Irreversible inhibition of CYP2D6 by (-)-chloroephedrine, a possible impurity in methamphetamine.

(+)- and (-)-Chloroephedrine, and their respective aziridines, cis- and trans-1,2-dimethyl-3-phenylaziridine, have been reported present in clandestinely synthesized methamphetamine. Since methamphetamine and structurally related compounds are potential substrates for human liver CYP2D6, the possible interaction of the chloroephedrines with human liver CYP2D6 was evaluated. Computational methods (using Flexidock and HINT in SYBYL) were used to determine the feasibility of (+)- or (-)-chloroephedrine and cis- or trans-1,2-dimethyl-3-phenylaziridine binding in the active site of a three dimensional CYP2D6 molecular model. Although modeling indicates both (+)- and (-)-chloroephedrine would bind comparably to methamphetamine, the binding energies of cis- or trans-1,2-dimethyl-3-phenylaziridine products indicate a preference for trans-1,2-dimethyl-3-phenylaziridine, the product formed from (-)-chloroephedrine. The effects of (+)- and (-)-chloroephedrine on the metabolism of dextromethorphan in human liver microsomes were then experimentally evaluated. (+)-Chloroephedrine (50 micro M) had no effect on human CYP2D6. (-)-Chloroephedrine appeared to be selective for human CYP2D6 versus CYP1A2 and CYP3A4/5. The inhibition of CYP2D6 was time-dependent, not dependent on metabolic activation, and irreversible. It appeared to bind at the active site of CYP2D6 with an apparent K(i) of 226 micro M, with a k(int) of 0.039 min(-1), and a t(1/2) of 23 min. Due to the irreversible nature of this inhibition, this impurity in clandestinely synthesized methamphetamine may be important and warrant further study.

Chloroephedrine: contaminant of methamphetamine synthesis with cardiovascular activity.

Chloroephedrine is an intermediate and possible contaminant formed when methamphetamine is manufactured using ephedrine or pseudoephedrine as precursors. The purpose of this study was to determine whether this contaminant has biological activity and might contribute to methamphetamine-induced cardiovascular toxicity. In conscious rats, the (-) and (+) isomers of chloroephedrine (0.1 and 1.0 mg/kg, i.v.) dose-dependently increased mean arterial pressure (MAP) and heart rate (HR). The potency of the pressor effects of (-) and (+)-chloroephedrine was between that of ephedrine and pseudoephedrine. The increases in HR elicited by the four stimulants were similar except that the tachycardia elicited by all doses of ephedrine and pseudoephedrine were preceded by a brief decrease in HR. The i.v. administration of 10 mg/kg of (+) or (-)-chloroephedrine produced biphasic (decrease followed by increase) the MAP and HR responses. Ephedrine and pseudoephedrine did not decrease MAP at any dose tested. The initial decrease in HR elicited by (-)-chloroephedrine was significantly reduced and the hypotensive response abolished by atropine, indicating that these components of the MAP and HR responses resulted from vagal activation. The secondary pressor response elicited by (-)-chloroephedrine was significantly reduced and the tachycardia significantly increased by pretreatment with phentolamine (3 mg/kg, i.v.). The increase in HR was reversed by propranolol. These results indicate that (-) and (+)-chloroephedrine have sympathomimetic properties similar to other known sympathomimetic stimulants. In addition, larger doses of chloroephedrine can activate the vagus nerve. The combination of (+)-methamphetamine and (-)-chloroephedrine did not markedly alter the magnitude of the MAP and HR responses of (+)-methamphetamine alone except at high doses of (-)-chloroephedrine (10 mg/kg). Contamination of illicit methamphetamine with chloroephedrine may have toxic consequences.

Dragon's Blood incense: misbranded as a drug of abuse?

