Clovoxamine kinetics in an early clinical trial.

Elimination kinetics of the new antidepressant clovoxamine were determined in a preliminary clinical trial in 10 depressed patients. When final oral doses of 50 mg clovoxamine fumarate were given, the mean peak steady-state plasma concentration was about 60 ng/ml after 3 hr. Clovoxamine elimination proceeded by an apparent single-phasic, first-order decline with a mean t1/2 of 9.5 +/- 2.8 hr. One subject had an unusually long t1/2 (31.5 hr) and had correspondingly high clovoxamine plasma concentrations. The mean apparent volume of distribution (Vd) calculated from oral dosage was 19.5 +/- 6 l/kg, but one atypical subject had an apparent Vd of 96 l/kg. The mean apparent oral clearance was 25.5 +/- 12.5 ml/min/kg. These parameters should be of assistance in planning dosage regimens and monitoring therapeutic blood levels according to kinetic principles. Atypical clovoxamine kinetics can be associated with abnormally high or low blood levels and could therefore lead to variability in clinical response.

Metabolism of lipid peroxidation product, 4-hydroxynonenal (HNE) in rat erythrocytes: role of aldose reductase.

Lipid peroxidation represents a significant source of erythrocyte dysfunction and aging. Because the toxicity of lipid peroxidation appears to be in part due to aldehydic end products, we examined, in rat erythrocytes, the metabolism of 4-hydroxy-trans-2-nonenal (HNE), one of the most abundant and toxic lipid-derived aldehydes. Packed erythrocytes, 0.1 ml, completely metabolized 20 nmoles of HNE in 20 min. The glutathione conjugate of HNE and 4-hydroxynonanoic acid (HNA) represented 70 and 25% of the total metabolism, respectively. Approximately 70% of the metabolites were extruded to the medium. Upon electrospray ionization mass spectrometry, the glutathione conjugate resolved into two distinct species corresponding to glutathionyl HNE (GS-HNE) and glutathionyl 1,4-dihydroxynonene (GS-DHN). The concentration of GS-DHN formed was twice that of GS-HNE. Inhibition of aldose reductase by sorbinil and tolrestat led to a selective decrease in the formation of GS-DHN, although the extent of HNE glutathiolation was unaffected. Inhibitors of aldehyde or alcohol dehydrogenase, i.e., cyanamide and 4-methyl pyrazole, had no effect on the formation of HNA and GS-DHN, indicating that these enzymes are not significant participants in the erythrocyte HNE metabolism. Thus, oxidation to HNA, conjugation with glutathione, and further reduction of the conjugate by aldose reductase appear to be the major pathways of HNE metabolism in erythrocytes. These pathways may be critical determinants of erythrocyte toxicity due to lipid peroxidation-derived aldehydes.

Identification and quantitation of N-(carboxymethyl)valine adduct in hemoglobin by gas chromatography/mass spectrometry.

A sensitive, specific and reproducible method was developed for the quantitation of the hemoglobin (Hb) adduct N-(carboxymethyl)valine (CMV). This adduct is one of various products from the Maillard reaction, involving reducing sugars and amino acids, proteins or other molecules with a free amino group. Such adducts, including N epsilon-(carboxymethyl)lysine (CML), are called advanced glycation end products (AGE) and have been correlated with aging and severity of diabetes in human tissues. This method was developed to examine the CMV-Hb adduct as a possible AGE formed by reaction of Hb with glucose or other oxidation products. CMV was cleaved selectively from isolated globin using pentafluorophenyl isothiocyanate (PFPITC) in a modified Edman degradation at pH 9.5. The carboxyl group of the adduct was derivatized to its methyl ester with diazomethane. The resulting derivative, 5-isopropyl-1-(methyl acetate)-3-pentafluorophenyl-2-thiohydantoin, was detected by gas chromatography/mass spectrometry with selected ion monitoring (GC/SIM/MS). Quantitation was based on the response factor of the derivative molecular ion (m/z 396) from synthesized CMV and N-(2-carboxyethyl)valine (molecular ion m/z 410) as internal standard. This method exhibits reproducibility and linearity in the range 0.2-100 ng CMV. The limit of quantitation (0.2 ng CMV) gave a signal-to-noise ratio greater than 5:1 using a 1:30 sample aliquot. The GC/SIM/MS method can detect CMV adduct in 5 mg globin samples with relative standard deviations less than 5%. This approach avoids tedious acid hydrolysis and interference from other amino acids. The molecular ion and other CMV derivative ion assignments from samples were confirmed by accurate mass determinations using GC/high resolution SIM/MS. Measurements from random mouse, rat and human globin samples gave mean CMV levels of about 6, 5 and 14 nmol g-1 Hb in these species, respectively.

