Measurement of disposition half-life, clearance, and residence times.

This unit provides an overview of the principles underlying the elimination process. Experimental design issues, methods of data analysis, and complications that may be encountered are discussed. A complete worked example is provided to exemplify the methods of analysis that are available.

Evaluation of a minimal experimental design for determination of enzyme kinetic parameters and inhibition mechanism.

The advent of combinatorial chemistry has led to a deluge of new chemical entities whose metabolic pathways need to be determined. A significant issue involves determination of the ability of new agents to inhibit the metabolism of existing drugs as well as its own susceptibility for altered metabolism. There is need to estimate the enzyme inhibition parameters and mechanism or mechanisms of inhibition with minimal experimental effort. We examined a minimal experimental design for obtaining reliable estimates of K(i) (and V(max) and K(m)). Simulations have been applied to a variety of experimental scenarios. The least experimentally demanding case involved three substrate concentrations, [S], for the control and one substrate-inhibitor pair, [S]-[I]. The control and inhibitor data (with 20% coefficient of variance random error) were simultaneously fit to the full nonlinear competitive inhibition equation [simultaneous nonlinear regression (SNLR)]. Excellent estimates of the correct kinetic parameters were obtained. This approach is clearly limited by the a prior assumption of mechanism. Further simulations determined whether SNLR would permit assessment of the inhibition mechanism (competitive or noncompetitive). The minimal design examined three [S] (control) and three [S]-[I] pairs. This design was successful in identifying the correct model for 98 of 100 data sets (20% coefficient of variance random error). SNLR analysis of metabolite formation rate versus [S] permits a dramatic reduction in experimental effort while providing reliable estimates of K(i), K(m), and V(max) along with an estimation of the mechanism of inhibition. The accuracy of the parameter estimates will be affected by the experimental variability of the system under investigation.

A simplified isolated perfused rat liver apparatus: characterization and measurement of extraction ratios of selected compounds.

A simplified isolated perfused rat liver (IPRL) preparation has been developed and evaluated. The liver is briefly perfused in situ prior to being placed into a 37 degrees C oven and suspended from a stand. This set-up takes about 5 min. A non-recirculatory or one-pass perfusion approach has been used. The performance of the apparatus was evaluated with use of three model compounds: antipyrine, lidocaine and ethanol. In addition, oxygen extraction was determined. The steady-state extraction ratio (ER) was determined for each compound (and oxygen) as a function of perfusate flow rate (15-35 ml/min) during sequential 45 min perfusion periods. Perfusion experiments lasted for up to 3 hr. The ERs (at 15 ml/min) of ethanol (0.65 +/- 0.15), lidocaine (0.91 +/- 0.01) and oxygen (0.65 +/- 0.10) were dependent upon perfusate flow; whereas, antipyrine ER (0.07 +/- 0.01) was independent of flow. The corresponding values for unbound intrinsic clearances (CLu,int) for antipyrine, ethanol, lidocaine and oxygen were: 1.6, 31.0, 158.0 and 27.5 ml/min, respectively. These findings are consistent with the known hepatic ER values for those compounds reported in the literature.

Human moricizine metabolism. II. Quantification and pharmacokinetics of plasma and urinary metabolites.

1. The metabolism of moricizine.HCl was studied in 12 male volunteers dosed with 250 mg (300 microCi) 14C-radiolabelled drug. 2. Moricizine was biotransformed to many metabolites in humans (at least 35 plasma and 51 urine metabolites). 3. Urine and faecal combined mean (range) recovery accounted for 90.2% (73.4-101.6%) of the administered radioactivity, with most of the recovered radioactivity present in faeces (mean 58.4%; range 45.6-64.7%). Mean (range) urinary recovery was 31.8% (26.2-36.9%), with <1% of the dose recovered as intact moricizine, and no one metabolite accounting for >2.5% of the dose. 4. Total radioactivity (TR) plasma t1/2 was 85.2 h, while that for moricizine was 2.4 h. Mean half-lives for plasma metabolites ranged from 2.9 to 23.6 h. The largest portion (11%) of TR AUC (area under the plasma concentration-time curve) was attributed to 2amino-10-glucuronophenothiazine. Each of the other metabolites accounted for less of the TR AUC than parent drug except for two unidentified peaks which had comparable areas (approximately 5% of the total radioactivity area). 5. Two identified moricizine metabolites, 2-amino-10-(3-morpholinopropionyl) phenothiazine and ethyl [10-(3-aminopropionyl) phenothiazin-2-yl] carbamate, possess the structural characteristics proposed for class 1 anti-arrhythmic activity (pendant amine functionality) and have plasma half-lives 4-7-fold longer than moricizine.

