a Caco-2 assay, a rat intestinal perfusion model and a microdosing study in healthy human volunteers, were conducted. RESULTS: The Caco-2 assay indicated that AFN-1252 in solution is well-absorbed and undergoes insignificant efflux, and its transport across the intestinal wall is probably passive. In the rat intestinal perfusion model, AFN-1252 exhibited high permeability potential across three segments, in the rank order of jejunum=ileum>colon. Taken together with the low aqueous solubility, the data from these studies indicate that AFN-1252 is a BCS Class II molecule with solubility-limited absorption. Analysis of the [(14)C]-AFN-1252 radioactivity concentration-time data indicated similar pharmacokinetics following intravenous and oral administration in the microdosing study in healthy volunteers. These included long terminal half-lives of ∼7 h and 83% bioavailability, indicating that there was little first-pass metabolism following oral dosing. AFN-1252 exhibited good distribution to skin and skin structures where its anti-staphylococcal activity may be required. Urinary and faecal excretion are major elimination routes for [(14)C]-AFN-1252 following intravenous or oral administration. CONCLUSIONS: AFN-1252 has the potential for both intravenous and oral administration, once- or twice-daily dosing and good tissue distribution in humans. Further safety, efficacy and pharmacokinetic studies in man are required to investigate therapeutically-relevant doses for this novel agent and its targeted selectivity and high potency against Staphylococcus spp.

Determination of novel nitrogen-containing metabolite after oral administration of swertiamarin to rats.

In a series of studies on the metabolism of iridoid compounds, we investigated the metabolic fate of swertiamarin (1) in Wistar rats. Liquid chromatography/ion trap mass spectrometry detected new nitrogen-containing metabolite gentiandiol (3) in rat plasma. The structure of the metabolite was unequivocally identified by comparing the retention time as well as the mass spectrum with those of authentic compound, which was synthesized from swertiamarin (1). The transformation of swertiamarin to nitrogen-containing metabolite gentiandiol (3) in vivo was verified for the first time. Understanding of this unique metabolic pathway may shed light on clinical efficacy of swertiamarin (1) and will also assist in studies for the metabolism of other natural iridoids in vivo.

Metabolites of the ellagitannin geraniin and their antioxidant activities.

Different types of ellagitannins are reported to have various biological activities, such as antioxidant, antiviral, and antitumor activities. However, there are few definitive studies on the absorption and metabolism of ellagitannins. This review compares the absorption and metabolism of ellagitannins, and the antioxidant properties of their metabolites in rats, with those of intact ellagitannins by means of IN VITRO and IN VIVO assays. We isolated 7 urinary and intestinal microbial metabolites in rats after the ingestion of geraniin, which is a typical ellagitannin isolated from GERANIUM THUNBERGII, an antidiarrheic remedy in Japan. The structures of these metabolites were determined to be dibenzopyran derivatives ( 1- 7), using NMR and mass spectroscopic data. Four major metabolites ( 1- 4) prepared by chemical synthesis were evaluated for their antioxidant activities by using 2,2-diphenyl-1-picrylhydrazyl radical scavenging and oxygen radical absorbance capacity (ORAC) methods. The metabolites exhibited more potent antioxidant activities in the ORAC assay than intact ellagitannins, such as geraniin and corilagin. Furthermore, plasma ORAC scores increased with increases in the plasma concentration of the metabolites after the oral administration of geraniin to rats. These findings suggest that these metabolites may contribute to the health benefits of ellagitannins as antioxidants in the body.

Characterization of cytochrome P450-mediated bioactivation of a compound containing the chemical scaffold, 4,5-dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide), to a chemically reactive p-chlorophenyl isocyanate intermediate in human liver microsomes.

Compound A (Cmpd A) was previously reported to form p-chlorophenyl isocyanate (CPIC), which was trapped by GSH to yield S- (N- [p-chlorophenyl] carbamoyl) glutathione adduct (SCPG) in the presence of human liver microsomes. In this study, P450 3A4 and 2C9 were demonstrated to be the enzymes mediating the activation of Cmpd A to CPIC in human liver microsomes based on inhibitory and correlation studies. Enzyme kinetics studies indicated that P450 3A4 was the primary enzyme involved in the activation of Cmpd A. In silico P450 3A4 active site docking of Cmpd A exhibited a low energy pose that orientated the pyrazole ring proximate to the heme iron atom, in which the distance between the C-3 and potential activated oxygen species was shown to be 3.4 A. Quantum molecular calculations showed that the electron density on C-3 was relatively higher than those on C-4 and C-5. These measurements suggested that the C-3 of Cmpd A was the preferred site of oxidation and hence predisposed Cmpd A in forming CPIC as previously proposed. The in silico prediction was corroborated by studies with the C-3 substituted analogue (methyl at C-3), which showed minimal conversion to CPIC in human liver microsomes. These results demonstrated a pivotal role for P450 3A4 in bioactivating Cmpd A by oxidizing at C-3 of the pyrazoline, hence facilitating the CPIC formation. Evidence of the bioactivation to CPIC in vivo was obtained by liquid chromatography-mass spectrometry (LC/MS) analysis of urine samples from human subjects administered a structural analogue of Cmpd A. The presence of S-(N-[p-chlorophenyl] carbamoyl) N-acetyl l-cysteine (SCPAC) as well as p-chlorophenyl aniline (CPA) was unequivocally demonstrated in the urine samples. The chemical scaffold, 4,5-dihydropyrazole-1-carboxylic acid-[(4-chlorophenyl)-amide], was demonstrated to possess potential metabolic liability in forming a reactive intermediate, CPIC, in humans. Bioactivation to CPIC may cause undesirable side effects through its reactivity and subsequent conversion to CPA, an established rodent carcinogen.

