Failure to detect amphetamine-induced dopamine release in the cortex with [11 C]FLB 457 positron emission tomography (PET): Methodological considerations.

Studies in nonhuman primates and humans have demonstrated that amphetamine-induced dopamine release in the cortex can be measured with [11 C]FLB 457 and PET imaging. This technique has been successfully used in recent clinical studies to show decreased dopamine transmission in the prefrontal cortex in schizophrenia and alcohol dependence. Here, we present data from a cohort of twelve healthy controls in whom an oral amphetamine challenge (0.5 mg kg-1 ) did not lead to a significant reduction in [11 C]FLB 457 BPND (i.e., binding potential relative to non-displaceable uptake). Two factors that likely contributed to the inability to displace [11 C]FLB 457 BPND in this cohort relative to successful cohorts are: (a) the acquisition of the baseline and post-amphetamine scans on different days as opposed to the same day and (b) the initiation of the post-amphetamine [11 C]FLB 457 scan at ∼5 hours as opposed to ∼3 hours following oral amphetamine. Furthermore, we show [11 C]FLB 457 reproducibility data from a legacy dataset to support greater variability in cortical BPND when the test and retest scans are acquired on different days as compared to the same day. These results highlight the methodological challenges that continue to plague the field with respect to imaging dopamine release in the cortex.

Selective and interactive effects of D2 receptor antagonism and positive allosteric mGluR4 modulation on waiting impulsivity.

BACKGROUND: Metabotropic glutamate receptor 4 (mGluR4) and dopamine D2 receptors are specifically expressed within the indirect pathway neurons of the striato-pallidal-subthalamic pathway. This unique expression profile suggests that mGluR4 and D2 receptors may play a cooperative role in the regulation and inhibitory control of behaviour. We investigated this possibility by testing the effects of a functionally-characterised positive allosteric mGluR4 modulator, 4-((E)-styryl)-pyrimidin-2-ylamine (Cpd11), both alone and in combination with the D2 receptor antagonist eticlopride, on two distinct forms of impulsivity. METHODS: Rats were trained on the five-choice serial reaction time task (5-CSRTT) of sustained visual attention and segregated according to low, mid, and high levels of motor impulsivity (LI, MI and HI, respectively), with unscreened rats used as an additional control group. A separate group of rats was trained on a delay discounting task (DDT) to assess choice impulsivity. RESULTS: Systemic administration of Cpd11 dose-dependently increased motor impulsivity and impaired attentional accuracy on the 5-CSRTT in all groups tested. Eticlopride selectively attenuated the increase in impulsivity induced by Cpd11, but not the accompanying attentional impairment, at doses that had no significant effect on behavioural performance when administered alone. Cpd11 also decreased choice impulsivity on the DDT (i.e. increased preference for the large, delayed reward) and decreased locomotor activity. CONCLUSIONS: These findings demonstrate that mGluR4s, in conjunction with D2 receptors, affect motor- and choice-based measures of impulsivity, and therefore may be novel targets to modulate impulsive behaviour associated with a number of neuropsychiatric syndromes.

A Novel NF-κB Inhibitor, Edasalonexent (CAT-1004), in Development as a Disease-Modifying Treatment for Patients With Duchenne Muscular Dystrophy: Phase 1 Safety, Pharmacokinetics, and Pharmacodynamics in Adult Subjects.

In Duchenne muscular dystrophy (DMD), NF-κB is activated in skeletal muscle from infancy regardless of the underlying dystrophin mutation and drives inflammation and muscle degeneration while inhibiting muscle regeneration. Edasalonexent (CAT-1004) is a bifunctional orally administered small molecule that covalently links 2 compounds known to inhibit NF-κB, salicylic acid and docosahexaenoic acid (DHA). Edasalonexent is designed to inhibit activated NF-κB upon intracellular cleavage to these bioactive components. Preclinical data demonstrate disease-modifying activity in DMD animal models. Three placebo-controlled studies in adult subjects assessed the safety, pharmacokinetics, and pharmacodynamics of single or multiple edasalonexent doses up to 6000 mg. Seventy-nine adult subjects received edasalonexent, and 25 received placebo. Pharmacokinetic results were consistent with the intracellular cleavage of edasalonexent to its active components. Food increased plasma exposures of edasalonexent and salicyluric acid, an intracellularly formed metabolite of salicylic acid. The NF-κB pathway and proteosome gene expression profiles in peripheral mononuclear cells were significantly decreased (P = .02 and P = .002, respectively) after 2 weeks of edasalonexent treatment. NF-κB activity was inhibited following a single dose of edasalonexent but not by equimolar doses of salicylic acid and DHA. Edasalonexent was well tolerated, and the most common adverse events were mild diarrhea and headache. In first-in-human studies, edasalonexent was safe, well tolerated, and inhibited activated NF-κB pathways, suggesting potential therapeutic utility in DMD regardless of the causative dystrophin mutation, as well as other NF-κB-mediated diseases.