Follicular fluid Lidocaine levels during transvaginal oocyte retrieval.

Lidocaine has been shown to have adverse effects on mouse oocyte fertilization and embryo development. We have demonstrated the presence of pharmacologic levels of lidocaine in human serum and follicular fluid obtained during ultrasound guided transvaginal oocyte retrieval. The significance of this finding is unclear, as four of the eight patients studied became pregnant, including the patient with the highest follicular fluid lidocaine levels. Further evaluation of the effect of lidocaine on human embryos is warranted.

Detoxification of endotoxin by endodontic irrigants and calcium hydroxide.

The effects of endodontic irrigants and calcium hydroxide on lipopolysaccharide (LPS; endotoxin) were analyzed using the highly selective technique of mass spectrometry/gas chromatography with selected ion monitoring. An aqueous solution of LPS was mixed with one of a variety of endodontic irrigants for 30 min. Because it is a commonly used interappointment dressing, calcium hydroxide was also applied to LPS for 1, 2, or 5 days. LPS inactivation was measured by quantitation of free fatty acid release. Water, EDTA, ethanol, 0.12% chlorhexidine, chlorhexidine + sodium hypochlorite, and sodium hypochlorite alone showed little breakdown of LPS. Long-term calcium hydroxide--as well as 30-min exposure to an alkaline mixture of chlorhexidine, ethanol, and sodium hypochlorite--did detoxify LPS molecules by hydrolysis of ester bonds in the fatty acid chains of the lipid A moiety.

Measurement of the hemoglobin N-(2-oxoethyl)valine adduct in ethyl carbamate-treated mice.

Ethyl carbamate (EC) is bioactivated by CYP2E1 through vinyl carbamate to its epoxide, a reactive electrophile. This carcinogen reacts with macromolecules, including hemoglobin (Hb). This report defines a method to examine levels of N-(2-oxoethyl) adduct on the N-terminal valine of Hb after EC treatment at carcinogenic doses. Concentrations were determined 24 hr following an oral dose of EC (1 mg/g body wt) to strains A/J and C57BL/6 mice. Globin samples were isolated by precipitation in acidified acetone, washed, dried, and stored frozen at -20 degrees C until analyzed. Weighed aliquots were treated with sodium borohydride to reduce the aldehyde of the 2-oxoethyl group to the N-(2-hydroxyethyl) adduct. The adduct valine was cleaved using phenylisothiocyanate to form a substituted phenylthiohydantoin derivative of N-(2-hydroxyethyl)valine in a modified Edman degradation. After reaction with N,O-bis(trimethylsilyl)trifluoroacetamide, the resultant product, 1-(2'-trimethylsilyloxy)ethyl-5-isopropyl-3-phenyl-2-thiohydantoin , was quantified by GC/MS with selected ion monitoring of the molecular ion using synthetic N-(3-hydroxypropyl)valine as an internal standard. No adducts were detected without NaBH4 reduction. Strain A/J mice treated with EC (1 mg/g, N = 10) yielded mean +/- standard deviation (SD) adduct level values of 13.3 +/- 1.03 nmol/g globin; saline-treated A/J controls (N = 7) gave background levels of 4.43 +/- 0.69 nmol/g globin. Strain C57BL/6 mice treated with EC (1 mg/g, N = 6) exhibited mean +/- SD values of 12.0 +/- 1.92 nmol/g globin, while control mice of this strain (N = 4) had adduct levels of 7.23 +/- 1.19 nmol/g globin. These results are consistent with findings of others that bioactivation of EC produces N-(2-oxoethyl)valine hemoglobin adducts. Although the difference between mouse strains in mean total adduct levels following EC treatment was not significant, the differences evident in comparisons within strains due to treatment, between strains in endogenous background levels, and between strains in estimates of mean increases in adduct concentrations resulting from EC treatment were highly significant (p < 0.01). This assay provides a biomarker system for assessment of production from EC of the electrophilic metabolites which are believed to be genotoxic following metabolic activation in vivo.