Moricizine bioavailability via simultaneous, dual, stable isotope administration: bioequivalence implications.

The relative bioavailability of a 200 mg film-coated tablet of [12C]moricizine.HCl in comparison to a 200 mg [13C6]moricizine.HCl oral solution was determined after simultaneous administration to 8 young healthy male subjects. Concentrations of [12C]moricizine.HCl and [13C6]moricizine.HCl were determined by thermospray liquid chromatography-mass spectrometry (LC-MS) using [2H11]moricizine.HCl as the internal standard. The mean absorption and disposition parameters of the tablet versus the solution were the following (%CV): maximum concentration, 0.83 (39%) versus 0.79 (39%) microgram/mL; time of maximum concentration, 0.81 (40%) versus 0.65 (28%) hours; area under the concentration-time curve (AUC), 1.58 (39%) versus 1.49 (37%) micrograms.h/mL; apparent oral clearance, 150.7 (52%) versus 158.1 (50%) L/h; and t1/2, 1.9 (42%) versus 1.9 (42%) hours. The AUC for the tablet averaged 106% of the solution, which likely reflects a greater first-pass effect with the oral solution. Partitioning sources of variation confirmed the low (< 6%) intrasubject coefficient of variation (cv epsilon) afforded via the single-period, dual-isotope design. In contrast, a previous study using the conventional two-period crossover design determined the cv epsilon about moricizine metrics to be in excess of 30%, resulting in classification of this drug as having highly variable absorption. The results of this study further illustrate the benefits of dual, stable isotopes to assess bioavailability and bioequivalence. This paradigm results in a reduction in experimental time and subject inconvenience and lower costs in comparison with the standard crossover study. Perhaps most important is the improved statistical power for the evaluation of bioavailability or bioequivalence in the absence of period and sequence effects that confound the assessment of intrasubject variation in the standard crossover design.

Sensitive and selective gas chromatographic methods for the quantitation of camphor, menthol and methyl salicylate from human plasma.

Analytical methods using gas chromatography-flame ionization detection (GC-FID) for the quantitation of camphor and menthol and GC-MS for the quantitation of methyl salicylate have been developed for measurement of low concentrations from human plasma. Anethole serves as the internal standard for camphor and menthol and ethyl salicylate serves as the internal standard for methyl salicylate. Plasma samples undergo multiple, sequential extractions with hexane in order to provide optimal recovery. For menthol and camphor, the extracting solvent is reduced in volume and directly injected onto a capillary column (Simplicity-WAX). Extracted methyl salicylate is derivatized with BSTFA prior to injection onto a capillary column (Simplicity-5). Between-day variation (% RSD) at 5 ng/ml varies from 6.2% for methyl salicylate to 13.5% for camphor. The limit of detection for each analyte is 1 ng/ml and the limit of quantitation is 5 ng/ml. These analytical methods have been used in a clinical study to assess exposure from dermally applied patches containing the three compounds.

Estimation of Ki in a competitive enzyme-inhibition model: comparisons among three methods of data analysis.