Chronic quercetin feeding decreases plasma concentrations of salicylamide phase II metabolites in pigs following oral administration.

We investigated acute effects and effects after chronic intake of the orally administered flavonol quercetin on pharmacokinetics of salicylamide metabolites (SAM) after oral administration of salicylamide in pigs. Salicylamide (8 mg/kg body weight) was orally administered to seven pigs either without or with quercetin (10 mg/kg body weight). Additionally, salicylamide was administered to five pigs that had received a diet supplemented with the flavonol for 1 week. Daily quercetin intake was 10 mg/kg in these animals. Co-ingestion of quercetin with the drug did not alter area under the concentration-time curve (AUC(0→∞)), time to achieve maximum plasma concentration (t(max)), mean residence time (MRT) or half-life (t(1/2)) of SAM. However, maximum plasma concentration (c(max)) of SAM was lower when quercetin was administered concomitantly. After quercetin pre-treatment for 1 week AUC(0→∞), t(1/2) and MRT of SAM were decreased, while other parameters investigated were not affected. Co-ingestions and dietary pre-treatment with quercetin influenced SAM metabolism after oral salicylamide intake. But effects seen after acute concomitant intake are rather explained by induced salicylamide excretion from the intestinal mucosa, whereas quercetin pre-treatment seemed to induce hepatic enzymes involved in phase-II metabolism and thereby enhanced elimination of SAM.

Simultaneous determination of salicylic acid and salicylamide in biological fluids.

A new methodology for the simultaneous determination of salicylic acid and salicylamide in biological fluids is proposed. The strong overlapping of the fluorescence spectra of both analytes makes impossible the conventional fluorimetric determination. For that reason, the use of fluorescence decay curves to resolve mixtures of analytes is proposed; this is a novel technique that provides the benefits in selectivity and sensitivity of the fluorescence decay curves. In order to assess the goodness of the proposed method, a prediction set of synthetic samples were analyzed obtaining recuperation percentages between 98.2 and 104.6%. Finally, a study of the detection limits was done using a new criterion resulting in values for the detection limits of 8.2 and 11.6 µg L(-1) for salicylic acid and salicylamide respectively. The validity of the method was tested in human serum and human urine spiked with aliquots of the analytes. Recoveries obtained were 96.2 and 94.5% for salicylic acid and salicylamide respectively.

Measurement of methylphenidate-induced change in extrastriatal dopamine concentration using [11C]FLB 457 PET.

[(11)C]FLB 457 is a very high-affinity radiotracer that allows the measurement of dopamine D(2/3) receptor availability in regions of the brain where densities are very low, such as the cerebral cortex. It is not known if [(11)C]FLB 457 binding is sensitive to the concentration of endogenous dopamine in humans in a manner analogous to [(11)C]raclopride and [(123)I]IBZM in the striatum. To test this possibility, extrastriatal [(11)C]FLB 457 binding was measured at baseline and after the oral administration of 40 to 60 mg of the psychostimulant methylphenidate (MP) in 12 healthy volunteers using positron emission tomography (PET) in a balanced-order, double-blind design. The dynamic PET data were quantified using a two-tissue compartment model with a metabolite-corrected arterial plasma input function. Two volunteers were excluded because of excessive head movement. In the remainder, MP caused significant reductions in the volume of distribution (VD) in temporal and frontal cortical regions and thalamus, suggesting that [(11)C]FLB 457 binding is sensitive to endogenous dopamine concentration. Moreover, the change in [(11)C]FLB 457 binding after MP correlated with the dose of MP (in mg/kg body weight) in all regions assessed. We conclude that MP in doses within the therapeutic range for the treatment of attention deficit hyperactivity disorder causes increases in dopamine concentrations in extrastriatal regions and that [(11)C]FLB 457 PET may be a useful tool for the assessment of change in dopamine concentration in these areas in humans.

Quantification of D2-like dopamine receptors in the human brain with 18F-desmethoxyfallypride.