Detoxification of vinyl carbamate epoxide by glutathione: evidence for participation of glutathione S-transferases in metabolism of ethyl carbamate.

Vinyl carbamate epoxide (VCO) is believed to be the metabolite of ethyl carbamate (EC) ultimately responsible for its carcinogenic effects. This study investigates the role of glutathione (GSH) in protection against VCO-mediated adduct formation, and the involvement of glutathione S-transferases (GSTs) in detoxification of VCO. Formation of 1,N6-ethenoadenosine from VCO and adenosine in vitro was employed as a measure of VCO toxicity. GSH inhibited formation of ethenoadenosine in a concentration-dependent manner at concentrations ranging from 1 to 8 mM. This effect was significantly enhanced by addition of rat liver GST. Mouse liver cytosol was also found to inhibit formation of ethenoadenosine in a concentration-dependent manner, and the inhibition was relieved by addition of S-octylglutathione, a competitive inhibitor of GST. Pretreatment of mice with 1% dietary (2(3)-tert-butyl-4-hydroxyanisole (BHA) caused parallel increases in cytosolic GST activity and cytosolic enhancement of detoxification of VCO by GSH. Furthermore, BHA increased hepatic steady-state concentrations of GSH greater than twofold. The effect of BHA on detoxification of EC in vivo was examined using formation of 2-oxoethylvaline (OEV) adducts of hemoglobin as a biomarker. Pretreatment with BHA decreased overall formation of OEV adducts 23%. The major conclusions of this study are (1) VCO can be detoxified by spontaneous conjugation with GSH, (2) conjugation of VCO with GST can be catalyzed by GST(s), (3) pretreatment with BHA protects against binding of active EC metabolites in vitro and in vivo, and (4) the protective effect of BHA against EC is mediated by increases in GST activity and GSH concentration.

Soluble cytokine receptors as carrier proteins: effects of soluble interleukin-4 receptors on the pharmacokinetics of murine interleukin-4.

Soluble interleukin-4 (IL-4) receptors (sIL-4R) can have either enhancing or inhibitory effects on the activity of IL-4 in vivo, depending on the relative concentration ratios of sIL-4R to IL-4. Whereas competition with membrane IL-4 receptors is the basis for their inhibitory action, the mechanisms responsible for the potentiation of IL-4 activity are not completely clear but may involve alterations in the half-life and biodistribution of IL-4 in vivo. To better understand the basis for the enhancing effect of sIL-4R, we have analyzed their effects on the pharmacokinetic properties of IL-4. Studies with radiolabeled recombinant IL-4 demonstrated that, when injected alone, IL-4 was rapidly cleared from the circulation and eliminated through the kidneys in a proteolytically degraded form. Administration of IL-4 in combination with increasing concentrations of sIL-4R resulted in a dose-dependent enhancement in the blood levels of IL-4 and a concomitant reduction in its clearance from circulation and excretion in the urine. Differences between measurements of IL-4 concentrations based on radioactivity and enzyme-linked immunosorbent assays indicated that the injected IL-4 was rapidly inactivated in vivo and that the presence of sIL-4R diminished this process. The inactivation of IL-4 was mediated through the membrane IL-4 receptors and required receptor internalization and intact lysosomal function. Taken together, these results suggest that sIL-4R are able to alter the pharmacokinetic properties of IL-4, prolonging its half-life in the circulation and reducing its clearance through diminished renal excretion and/or interference with inactivation. These effects are consistent with the ability of sIL-4R to potentiate IL-4 activity in vivo.

Quantification of amitriptyline, nortriptyline, and 10-hydroxy metabolite isomers in plasma by capillary gas chromatography with nitrogen-sensitive detection.