There are a variety of methods available to calculate the inhibition constant (Ki) that characterizes substrate inhibition by a competitive inhibitor. Linearized versions of the Michaelis-Menten equation (e.g., Lineweaver-Burk, Dixon, etc.) are frequently used, but they often produce substantial errors in parameter estimation. This study was conducted to compare three methods of analysis for the estimation of Ki: simultaneous nonlinear regression (SNLR); nonsimultaneous, nonlinear regression, "KM,app" method; and the Dixon method. Metabolite formation rates were simulated for a competitive inhibition model with random error (corresponding to 10% coefficient of variation). These rates were generated for a control (i.e., no inhibitor) and five inhibitor concentrations with six substrate concentrations per inhibitor and control. The KM/Ki ratios ranged from less than 0.1 to greater than 600. A total of 3 data sets for each of three KM/Ki ratios were generated (i.e., 108 rates/data set per KM/Ki ratio). The mean inhibition and control data were fit simultaneously (SNLR method) using the full competitive enzyme-inhibition equation. In the KM,app method, the mean inhibition and control data were fit separately to the Michaelis-Menten equation. The SNLR approach was the most robust, fastest, and easiest to implement. The KM,app method gave good estimates of Ki but was more time consuming. Both methods gave good recoveries of KM and VMAX values. The Dixon method gave widely ranging and inaccurate estimates of Ki. For reliable estimation of Ki values, the SNLR method is preferred.

Simultaneous quantitative analysis of methyl salicylate, ethyl salicylate and salicylic acid from biological fluids using gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-microl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH2PO4 (pH 7.4) buffer and from 100 microl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC-MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.

Enantiomeric determination of amphetamine and methamphetamine in urine by precolumn derivatization with Marfey's reagent and HPLC.

An analytical method was developed for enantiomeric determination of amphetamine and methamphetamine in human urine. The enantiomers were isolated from urine by solid-phase extraction, and diastereomers were formed by derivatization with the chiral Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-aniline amide). The diastereomers were separated by reversed-phase high-performance liquid chromatography in a water/methanol mobile phase and detected by absorbance spectrophotometry at 340 nm. Linear standard curves were obtained for all four enantiomers over a concentration range of 0.16-1.00 mg/L in urine. The detection limit was 0.16 mg/L urine for each enantiomer, and the limit of quantitation was 0.40 mg/L. The urine of 10 decedents was analyzed by this method and by a previously published precolumn derivatization procedure using (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as the derivatizing agent and fluorescence detection. Comparison of the results of the two methods by linear regression showed comparable results for both d-amphetamine and d-methamphetamine. Neither method detected the presence of the l-enantiomers in the urine samples.

The influence of cimetidine on the disposition kinetics of the antidepressant venlafaxine.

The influence of cimetidine on the disposition pharmacokinetics of the antidepressant drug, venlafaxine, and its active metabolite, O-desmethylvenlafaxine, was examined in 18 healthy young men and women. The steady-state pharmacokinetic profiles of venlafaxine and O-desmethylvenlafaxine were evaluated during a 24-hour period after 5 days of treatment with venlafaxine (50 mg three times a day) and during a second 24-hour period after 5 days of combination treatment with venlafaxine (50 mg three times a day) and cimetidine (800 mg once a day). The apparent oral clearance of venlafaxine decreased significantly in the presence of cimetidine and the average steady-state plasma concentration of venlafaxine increased significantly in the presence of cimetidine, but there were no changes in the corresponding concentrations of the active metabolite. However, O-desmethylvenlafaxine exhibits pharmacologic activity that is approximately equimolar to that of venlafaxine, and the sum of venlafaxine plus O-desmethylvenlafaxine plasma concentrations was increased by an average of only 13%. Therefore, the effect of cimetidine coadministration is not expected to result in clinically important alterations in the response to venlafaxine in patients with depression. This may not be true, however, for patients with compromised hepatic metabolic function.

In vitro transesterification of cocaethylene (ethylcocaine) in the presence of ethanol. esterase-mediated ethyl ester exchange esterase-mediated ethyl ester exchange.