Steady-state disposition of the nonpeptidic protease inhibitor tipranavir when coadministered with ritonavir.

The pharmacokinetic and metabolite profiles of the antiretroviral agent tipranavir (TPV), administered with ritonavir (RTV), in nine healthy male volunteers were characterized. Subjects received 500-mg TPV capsules with 200-mg RTV capsules twice daily for 6 days. They then received a single oral dose of 551 mg of TPV containing 90 microCi of [(14)C]TPV with 200 mg of RTV on day 7, followed by twice-daily doses of unlabeled 500-mg TPV with 200 mg of RTV for up to 20 days. Blood, urine, and feces were collected for mass balance and metabolite profiling. Metabolite profiling and identification was performed using a flow scintillation analyzer in conjunction with liquid chromatography-tandem mass spectrometry. The median recovery of radioactivity was 87.1%, with 82.3% of the total recovered radioactivity excreted in the feces and less than 5% recovered from urine. Most radioactivity was excreted within 24 to 96 h after the dose of [(14)C]TPV. Radioactivity in blood was associated primarily with plasma rather than red blood cells. Unchanged TPV accounted for 98.4 to 99.7% of plasma radioactivity. Similarly, the most common form of radioactivity excreted in feces was unchanged TPV, accounting for a mean of 79.9% of fecal radioactivity. The most abundant metabolite in feces was a hydroxyl metabolite, H-1, which accounted for 4.9% of fecal radioactivity. TPV glucuronide metabolite H-3 was the most abundant of the drug-related components in urine, corresponding to 11% of urine radioactivity. In conclusion, after the coadministration of TPV and RTV, unchanged TPV represented the primary form of circulating and excreted TPV and the primary extraction route was via the feces.

Kinetics of kavain and its metabolites after oral application.

Kavain metabolism in humans was the target of this current investigation. In the present study a high-performance liquid chromatographic (HPLC-DAD) assay method for the simultaneous determination of kavain and its main metabolites (p-hydroxykavain, p-hydroxy-5,6-dehydrokavain and p-hydroxy-7,8-dihydrokavain) in serum and urine was developed and validated. The metabolites were mainly excreted in the form of their conjugates. All kavain metabolites were detectable in serum and urine, except for p-hydroxy-7,8-dihydrokavain, which was found in urine only. Confirmation of the results and identification of the metabolites were performed by LC-MS or LC-MS-MS. Kinetics of kavain and its metabolites in serum were investigated after administration of a single oral dose (800 mg kavain). Within 1 and 4 h after uptake, the serum concentrations ranged between 40 and 10 ng/ml for kavain, 300 and 125 ng/ml for p-hydroxykavain, 90 and 40 ng/ml for o-desmethyl-hydroxy-5,6-dehydrokavain, and 50 and 30 ng/ml for 5,6-dehydrokavain.

Microbial transformation of kawain and methysticin.

The styryl alpha-pyrones, d-kawain (1) and d-methysticin (2) are two of the major kavalactone constituents of the anxiolvtic herb Piper methysticum, commonly known as kava. The use of fungal models to mimic the mammalian metabolism of 1 resulted in the production of 4'-hydroxykawain (1a) from the culture broth of Cunninghamella elegans (ATCC 9245), the same metabolite identified in rat urine. The fungus Torulopsis petrophilum (ATCC 20225) biotransformed 2 to 3'-hydroxy-4'-methoxykawain (2c) which is analogous, but not identical, to a known rat metabolite of methysticin.

Mass spectral characterization of urinary metabolites of D,L-kawain.

The urinary metabolism of D,L-kawain was studied in humans after an oral dose of 200 mg. Ten metabolites of kawain could be identified by gas chromatography-mass spectrometry with electron impact and chemical ionization. The main metabolic pathways were hydroxylation of the phenyl ring, reduction of the 7,8-double bond, hydroxylation of the lactone ring with subsequent dehydration and opening of the lactone ring. The metabolites were mainly excreted in the form of their conjugates.

Metabolism of some kava pyrones in the rat.

1. The metabolism in rats of several kava pyrones from Piper methysticum Forst. was studied. The compounds were the 5,6-dihydro-alpha-pyrones, dihydrokawain, kawain and methysticin, and the alpha-pyrones, 7,8-dihyroyangonin and yangonin. 2. Approx. half the dose (400 mg/kg, p.o.) of dihydrokawain was found in the urine in 48 h. About two-thirds of this was hydroxylated metabolites (three mono- and three di-hydroxylated derivatives), of which p-hydroxydihydrokawain was the most abundant. The remaining third consisted of metabolites formed by scission of the 5,6-dihydro-alpha-pyrone ring and included hippuric acid (9--13% dose). 3. Lower amounts of urinary metabolites were excreted when kawain was given, but both hydroxylated and ring-opened products were formed. 4. Methysticin gave rise to only small amounts of two urinary metabolites formed by demethylenation of the methylenedioxyphenyl moiety. 5. Urinary metabolites of the alpha-pyrones, 7,8-dihydroxyangonin and yangonin, were formed via omicron-demethylation. No ring-opened products were detected. 6. These lipophilic kava pyrones have extremely low solubility in water, which would be expected to reduce their absorption rates and appears to be responsible for the variable and low extent of metabolism observed.