UNLABELLED: Substituted benzamides such as (11)C-raclopride or (123)I-iodobenzamide are selective radiotracers for PET and SPECT imaging of D(2)-like dopamine (DA) receptors. (18)F-Desmethoxyfallypride ((18)F-DMFP) is a benzamide tracer with the advantage of an (18)F label. We optimized the synthesis and evaluated (18)F-DMFP in PET studies on healthy human volunteers. METHODS: The affinity of DMFP for D(2)-like DA receptors was characterized in vitro using membrane preparations from rat striatum and the DA receptor ligand (3)H-spiperone. PET studies on 10 healthy human volunteers were performed using a whole-body PET scanner after injection of 214 +/- 54 MBq (mean +/- SD) (18)F-DMFP. Brain images were acquired dynamically over 124 min, and metabolite-corrected plasma activity was used as the input function. Data analysis was performed using several different approaches (compartmental, graphical, equilibrium methods). RESULTS: The mean inhibition constant (K(i)) of DMFP was 15 +/- 9 nmol/L. In human brain, the striatum-to-cerebellum ratio reached a maximum of about 4 between 60 and 120 min. When specific binding in the striatum was expressed as the difference between binding in the striatum and the cerebellum, it reached a maximum at approximately 60 min after injection and remained almost constant until the end of data acquisition. The ratio of specific striatal to nonspecific cerebellar binding was about 3:1 at 120 min after injection. A small, but significant specific tracer binding could also be detected in the thalamus. Treatment of a schizophrenic patient with a high dose (1,000 mg/d) of another substituted benzamide, amisulpride, resulted in a reduction of specific tracer uptake of about 90% in striatal regions. With regard to measured distribution volumes and binding potentials, there was an excellent agreement between all applied analytic methods. CONCLUSION: Our study demonstrates that (18)F-DMFP is a highly reliable tracer for PET imaging of D(2)-like DA receptors. It offers the major advantage that it can be used independently of an on-site cyclotron within a PET satellite network. Noninvasive analytic methods without blood sampling provide valid measurements of receptor quantities in human striatum. Because of the (18)F label and the favorable imaging properties, (18)F-DMFP could become an efficient substitute for (11)C-raclopride in a clinical context.

Sensitive measurement of positron emitters eluted from HPLC.

For sensitive analysis of the radioactive-metabolite in human PET, a radio-HPLC system coupled to a newly designed positron detector was constructed. The detector had the advantages of low noise level (1.7 +/- 1.0 cpm) and high sensitivity (32 +/- 1%) due to coincidence counting and large BGO crystals. Furthermore, the detector was easy to move, since a pair of the BGO housings coupled to photomultipliers was effectively arranged in parallel and a HPLC cell with different volume could be inserted between the BGO housing. This radio-HPLC system was useful for analyzing samples with low radioactivity. It was applied to the measurement of [11C]FLB457 in plasma, having high affinity and high selectivity with dopamine D2 receptors. Extremely low radioactivity of [11C]FLB457 (2500 dpm) could be analyzed by using the radio-HPLC system. The performance of this detector was compared with those of commercially available systems that had been used as sensitive detectors for HPLC.

Chiral analysis of selected dopamine receptor antagonists in serum using capillary electrophoresis with cyclodextrin additives.

Dopamine D-2 receptor antagonists' eticlopride and sulpiride were determined in serum using capillary electrophoresis with cyclodextrin additives. Chiral resolution of S(-) and R(+) sulpiride and eticlopride were achieved using 2% sulfated-beta-cyclodextrin (S-beta-CD) in 20 mM citrate run buffer (pH 2.90). A 72-cm fused silica capillary operated in the reversed polarity mode voltage of 20 kV was used for the analysis. The analytes of interest were isolated from serum using a solid phase extraction procedure with recoveries in excess of 85% for all four enantiomers. The D-2 receptor antagonist (-) butaclamol was used as internal standard. The limits of detection were 0.3 and 0.1 microg/ml for S(-) and R(+) eticlopride and for S(-) and R(+) sulpiride, respectively, in 1 ml of serum. The limits of quantitation were 2 and 1 microg/ml for S(-) and R(+) eticlopride, and for S(-) and R(+) sulpiride, respectively. Calibration curves were linear over the 2-20 micro/ml range for eticlopride and 1-20 microg/ml range for sulpiride. The coefficients of determination were greater than 0.99 (n = 12 for eticlopride and n = 15 for sulpiride). Precision and accuracy of the method were 0.27-6.38 and 0.20-3.60% for S(-) eticlopride, 2.33-4.28 and 0.80-5.73%, for R(+) eticlopride, 3.46-6.84 and 0.80-4.26%, for S(-) sulpiride; and 4.71 -6.47 and 2.00-6.67%, for R(+)-sulpiride, respectively.