A selective, sensitive method for the determination of amitriptyline and its metabolites is described. This method involves liquid-liquid extraction and capillary gas chromatography with nitrogen-sensitive detection. The detection limits of amitriptyline, nortriptyline, 10-hydroxy(E)amitriptyline, 10-hydroxy(E)nortriptyline, and 10-hydroxy(Z)nortriptyline were slightly less than 0.5 ng/ml in 1.0-ml plasma samples. The coefficients of variation for within-run and between-run analyses of samples containing 100 ng/ml were less than 12% and 9%, respectively. The method offers rapid analysis of individual isomers, increased sensitivity over high-performance liquid chromatographic methodology and the conveniences of the gas chromatographic technique.

Measurement of ethyl carbamate in blood by capillary gas chromatography/mass spectrometry using selected ion monitoring.

Methodology is presented for convenient, reproducible and direct measurement of blood concentrations of ethyl carbamate, an experimental animal carcinogen. Extraction techniques requiring 20 microliters of blood and selected ion monitoring using ethyl (13C, 15N)carbamate as internal standard enabled quantification of ethyl carbamate concentrations ranging from 50 ng ml-1 to 100 micrograms ml-1. Coefficients of variation at several representative concentrations averaged less than 4%. The method was used to determine the time course of elimination of ethyl carbamate from mice receiving doses of 125 mumol kg-1.

Determination of ethyl carbamate in distilled alcoholic beverages by gas chromatography with flame ionization or mass spectrometric detection.

Quantitative methods are detailed for determination of ethyl carbamate in distilled alcoholic beverages by capillary gas chromatography with flame ionization detection (GC/FID) and by packed-column gas chromatography/mass spectrometry (GC/MS) using selected ion monitoring. Five g samples of distillate of known ethanol concentration are diluted with water to 25% ethanol (v/v), washed with petroleum ether, and extracted with dichloromethane prior to GC/FID or GC/MS analysis. As necessary, sample extracts that exhibit GC/FID interference are passed through alumina for additional cleanup. When internal standards (tert-butyl carbamate and n-butyl carbamate for GC/FID, or ethyl 13C-15N-carbamate for GC/MS) were used for quantitation, the limit of detection for ethyl carbamate was in the range of 5-25 ppb. Coefficients of variation ranged from 3.5 to 6.0% for GC/FID determinations, and from 1.4 to 3.2% for GC/MS. Correlation between methods for 22 random distillate samples ranging in concentration from approximately 40 to 800 ppb gave a correlation coefficient (r) of 0.996.

Toxicokinetics of caffeine elimination in an infant.

The kinetics of caffeine elimination were followed in a ten month old female acutely intoxicated on a street form of the drug. Urine drug screening by gas chromatography-mass spectrometry also showed the presence of ephedrine, phenylpropanolamine, caffeine, theophylline and theobromine. Blood analyses using high pressure liquid chromatography gave evidence for the presence of theobromine, theophylline, caffeine and 1,7-dimethylxanthine. Sequentially taken blood samples determined that the initial stage of caffeine elimination was nonlinear. The Vmax = 27.6 micrograms/ml/hr and Km = 284.6 micrograms/ml. At a plasma level of approximately 30 micrograms/ml the elimination became first order with ke = 0.097 hr-1 and t 1/2 = 7.1 hr. The metabolic generation of theophylline and its elimination were also studied. Theophylline ke = 0.069 hr-1 and t 1/2 = 10.0 hr. Elimination of both drugs was anomalously long for a child of this age.

Studies on induction of metabolism of ethyl carbamate in mice by ethanol.