An unknown red substance was being sold and used with other drugs of abuse in Virginia (often being used in conjunction with marihuana). The red substance was identified as Dragon's Blood incense from Daemonorops draco. In bioassays, Dragon's Blood incense exhibited a low, but measurable cytotoxicity in in vitro cell lines. Dragon's Blood incense or Volatilized Dragon's Blood had no adverse effect on mouse motor performance based on the inclined screen and rotorod tests. delta(9)-Tetrahydrocannibinol (THC) produced a dose-related decline in mouse performance on the rotorod test. The combination of Dragon's Blood incense or Volatilized Dragon's Blood with delta(9)-THC did not contribute further to the impairment of the mice on the rotorod. This data suggests that the abuse potential for Dragon's Blood incense alone or in combination with marihuana is minimal.

HPLC analysis for amobarbital N-glycosides in urine.

A study was undertaken to determine if humans excrete both amobarbital N-glucuronides and N-glucosides in urine after an oral dose of amobarbital. Amobarbital N-glucuronides were synthesized and characterized. A reverse phase LC method using post-column pH adjustment and UV detection at 240 nm was developed and used for the quantification of the amobarbital N-glucosides and N-glucuronides in human urine. Amobarbital was administered orally to seven male subjects and the total urine was collected for a period of 48-53 h after dosing. After filtration, the urine was injected directly onto the HPLC column to analyze for the presence of metabolites. The previously identified (5S)-amobarbital N-glucoside was detected in all seven subjects. The (5R)-amobarbital N-glucoside was detected at lower concentrations in only four of the subjects. At the levels at which amobarbital N-glucosides were detected, there was no evidence for the formation and excretion of the amobarbital N-glucuronides. Amobarbital N-glucuronidation is not a quantitatively significant pathway for the biodisposition of amobarbital in humans.

High-performance liquid chromatographic analysis of phenobarbital and phenobarbital metabolites in human urine.

A HPLC assay using UV detection and post-column alkalinization was developed to quantify possible urinary excretion products of phenobarbital in human urine. After filtration the urine was injected directly onto the HPLC column for analysis of phenobarbital, p-hydroxyphenobarbital, phenobarbital N-glucosides and phenobarbital N-glucuronides. The accuracy and precision of the assay were within +/- 15% and the limit of detection (LOD) was 1 microM, suitable for pharmacokinetic studies. Phenobarbital was administered orally to five male subjects and urine was collected for a period of 96-108 h. Phenobarbital, p-hydroxyphenobarbital, and phenobarbital N-glucosides were detected and quantified in the urine of all five subjects. The phenobarbital N-glucuronides were not detected in the urine. This assay provides a rapid method with improved selectivity to analyze urine for phenobarbital and its metabolites.

alpha-Benzyl-N-methylphenethylamine (BNMPA), an impurity of illicit methamphetamine synthesis: I. Physical characterization and GC-MS analysis of BNMPA and anticipated metabolites in urine.

alpha-Benzyl-N-methylphenethylamine (BNMPA) is an impurity of illicit methamphetamine synthesis. We synthesized BNMPA and three of its anticipated metabolites: N-demethyl-alpha-benzyl-phenethylamine, 1,3-diphenyl-2-propanone, and 1,3-diphenyl-2-propanol. The purity and structure of these compounds and their heptafluorobutyric anhydride (HFBA) derivatives were confirmed by melting point, gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance. A GC-MS method to detect these compounds in urine, using liquid-liquid extraction and derivatization with HFBA, was developed. Interference studies showed BNMPA and its proposed metabolites to be well-resolved from other common phenethylamine drugs and Health and Human Services-Forensic Urine Drug Testing required analytes. The limit of detection of BNMPA and its metabolites was 2.5 ng/mL; the limit of quantitation (LOQ) of the four compounds was 25 ng/mL. The calibration curves were generally linear from 25 to 500 ng/mL. Typical within-run coefficients of variation (CVs) at the LOQ ranged from 13 to 20% (n = 8). Between-run CVs over 1 month at 25 ng/mL were from 9 to 28%, and at 500 ng/mL, they were from 2.6 to 3.9%. The detection of BNMPA or its metabolites in urine samples may provide a marker of use of illicitly synthesized methamphetamine.

Identification of phenobarbital N-glucuronides as urinary metabolites of phenobarbital in mice.