High sensitivity determination of the remoxipride hydroquinone metabolite NCQ-344 in plasma by coupled column reversed-phase liquid chromatography and electrochemical detection.

An HPLC method for the determination of NCQ-344, a remoxipride metabolite with a hydroquinone structure, in human plasma is described. Special precautions for the sampling were necessary as the compound rapidly decomposes. An efficient clean-up of the plasma samples was necessary to make use of the inherent sensitivity of the electrochemical detector. This was accomplished by a fast and simple liquid-liquid extraction at pH 7.05 combined with further cleaning on-line by using a short cyanopropyl column as the first column in a column switching system. A heart-cut from the cyanopropyl column containing the NCQ-344 fraction was then injected onto the analytical octadecyl silica column and NCQ-344 was detected at an oxidation potential of 0.70 V. The absolute recovery was > 95% and concentrations down to 0.10 nM could be determined with acceptable precision. The NCQ-344 levels in a limited number of samples from patients given remoxipride were found to be between 0.10 and 1 nM. The remoxipride concentrations in the same samples were 5,000-20,000 nM.

Simultaneous determination of salicylamide and salsalate in serum and urine by first derivative variable-angle synchronous fluorescence spectrometry.

The determination of salicylamide and salsalate in human serum and urine is performed using a simple, rapid, sensitive, and selective method. The broad-band overlapping conventional spectra of both compounds are resolved by means of first derivative variable-angle synchronous fluorescence spectrometry. The method is based on the intrinsic fluorescence of both drugs in chloroformic solution. The measurements are performed in an alkaline medium, which is adjusted by adding 0.40 M pyrrolidine chloroformic solution to the organic phase. The method was applied for the simultaneous determination of salicylamide and salsalate, at concentrations between 0.100 and 1. 000 microg mL-1 for both components, by means of absolute values of a first derivative variable-angle synchronous scan at the emission/excitation wavelengths of 410/299 nm for salicylamide and 440/307 nm for salsalate. Serum and urine are extracted with chloroform, by adding acetate buffer solution to provide pH 4.8 in the aqueous phase. Finally, pyrrolidine chloroformic solution is added to organic phase, where both components are determined, without the need for a reextraction step to an aqueous phase.

Effects of intermittent and continuous subchronic administration of raclopride on motor activity, dopamine turnover and receptor occupancy in the rat.

With the purpose of finding means to circumvent the marked pharmacokinetic differences of raclopride between rats and man, the effects of intermittent and continuous administration of raclopride were compared in rats. Intermittent administration of raclopride via subcutaneous injections resulted in a prompt increase of dopamine (DA) turnover and decrease of motor activity but these effects were of short duration, probably due to rapidly decreasing raclopride DA D2 receptor occupancy. In contrast, but similar to schizophrenic patients on raclopride treatment, stable plasma raclopride levels and a steady DA D2 receptor occupancy above 70% were produced in the caudate-putamen and nucleus accumbens/olfactory tubercle, when raclopride was administered continuously via minipumps at daily doses above 2 mg/kg. Tolerance to the acute effects of raclopride on DA turnover and locomotion was found with both routes of administration but it was more marked with continuous administration. At continuous raclopride administration, tolerance to the effects of raclopride on DA turnover and spontaneous motor activity as well as supersensitivity to amphetamine-induced motor activity occurred when 70% or more of DA D2 receptor sites were occupied, i.e. the same degree of receptor occupancy as found in patients given therapeutic doses of raclopride.

Central D2-dopamine receptor occupancy in relation to antipsychotic drug effects: a double-blind PET study of schizophrenic patients.