This study reports that cocaethylene undergoes an esterase-mediated ethyl ester exchange with ethanol, resulting in an increase in the apparent in vitro t1/2, compared with control conditions. Homogenized liver from male Sprague Dawley rats in pH 7.4 phosphate buffer was centrifuged at 9000g, and the resulting supernatant (S9) fraction was collected. Tubes containing the rat S9 fraction and 50 microM cocaethylene plus aqueous buffer (control), 50 mM ethanol, or 51. 3 mM 2H6-ethanol were incubated at 37 degrees C for 4 hr. Samples were collected from the incubation tubes at various times, extracted with a solid-phase extraction system, and assayed for cocaethylene and 2H5-cocaethylene by GC/MS. Concentration-time profiles were constructed and kinetic parameters were determined. The experiment was repeated in the presence of specific and nonspecific esterase inhibitors. Enzyme kinetic parameters were also determined. Cocaethylene underwent ethyl ester exchange, being converted to 2H5-cocaethylene in the presence of 2H6-ethanol. The average apparent in vitro t1/2 value for cocaethylene (13.0 +/- 1.4 min) incubated with the S9 fraction and buffer only was increased approximately 5-fold (67.8 +/- 0.3 min) in the presence of ethanol. Formation of 2H5-cocaethylene was totally blocked with the addition of bis-(p-nitrophenyl)phosphate but was unaffected by physostigmine. The intrinsic metabolite formation clearance of 2H5-cocaethylene from cocaethylene and 2H6-ethanol (1.92 +/- 0.03 microl/min.mg protein) was several times greater than the corresponding value for cocaethylene formation from cocaine and ethanol (0.94 +/- 0.01 microl/min.mg protein) or 2H6-ethanol (0.87 +/- 0.04 microl/min.mg protein).

Absorption, disposition, and metabolism of trans-methyl styryl ketone in female B6C3F1 mice.

trans-Methyl styryl ketone (MSK; trans-4-phenyl-3-buten-2-one) is a beta-unsaturated ketone that has a wide range of uses in industry, as well as consumer products. MSK does not appear to be overtly toxic in animal models, however, it has been shown to be mutagenic in several in vitro assays after S-9 activation. In this study experiments were conducted to characterize MSK absorption, distribution, metabolism, and elimination after iv, oral, and topical administration to female B6C3F1 mice. After iv administration, [14C]MSK (20 mg/kg; 120 microCi/kg) was rapidly cleared from the blood as evidenced by the following pharmacokinetic values (mean +/- SD): terminal disposition half-life (t1/2), 7.98 +/- 1.72 min; mean residence time, 5.6 +/- 1.7 min; steady-state apparent volume of distribution (Vss), 3.33 +/- 0.75 liters/kg; and systemic body clearance (CLs), 0.53 +/- 0.05 liters/min/kg. Within 48 hr, 92.4% of the dose was excreted in the urine and 3.5% in the feces. The major blood metabolites after iv administration were identified by GC-MS as the 4-phenyl-3-buten-2-ol (methyl styryl carbinol), 4-hydroxy-4-phenyl-2-butanone, and benzyl alcohol. After oral administration of [14C]MSK (200 mg/kg; 100 microCi/kg), 95% of the dosed radioactivity was recovered in the urine and 1.2% in the feces within 48 hr. Major urinary metabolites were identified by LC-MS/MS as N-phenylacetyl-l-glycine (35.1% of dose) and N-benzyl-L-glycine (19.1% of dose). Only a small amount of MSK was detected in the blood after oral administration ( approximately 0.73 microg/ml at 10 min), and [14C]-equivalents in the blood never exceeded 2.8% of the dose. Ater topical application of [14C]MSK (250 mg/kg; 50 microCi/kg), approximately 40% of the dose was absorbed and 84.5% of the absorbed dose was excreted into the urine (36% of the total dose). Urinary metabolites were similar to those described for oral administration. Importantly, [14C]-equivalents were not detected in the blood at any time after dermal administration. These results indicate that the rate of MSK clearance is equivalent to its rate of absorption, and tissue exposure to intact MSK is expected to be limited.

p-Hydroxymethamphetamine enantiomer pharmacokinetics and metabolism in rats: absence of N-demethylation.