Mice were evaluated for their ability to form phenobarbital N-glucuronides. Following oral administration of [14C]phenobarbital to mice, a radiolabeled phenobarbital metabolite cochromatographed with synthetic standards of phenobarbital N-glucuronides. The phenobarbital N-glucuronides were partially purified from the mouse urine as phenobarbital N-methylglucuronates. The phenobarbital N-methylglucuronates isolated from mouse urine had similar chromatographic and spectroscopic properties as synthetic standards. The diastereomers of phenobarbital N-glucuronides and phenobarbital N-glucosides accounted for 7.8 +/- 2.3% and 1.6 +/- 0.6%, respectively, of the radioactivity excreted in mouse urine in the first 48 hr after dosing. This study indicates that the mouse may be a suitable species to study both N-glucosidation and N-glucuronidation simultaneously as metabolic pathways for barbiturates.

Synthesis of N-beta-D-glucopyranosyluronate derivatives of barbital, phenobarbital, metharbital, and mephobarbital.

The synthesis and characterization of barbital, phenobarbital, metharbital, and mephobarbital glucuronides is reported. The condensation of per(trimethylsilyl)-barbital and -phenobarbital with methyl 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranuronate in the presence of trimethylsilyl trifluoromethanesulfonate gave moderate yields of the N1-(beta-D-glucopyranosyluronate) barbiturate derivatives. The diastereomers of the phenobarbital derivatives were resolved by use of C18 reversed-phase HPLC. The homologous N3-methyl barbiturate N1-glucuronates were prepared by reaction of the barbital and phenobarbital N1-glucuronate derivatives with diazomethane. The absolute configuration of the phenobarbital N1-beta-D-glucopyranuronate epimers was determined by oxidative removal of the glycon from the mephobarbital N1-beta-D-glucopyranuronate epimers to give the optical isomers of mephobarbital. The spectroscopic data for this series of compounds will facilitate the characterization of N-glycosylated imide xenobiotics that may be detected as mammalian metabolites in biodisposition studies.

Identification of the diastereomers of pentobarbital N-glucosides excreted in human urine.

A study was undertaken to determine if humans excreted pentobarbital N-glucosides as urinary metabolites following oral administration of pentobarbital. (1'RS,5RS)-1-(beta-D-Glucopyranosyl)pentobarbital ((1'RS,5RS)-PTBG) was isolated from the urine of one subject. The two diastereomers, (1'RS,5R)-PTBG and (1'RS,5S)-PTBG were separated and found to be identical to synthetic standards when compared using HPLC retention times coupled with UV (with and without post-column ionization) and mass spectrometry (HPLC/MS). A HPLC method was developed for detecting and quantifying (1'RS,5R)-PTBG, (1'RS,5S)-PTBG and pentobarbital in urine. Following a single oral dose of sodium pentobarbital to male subjects (n = 6), 1.6-6.2% of the pentobarbital dose was excreted as (1'RS,5S)-PTBG over 60 hours. (1'RS,5R)-PTBG was also detected in one subject and accounted for 0.3% of the pentobarbital dose. Using a modified HPLC system, the four pentobarbital N-glucosides were resolved and analysis of a partially purified pentobarbital N-glucoside extract from one subject indicated that only (1'R,5R)-PTBG and (1'S,5S)-PTBG could be detected as urinary excretion products. These results indicate that the side chain chirality of pentobarbital may influence the observed enantioselectivity for the formation and/or urinary excretion of the pentobarbital N-glucosides.

Initial studies on the N-glucosylation of phenobarbital by mouse liver microsomes using a radiochemical high-performance liquid chromatographic (HPLC) method.