The relationship between central D2-dopamine receptor occupancy and antipsychotic drug effects was examined in a double-blind study. Raclopride was the compound used to induce a selective occupancy of the D2-dopamine receptors. In addition, 11C-labeled raclopride was the radioligand used to measure occupancy by positron emission tomography (PET). Seventeen schizophrenic patients were randomly assigned to one of three parallel groups treated for 4 weeks with daily doses of 2, 6, or 12 mg of raclopride. D2-receptor occupancy was determined by PET at steady-state conditions in 13 patients who completed the study. A statistically significant relationship was demonstrated between antipsychotic effect and degree of D2-receptor occupancy (p < 0.05). Patients with extrapyramidal side effects had significantly higher D2-receptor occupancy than those without (p = 0.02). The finding of a relationship between selective occupancy of the D2-dopamine receptors and clinical effects in schizophrenic patients principally provides new support for the dopamine hypothesis of antipsychotic drug action.

Dopamine D2 blocking activity and plasma concentrations of remoxipride and its main metabolites in the rat.

Remoxipride and its active metabolites, the phenolic compounds FLA797(-) and FLA908(-) and the catecholic NCQ436(-) and haloperidol, were examined for their ability to block hypothermia in the rat induced by dopamine (DA) D2 receptor stimulation. In addition, plasma levels of remoxipride and its active metabolites were measured using HPLC methods. Remoxipride (1 mumol/kg), given 30 or 15 min prior to, or 5 and 15 min after, the DA agonists, blocked the hypothermia induced by the DA D2 receptor agonists quinpirole (0.25 mg/kg s.c.) and pergolide (0.1 mg/kg s.c.). Administration of remoxipride by the i.v. or s.c. routes was more effective than by the i.p. route. FLA797(-), FLA908(-), and haloperidol were more effective than remoxipride in preventing the hypothermia caused by quinpirole, while NCQ436(-) was less effective than remoxipride. The variation in time of remoxipride's action and effectiveness in blocking the induced hypothermia followed the variations in plasma concentrations. The plasma concentrations of the active metabolites were below the limit of determination (< 2 nmol/l). Based on estimation of free brain concentrations at effective dose levels together with in vitro affinities for the DA D2 receptor it was concluded that the metabolites FLA797(-), FLA908(-), and NCQ436(-) do not appear to contribute to the antagonism of DA D2 mediated neurotransmission following a low remoxipride dose (1 mumol/kg).

An assessment of salicylic acid-induced mucosal damage in vivo by measuring the metabolism of salicylamide in rabbit intestine.

An assessment of salicylic acid-induced mucosal damage in vivo by measuring the metabolism of salicylamide (SAM) was investigated in rabbit intestine. The intestinal first-pass metabolism of SAM was studied using in situ intestinal sacs with complete mesenteric venous blood collection. The appearance of both SAM and its metabolites into the mesenteric venous blood was measured directly by cannulating the mesenteric vein of exposed intestine and collecting all venous blood draining from the absorbing region. Following oral pretreatment with salicylic acid, the appearance of SAM glucuronide (SAMG) in the mesenteric venous blood was significantly increased compared with the control. The increased blood concentration of SAMG following intraduodenal administration of SAM in vivo was observed in rabbits pretreated with salicylic acid orally. The blood concentration of SAMG after the intravenous administration of SAM was not increased compared with the control. We suggest that the change in the intestinal first-pass metabolism of SAM may be due to the intestinal mucosal damage induced by oral pretreatment with salicylic acid. The measurement of SAM metabolites may be of value in the assessment of intestinal mucosal damage in vivo.

Optimization of a peak compression system for a remoxipride metabolite (FLA797) and its application to bioanalysis.

A peak compression system is optimized for FLA797 (I), a phenolic tertiary amine and a metabolite to the antipsychotic drug remoxipride. An application is described where this effect is used to give a 6-7-fold improvement of the quantification limit in an assay of I in plasma. The liquid chromatographic system is constructed so that the injection of I dissolved in a solution of a competing amine gives a very high and narrow analyte peak with an apparent efficiency of 1.5 x 10(6) plates/m. When the levels of I in plasma are determined, an internal standard, giving a normal isocratic peak, is added to the plasma sample before the extraction. Concentrations of I down to 0.5-1.0 nM can be determined with reasonable precision. FLA908, another phenolic remoxipride metabolite and a regioisomer to I, eluting as a normal isocratic peak, can be determined simultaneously although only at concentrations higher than 10-15 nM.

Pharmacokinetics of raclopride formulations. Influence of prolactin and tolerability in healthy male volunteers.