p-hydroxymethamphetamine (OHMAP) is one of the major metabolites of the widely abused drug methamphetamine (MAP). The demethylation of OHMAP to p-hydroxyamphetamine (OHAP) has been shown in vitro but has never been reported in vivo. The disposition kinetics as well as the metabolism of OHMAP was investigated employing a sensitive HPLC method which can separate the enantiomers of OHMAP and OHAP. Both conjugated and unconjugated forms of these compounds can be quantitated. Male Sprague-Dawley rats were given an iv bolus of racemic OHMAP (20 mg kg-1) and serum and urine samples were collected at selected times. The serum concentration-time data for OHMAP enantiomers could be described by a biexponential equation. The clearance of D-OHMAP (93.5 mL min-1 kg-1) was slightly, but statistically significantly, greater than that of the L-enantiomer (83.9 mL min-1 kg-1). The steady-state volumes of distribution of L- and D-OHMAP were (mean +/- SD) 3.15 +/- 0.84 and 4.23 +/- 1.76 L kg-1, respectively. No significant concentrations or amounts of OHAP enantiomers could be detected in any serum or urine sample. Rats excreted more unchanged L-OHMAP (34%) than D-OHMAP (29%). In contrast, more conjugated D-OHMAP (57%) was recovered compared to the conjugated L-OHMAP (52%). The results suggest that there is slight stereoselectivity in the disposition of OHMAP enantiomers. The N-demethylation product (OHAP) was not produced in vivo.

Oral and topical absorption, disposition kinetics, and the metabolic fate of trans-methyl styryl ketone in the male Fischer 344 rat.

trans-Methyl styryl ketone (MSK; trans-4-phenyl-3-buten-2-one) is a beta-unsaturated ketone that has a wide range of uses in industry and is present in numerous consumer products. Although MSK has been shown to be positive in several in vitro mutagenic assays, it does not seem to be overtly toxic in animal models. This lack of toxicity may relate to its poor absorption and/or rapid elimination. However, little is known about the fate of MSK in the body. Studies were conducted to characterize the absorption, and disposition kinetics of MSK after intravenous, oral, and topical administration to male Fischer 344 rats. After intravenous administration of [14C]MSK (20 mg/kg, 120 microCi/kg), blood concentration-time data could be characterized with a biexponential equation and apparent first-order elimination kinetics. The following pharmacokinetic parameter values were obtained (mean +/- SD): terminal disposition half-life, 17.7 +/- 0.08 min; apparent steady-state volume of distribution, 0.89 +/- 0.14 liters/kg; systemic body clearance, 68.9 +/- 10.0 ml/kg *min; and mean residence time, 13.1 +/- 2.2 min. Within 48 hr, 95.5% of the dose was excreted in the urine and 2.7% in the feces. The major blood metabolite after intravenous administration was identified by GC/MS as the 4-phenyl-3-buten-2-ol (methyl styryl carbinol). After oral administration of [14C]MSK (200 mg/kg, 100 microCI/kg), approximately 96.6% of the dosed radioactivity was recovered in the urine and 4.8% in the faces within 48 hr. Major urinary metabolites identified by LC-MS/MS and quantified by HPLC radioassay were N-phenylacetyl-L-glycine (64.9% of dose) and N-benzyl-L-glycine (9.9% of dose). Parent compound could not be detected in the blood after oral administration, and 14C-equivalents in the blood never exceeded 1.3% of the dose. Results suggest near-total presystemic elimination of the oral dose. After topical application of [14C]MSK (250 mg/kg, 50 microCi/kg), > 60% of the dose was absorbed, and the majority of the dose was excreted into the urine (55% of dose) in the form of metabolites. Urinary metabolites were similar to those described after oral administration. 14C-equivalents were not detected in the blood at any time after topical administration. These results indicate that MSK is almost totally metabolized before systemic distribution after oral or topical administration. The systemic exposure dose of MSK seems to be exceedingly low at the doses studied herein.

Absorption, disposition kinetics, and metabolic pathways of cyclohexene oxide in the male Fischer 344 rat and female B6C3F1 mouse.