Acute administration of ethanol, acetaldehyde, dimethyl sulfoxide and several other compounds has been reported previously by this laboratory to inhibit the metabolism of ethyl carbamate (E.C.) in mice. Since many enzyme systems that are inhibited by a compound are also induced by that chemical, the effect of chronic administration of ethanol on the metabolism of E.C. was studied in male, A/JAX mice. Ethanol was given in three pretreatment schedules: 1, 5% in drinking water for 7 days with a 24-hr washout before E.C.; 2, 10% in drinking water 48-12 hr before E.C.; 3, 5 g/kg orally as 10% in saline 48 and 24 hr before E.C. E.C. (11.125 mg/kg) in saline was administered orally and blood samples taken at frequent intervals for analysis of E.C. by a GC/MS technique developed in this laboratory. AUCs of E.C. concentration vs. time were calculated by trapezoidal estimation. From these data, E.C. blood clearance values (dose/AUC; ml hr-1kg-1) were calculated: control, 751 +/- 49.7; group 1,803 +/- 43.5; group 2, 1225 +/- 24.6; group 3, 815 +/- 75.4. Only group 2 was significantly different (p less than 0.01) from control and other groups by Newman-Keuls test. These results indicate that ethanol may be an inducer of E.C. metabolism only under certain limited conditions. The induction may be detected 12 hr after ethanol administration but is not apparent at 24 hr after ethanol pretreatment.

Studies on inhibition and induction of metabolism of ethyl carbamate by acetone and related compounds. Evidence for metabolism by cytochromes P-450.

Ethanol and a variety of other compounds previously have been shown to acutely inhibit the metabolism of ethyl carbamate (EC) when given concurrently in mice. On the other hand, ethanol pretreatment (10% in drinking water for the period 48 to 12 hr prior to EC treatment) is known to have the opposite effect and enhance the clearance of EC from blood of mice. In the present work, acetone has been shown to act similarly. Concurrent acetone treatment inhibits the metabolism of EC (11.1 mg/kg po) in male A/JAX mice in a dose-response manner. Blood clearance (Cl) of this po dose of EC from mice following concurrent acetone treatment (50 mg/kg, 0.86 mmol/kg ip) averaged 185 +/- 5.4 (SE) ml hr-1 kg-1 vs. controls of 804 +/- 24.6 ml hr-1 kg-1. Comparing doses that produce equal effects on the blood clearance values of EC, acetone is approximately 50-fold more potent as an inhibitor than ethanol. Pretreatment of mice with acetone (2 g/kg ip) 48 hr and 24 hr before EC administration po increased the clearance of EC approximately 3-fold (CI = 2623 +/- 123 ml hr-1 kg-1). 2-Propanol was found to be at least as potent as inhibitor as acetone, but with a longer duration of inhibition; this longer duration was explained by the longer persistence of acetone in blood from conversion of 2-propanol to acetone.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of clovoxamine concentration in human plasma by electron capture gas chromatography.

The antidepressant drug clovoxamine can be specifically and sensitively quantitated in human plasma by electron capture gas chromatography. Clovoxamine and the internal standard fluvoxamine are extracted into ethyl acetate from plasma at pH 12, back-extracted into phosphoric acid, and hydrolyzed at 90 degrees C to form ketone derivatives, which are then re-extracted into hexane for injection into the chromatograph. As little as 1 microgram of clovoxamine per liter of plasma can be measured. The coefficients of variation (CV) for th analysis at concentrations of 10 and 100 micrograms/L are respectively: within-run, 5.4% and 2.7%; between-run, 17.5% and 7.0%. When this assay was applied to plasma from individuals involved in an early clinical trial of clovoxamine, steady-state plasma concentrations ranged from 50 to 77 micrograms/L 3 h after a 50-mg oral dose of clovoxamine fumarate.

Inhibition of the metabolism of urethane by ethanol.

Ethanol has been shown to inhibit the localization of [ethyl-1-14C] urethane in the male mouse, but the effect of ethanol on the metabolism of urethane has not been clarified. Consequently, the concentration of unchanged urethane was determined in the blood of male mice up to 11 hr after oral administration of urethane with or without ethanol. A high and constant blood level of urethane persisted for 8 hr after the administration of an ethanolic solution of [ethyl-1-14C] urethane (125 mumol/kg, 10 muCi/20 g of mouse, 5 g of ethanol per kg, po); the blood level of ethanol was at or above 150 mg/dl during these 8 hr. In contrast, rapid clearance of radioactivity was observed in mice treated with [ethyl-1-14C]urethane dissolved in water. Coadministration of ethanol with urethane decreased the rate of 14CO2 expiration; furthermore, covalent binding with liver protein was delayed about 8 hr and was less than that in the group treated with urethane in water. The metabolism of urethane and production of 14CO2 from [carbonyl-14C]urethane by mouse liver homogenate in vitro were inhibited by the presence of ethanol (greater than 10 mM); these concentrations of ethanol in vitro are about the same as those that are inhibitory in vivo, but the extent of inhibition suggests that the liver is not the only site of metabolism of urethane. These results indicate that ethanol can inhibit the initial metabolism of urethane, prevent the formation of active metabolites, and allow urethane to persist in blood.