A method is described for the assay of phenobarbital N-glucosylation using UDP-D-[6-3H]glucose. The radioactive phenobarbital N-glucoside conjugates [(5R)-PBG, (5S)-PBG] formed during the incubations were resolved from each other and from uncharacterized radioactive products by semipreparative HPLC. The product ratio of the N-glucosides of (5R)-PBG/(5S)-PBG was 2.9 for the crude liver homogenate and 3.0 +/- 0.5 for the microsomes. Magnesium was necessary for optimal activity. The Km values for formation of (5R)-PBG, (5S)-PBG, and (5R + 5S)-PBG were 1.55 +/- 0.35, 1.27 +/- 0.14, and 1.47 +/- 0.21 mM, respectively. The Vmax values for formation of (5R)-PBG, (5S)-PBG, and (5R + 5S)-PBG were 1.34 +/- 0.05 x 10(-6), 0.43 +/- 0.01 x 10(-6), and 1.77 +/- 0.04 x 10(-6) mumol/min/mg microsomal protein, respectively. It was observed that at concentrations greater than 5 mM sodium phenobarbital, inhibition of formation of phenobarbital N-glucosides occurred. The product ratio of (5R)-PBG/(5S)-PBG is comparable to that observed in the urinary excretion studies with the mouse and opposite to that observed in urinary excretion studies in humans.

Identification of 5-ethyl-5-(2-methylbutyl)barbituric acid as an impurity of manufacture in amobarbital.

Amobarbital [5-ethyl-5-(3-methylbutyl)barbituric acid], USP, was found to contain an impurity that was not associated with hydrolysis and decomposition of the barbiturate ring. The impurity was isolated by semipreparative HPLC and was identified as 5-ethyl-5-(2-methylbutyl)barbituric acid (1) by MS (electron impact and chemical ionization) and 1H NMR. The substitution pattern on the alkyl side chain was verified by using the achiral NMR shift reagent tris(6,6,7,7,8,8,8-heptafluoro-2,2- dimethyl-3,5-octanedionato)europium(III). Older samples of amobarbital, USP, contained greater than 6% of 1, whereas recent samples of amobarbital, USP, contained less than 1% of 1. Because the pharmacological profiles of 1 and amobarbital in rodents are comparable, the impurity probably does not constitute a clinically significant problem for humans.

Differentiation of side chain isomers of ring-substituted amphetamines using gas chromatography/infrared/mass spectrometry (GC/IR/MS).

Common analytical methods used for identifying samples obtained from clandestine laboratories were evaluated for their ability to differentiate between possible amphetamine isomers and homologs. A series of ring-substituted (4-methyl, 4-methoxy, and 3,4-methylenedioxy) amphetamine and N-methylphenethylamine isomers was analyzed using color tests, thin-layer chromatography, gas chromatography/mass spectrometry (GC/MS) and GC/infrared (GC/IR). The N-acetyl derivatives of the isomers were analyzed using GC/IR/MS. GC/IR/MS readily differentiated the 4-methylphenylalkylamine isomers. MS and IR spectra were also obtained for each pair of the 4-methoxyphenylalkylamine isomers and the 3,4-methylenedioxyphenylalkylamine isomers, but differentiation via GC/IR/MS was difficult. The N-acetyl derivatives of each pair of isomers could be readily differentiated using GC/IR/MS. Good library researchable spectra for N-acetylamphetamine could be obtained for IR identification with 10 ng (on-column) and MS identification with 2 ng. The spectrometrically independent IR and MS data obtained for the N-acetyl derivatives indicated that the combination of GC/IR/MS can add a significant level of confidence in the analysis of ring-substituted arylalkylamines.

Identification of phenobarbital N-glucosides as urinary metabolites of phenobarbital in mice.

Previously, the N-glucosylation of phenobarbital had been observed only in humans. The results of a species screen (mouse, rat, guinea pig, rabbit, cat, dog, pig, and monkey) found that only mice excreted the N-glucosides of phenobarbital in urine after ip administration of sodium phenobarbital. The major diastereomer excreted by the mouse had the R configuration at the C-5 position of the barbiturate ring. The N-glucoside metabolites accounted for a small percentage of the dose (approximately 0.5%). Following ip dosing of the mouse with the phenobarbital N-glucosides, free phenobarbital could be detected in the urine. Upon ip or intercerebroventricular (icv) injection of the phenobarbital N-glucosides, minimal CNS activity was observed in the mouse.

Barbital N-glucoside is not detected as a urinary excretion product of barbital in humans.