Cyclohexene oxide (CHO) is a monomer intermediate used in the synthesis of pesticides, pharmaceuticals, and perfumes. Although CHO has a variety of industrial uses where direct human exposure is possible, very little is known about its fate in the body. Therefore, the objectives of this study were to determine the absorption, distribution, metabolism, and excretion of cyclohexene oxide after oral, intravenous, and dermal exposure in male Fischer 344 rats and female B6C3F, mice. After intravenous administration of [14C]CHO (50 mg/kg), CHO was rapidly distributed, metabolized, and excreted into the urine. Plasma concentrations of CHO rapidly declined and were below the limit of detection within 60 min. Average (+/- SD) values for terminal disposition half-life, apparent volume of distribution at steady-state, and systemic body clearance were: 19.3 +/- 1.6 min; 0.44 +/- 0.08 liter/kg; and 31.3 +/- 0.5 ml/kg * min, respectively. After oral administration of [14C]CHO (10 and 100 mg/kg), it was found that 14C-equivalents were rapidly excreted in the urine of both species. At 48 hr, the majority of the dose (73-93%) was recovered in urine, whereas fecal elimination accounted for only 2-5% of the dose. At no time after oral administration was parent CHO detected in the blood. However, its primary metabolite cyclohexane-1,2-diol was present for different lengths of time depending on the dose. Four metabolites were detected and identified in mouse urine by MS: cyclohexane-1,2-diol; cyclohexane-1,2-diol-O-glucuronide; N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine; and cyclohexane-1,2-diol-O-sulfate. The sulfate conjugate was not present in rat urine. Topical application of [14C]CHO (60 mg/kg) provided poor absorption in both species. The majority of 14C-equivalents applied dermally were recovered from the charcoal skin trap (approximately 90% of the dose). Only 4% of the dose was absorbed, and the major route of elimination was via the urine. To evaluate the toxicity of CHO, animals were given daily doses of CHO orally and topically for 28 days. No statistically significant changes in final body weights or relative organ weights were noted in rats or mice treated orally with CHO up to 100 mg/kg or up to 60 mg/kg when given topically. Very few lesions were found at necropsy, and none were considered compound related. In conclusion, regardless of route, CHO is rapidly eliminated and excreted into the urine. Furthermore, after either oral or dermal administration, it is unlikely that CHO reaches the systemic circulation intact due to its rapid metabolism, and is therefore unable to cause toxicity in the whole animal under the test conditions used in this study.

Comparative pharmacokinetics and interspecies scaling of amphotericin B in several mammalian species.

This study employed several interspecies scaling methods, to evaluate the applicability of extrapolating to man, pharmacokinetic information obtained from animals for amphotericin B, an anti-fungal drug. Pharmacokinetic parameters from four animal species (mouse, rat, monkey and dog) and man were obtained from the literature or from analysis of data reported in the literature. The allometric relationships (obtained from four animal species) as a function of species body weight (W; kg) for systemic clearance per maximum life span potential (CLS/MLP), steady-state volume of distribution (VSS), apparent volume of distribution (V beta) and volume of the central compartment (VC) were: 5691W1.096; 2.46W0.839; 3.08W0.948 and 1.07W0.965, respectively. The allometric relationships for half-life (h) and mean residence time (h) did not scale well with body weight. The prediction of pharmacokinetic parameters in man from the allometric equations do not always agree with those reported in the literature which are based upon a limited number of studies with few human subjects. The plasma concentration-time profiles from these animals were adjusted by normalizing the concentration with dose/W0.948, and re-plotted on different pharmacokinetic time scales. The syndesichrons plot produced an almost superimposable profile of adjusted concentrations as a function of adjusted time among the four species.

Simple apparatus for serial blood sampling in rodents permitting simultaneous measurement of locomotor activity as illustrated with cocaine.

A simple device has been developed for serial venous blood sampling which permits the simultaneous measurement of locomotor activity in the freely moving rat. The device can be easily constructed from routine laboratory material and it does not interfere with the light beams used to measure locomotor activity. The device, in conjunction with an activity cage, has been applied to the combined pharmacokinetic and pharmacodynamic modeling of cocaine. The relationship between the locomotor activity following a single short iv infusion of cocaine (5 mg/kg) and cocaine plasma concentrations can be adequately described by the Sigmoid-E(max) model. Further, the relationship between activity and time can be described by the same model coupled with an effect compartment. These results suggest the applicability of the device in facilitating pharmacokinetic/pharmacodynamic modeling of drugs that affect locomotor activity.