Inhibition of the metabolism of ethyl carbamate by acetaldehyde.

Ethyl carbamate is an animal carcinogen when administered in large doses; it is naturally present in minute concentrations in fermented foods and beverages. Previous studies from this laboratory have demonstrated that ethanol, in vivo, inhibits the metabolism of ethyl carbamate in mice, but the enzyme system has not been identified. In an effort to further characterize the enzyme system responsible, the metabolic products of ethanol metabolism were studied to determine whether ethanol or either of its metabolites is inhibitory. Acetaldehyde (400 mg/kg) is a potent inhibitor of ethyl carbamate metabolism for about 2 hr in vitro, but sodium acetate is not. Paraldehyde (250 mg/kg) has a slower onset and longer duration of inhibition, suggesting that its conversion to acetaldehyde produces the inhibitory molecule. Disulfiram (200 mg/kg) has a prolonged inhibitory effect; this effect is enhanced and extended when the disulfiram is combined with acetaldehyde (400 mg/kg). D-Penicillamine, given in a regimen of 1.2 g/kg 0.5 hr before and 0.6 g/kg 1.5 and 3.5 hr after ethyl carbamate, is not inhibitory; however, it abolishes the inhibitory effect of acetaldehyde, presumably from sequestration of acetaldehyde. These studies demonstrate that acetaldehyde is an inhibitor of the metabolism of ethyl carbamate and suggest that acetaldehyde is one, and perhaps the only, molecule responsible for the inhibition seen when ethanol is administered to mice. In vitro incubation studies determined that ethyl carbamate was not metabolized by human plasma.

Ethyl carbamate metabolism: in vivo inhibitors and in vitro enzymatic systems.

The metabolism of ethyl carbamate and the localization of its metabolites have been shown to be almost completely inhibited by ethanol in the mouse [Waddell, Marlowe, Pierce: Food Chem. Toxicol.25, 527 (1987); Yamamoto, Pierce, Hurst, Chen, Waddell: Drug Metab. Dispos. 16, 355 (1988)]. The enzyme system catalyzing this metabolism which is inhibited by ethanol now has been further investigated in both in vivo and in vitro studies. There is a direct, highly significant relationship between the extent of metabolism of ethyl carbamate and covalent binding of metabolites to liver protein. Paraoxon, carbaryl, CCl4 ethanol, methimazole, 4-methylpyrazole, diethyl maleate, ethyl N-hydroxycarbamate, and t-butyl carbamate inhibit, to different extents, the metabolism of ethyl carbamate in vivo; SKF-525A, CoCl2, Cacyanamide, chloral hydrate, 2-oxo-4-thiazolidine carboxylic acid, allopurinol, and methyl carbamate do not. Porcine liver esterase, yeast aldehyde dehydrogenase and mouse liver catalase catalyzed the metabolism in vitro; dog or bovine catalase, acid phosphatase, alcohol dehydrogenase, or carbonic anhydrase did not under the conditions tested. Paraoxon, 4-methylpyrazole, carbaryl, and NaF significantly inhibited the hydrolytic activity of mouse liver homogenates toward p-nitrophenyl acetate; ethanol or ethyl carbamate did not. However, each of these, except 4-methylpyrazole, inhibited the metabolism of ethyl carbamate by mouse liver homogenate or porcine liver esterase to about the same extent. Ion exchange chromatography of mouse liver cytosol revealed that the fraction with ability to metabolize ethyl carbamate co-chromatographed almost exactly with the ability to hydrolyze p-nitrophenyl acetate. It is proposed that ethyl carbamate is metabolized in the mouse, at least partially, by esterases; however, metabolism by other enzyme systems cannot be excluded.