A study was undertaken to determine if humans excreted barbital N-glucoside as a urinary metabolite following oral administration of barbital. A liquid chromatography method using gradient elution was developed for detecting and quantifying barbital N-glucoside and barbital in urine. Following a single oral dose of barbital to male caucasian and oriental subjects that had previously been shown to excrete amobarbital and phenobarbital N-glucosides, no barbital N-glucoside conjugate was observed in the urine. This result indicates that N-glucosylation of barbiturates is not a general pathway for the biodisposition of barbiturates in man.

Stereochemical characterization of the diastereomers of the amobarbital N-glucosides excreted in human urine.

The stereochemistry associated with the amobarbital N-glucoside diastereomers (1a and 1b) that are excreted by humans in urine is unknown. Using X-ray crystallography, the absolute configuration of 1b was determined to be S (C-5 position of the barbiturate ring). Following oral administration of amobarbital to Caucasians and Orientals, from 5 to 25% of the dose of amobarbital was excreted in the urine as 1b. The other diastereomer, 1a, accounted for less than 0.1 to 0.2% of the dose in four individuals, with none detected in nine individuals. The rate constants, kf,1b, determined from the urinary excretion of 1b were lower than those previously reported for unresolved amobarbital N-glucosides. However, based on the urinary excretion of 1b, the rate constants, K, for elimination of amobarbital in Caucasians and Orientals were similar to those previously determined from the serum levels of amobarbital and the urinary excretion of unresolved amobarbital N-glucosides. In previous studies of the N-glucosylation of amobarbital, it is likely that a single N-glucose diastereomer, 1b, was being observed.

Stereochemical characterization of the diastereomers of the phenobarbital N-beta-D-glucose conjugate excreted in human urine.

The absolute configuration of the N-beta-D-glucoside metabolites of phenobarbital was determined by methylation of the diastereomers to make mephobarbital N-beta-D-glucosides, followed by oxidative removal of glucose to give the optical isomers of mephobarbital. Following a single oral dose of phenobarbital to two male subjects, both phenobarbital N-beta-D-glucosides were excreted in the urine. The absolute configuration (C-5 position) of the major phenobarbital N-beta-D-glucoside excreted in the urine was the S form. A pronounced stereoselective formation and/or urinary excretion occurs for the N-glucoside conjugates of phenobarbital in humans.

LC determination of the diastereomers of 1-(beta-D-glucopyranosyl)phenobarbital in human urine.

The "product enantioselectivity" associated with the urinary excretion of the phenobarbital N-glucoside conjugates has not been determined previously. A liquid chromatography method using gradient elution was developed for quantifying both phenobarbital N-glucoside conjugates, phenobarbital, and p-hydroxyphenobarbital. Following a single oral dose of phenobarbital to male Caucasian and Oriental subjects, both phenobarbital N-glucoside conjugates were observed in the urine. In seven subjects, 3.3-10.6% of the phenobarbital dose was detected as a single phenobarbital N-glucoside (S configuration at the C-5 position of the barbiturate ring). The other phenobarbital N-glucoside diastereomer accounted for less than 1.5% of the phenobarbital dose. The urinary excretion of the major phenobarbital N-glucoside diastereomer paralleled the urinary excretion of phenobarbital and was comparable in both Caucasian and Oriental subjects. These results indicate a pronounced selectivity for the formation and/or urinary excretion of the phenobarbital N-glucosides.

Synthesis of N-beta-D-glucopyranosyl derivatives of barbital, phenobarbital, metharbital, and mephobarbital.

The condensation of per(trimethyl)silylbarbital and -phenobarbital with 1,2,3,4,6-penta-O-acetyl-beta-D-glucopyranose in the presence of stannic chloride in dichloroethane gave moderate yields of the beta-coupled barbiturate N-D-glucopyranosyl derivatives. Reaction of metharbital and mephobarbital under the same conditions was unsuccessful. The homologous N-methylglucosides were prepared by reaction of the barbital and phenobarbital N-glucosyl derivatives with diazomethane. The diastereomers of the phenobarbital and mephobarbital derivatives were resolved by use of C-18 reverse-phase h.p.l.c. 1H- and 13C-n.m.r. spectroscopy, and thermospray 1.c.-m.s. proved to be the most useful methods for characterizing the barbiturate glucosides.