The pharmacokinetic and pharmacodynamic properties of raclopride, a new antipsychotic, were investigated in 16 healthy men. Single 4 mg doses were administered as intravenous infusion, oral solution and 2 extended release (ER) formulations. Total plasma clearance was about 100 ml/min (6.0 L/h), of which renal clearance accounted for 0.2 ml/min, indicating extensive metabolism. The volume of distribution was 1.5 L/kg; mean absolute bioavailability was 65 to 67% following the oral solution and the ER formulations. A transient increase in plasma prolactin levels followed both the intravenous infusion and the oral solution. The ER formulations resulted in a lower increase, which appeared later. However, the area under the prolactin level curve was similar after administration of all dosage forms. The frequency and severity of the most commonly reported side effects (tiredness and restlessness) were higher after the intravenous infusion than after the ER capsules. These findings indicate that such capsules may be advantageous for clinical antipsychotic treatment with raclopride.

Pharmacokinetics of the analgesic o-carbamoylphenoxyacetic acid.

The pharmacokinetics of o-carbamoylphenoxyacetic acid was studied in 9 male healthy subjects following a single i.m. administration of 1000 mg of this compound in form of its sodium salt. Venous blood specimens were drawn up to the 10th h p.a. and the plasma concentration of o-carbamoylphenoxyacetic acid and its metabolite salicylamide were determined using a specific HPLC-assay. Following injection, o-carbamoylphenoxyacetic acid was rapidly distributed in the body since the maximal plasma concentration occurred within 0.37 +/- 0.08 h and was determined with 23.5 +/- 7.51 micrograms/ml in mean. In the following, the plasma concentration declined rapidly as well according to the mean terminal elimination half-life of 0.93 +/- 0.23 h. By contrast, plasma concentration of the metabolite salicylamide was comparatively low. Mean maximal plasma concentration amounted to only 0.18 +/- 0.03 microgram/ml and the peak concentration occurred 1.08 +/- 0.43 h after injection. The mean of the individual areas under the plasma concentration time profiles AUC(O-t) was 30.2 +/- 3.96 micrograms x h/ml for the parent compound and 0.69 +/- 0.14 microgram x h/ml for the metabolite. The relative mass portion of salicylamide vs o-carbamoylphenoxyacetic acid in systemic circulation was estimated with 0.008 comparing mean Cmax and with 0.023 comparing the mean areas AUC(O-t). A possible interpretation of these findings might be that a relatively stable ether bond exists in o-carbamoylphenoxyacetic acid which has to be cleaved during the formation of salicylamide. Due to the moderate rate of metabolic conversion, only minor quantities of salicylamide appeared in the systemic circulation. Furthermore, its peak concentration occurred at a time 2-3 times later than that of the parent compound.

Sequential metabolism of salicylamide exclusively to gentisamide 5-glucuronide and not gentisamide sulfate conjugates in single-pass in situ perfused rat liver.

Gentisamide (GAM), the hydroxylated metabolite of salicylamide (SAM), underwent sequential metabolism, and GAM-5-glucuronide was the only metabolite detected in the single-pass in situ rat liver preparation (10 ml/min/liver) perfused at varying SAM steady-state input concentrations (CIn). The exclusive formation of GAM-5-glucuronide was unexpected, because GAM, when administered to the rat liver, formed predominantly monosulfate conjugates at the 5- and 2-positions (Morris et al., J. Pharmacol. Exp. Ther. 245: 614-624, 1988). Kinetic parameters obtained from single-pass studies for SAM sulfation (CIn increasing from 31 to 347 microM) and glucuronidation and hydroxylation (CIn decreasing from 1383 to 130 microM, with 0.85 mM SO4(2-), and CIn increasing from 32 to 800 microM in an absence of SO4(2-), with 2,6-dichloro-4-nitrophenol, a sulfation inhibitor) revealed sulfation as a high-affinity, high-capacity pathway, glucuronidation as a lower-affinity, high-capacity pathway and hydroxylation as a low-affinity, low-capacity pathway. However, these parameters would not explain the exclusive glucuronidation of GAM when generated from SAM. Rather the zonal localization of metabolizing activities [a periportal sulfation, evenly distributed glucuronidation, and perivenous hydroxylation system (Xu and Pang, J. Pharmacokinet. Biopharm. 17: 645-671, 1989; Morris et al., J. Pharmacokinet. Biopharm. 16: 633-656, 1988)] could explain the complete lack of sulfation in SAM sequential metabolism. The different metabolite patterns arising from the administrations of a metabolite precursor (SAM) versus a preformed metabolite (GAM), due to the proximity of enzymes for formation and sequential metabolism, may serve to explain the differential toxic or pharmacologic effects observed in other precursor-metabolite pairs.