Cytochrome P450 2D6 and treatment of codeine dependence.

Oral opioid analgesics such as codeine are used extensively worldwide and are frequently misused. Codeine is a substrate of CYP2D6, a genetically polymorphic P450 enzyme, and is metabolized to the more potent drug morphine. CYP2D6 activity can be inhibited by fluoxetine, and the inhibition of morphine formation may help individuals reduce their use of codeine. Fourteen long-term users of oral opiates (principally codeine) were assessed for an open-label pilot treatment study of fluoxetine 20 mg/day combined with a brief behavioral intervention and structured tapering of the opiate. Eight subjects entered and completed the 8-week treatment. Opiate use decreased by 30% to 100% of baseline use (p < 0.0001) in parallel with a decrease in CYP2D6 activity. Fluoxetine may have a role in the treatment of opiate dependence by decreasing opiate-reinforcing properties.

Inhibitors of cytochrome P450 differentially modify discriminative-stimulus and antinociceptive effects of hydrocodone and hydromorphone in rhesus monkeys.

The present study was conducted to investigate the role of cytochrome P450 in the discriminative-stimulus and antinociceptive effects of hydrocodone (HC) and hydromorphone (HM) in rhesus monkeys. In morphine-deprived monkeys, morphine dose-dependently reversed naltrexone-lever responding, an effect also produced by HC and HM. HC and HM also produced antinociception in a warm-water tail withdrawal procedure. Budipine and naltrexone shifted the dose-effect curves for the discriminative-stimulus effects of HC and HM to the right. In contrast, naltrexone, but not budipine (10.0 mg/kg) or quinidine (10.0 mg/kg), dose-dependently antagonized the antinociceptive effects of HC. Budipine and quinidine decreased the concentration of HM in plasma without significantly affecting the levels of HC, suggesting that these CYP2D6 inhibitors decreased the conversion of HC HM. Thus, some behavioral effects of HC are not modified by a marked inhibition of CYP2D6, suggesting that these effects of HC are not due to its conversion to HM but, rather, that both HC and HM act directly on mu receptors.

Comparison of three CYP2D6 probe substrates and genotype in Ghanaians, Chinese and Caucasians.

The ability to metabolize CYP2D6 substrates sparteine, debrisoquine, and dextromethorphan was studied in healthy Caucasian (n = 20), Ghanaian (n = 21), and Chinese (n = 22) CYP2D6 extensive metabolizers. Genotype analysis for the CYP2D6*1, *3, *4, *5, *9, *10, and *17 alleles was performed. Interethnic differences in the disposition of the probe drugs were found among the extensive metabolizers; extensive metabolizer status was confirmed by phenotype and genotype analysis. The mean metabolic rate was lower for Caucasians than for Ghanaians for sparteine (P < 0.02) and for both Ghanaians and Chinese for debrisoquine (P < 0.02). Correlation comparisons resulted in lower pairwise correlation coefficients in Ghanaians compared with Chinese and Caucasians for every combination of probe substrates. In addition, in Chinese and Caucasians, metabolic rates for each pair of probe drugs were significantly correlated (P < 0.002), but in Ghanaians the dextromethorphan metabolic rates were not correlated to either sparteine or debrisoquine (P < 0.05). Even when only those with a CYP2D6*1/*1 genotype were included in the correlation calculations, the Ghanaians had very low correlation coefficients (r(s) - 0.02-0.2, n = 9); much lower than those found in Caucasian (r(s) 0.78-0.92, n = 14) or Chinese (r(s) 0.54-0.96, n = 7) individuals. Quinidine had significantly less affect on sparteine metabolic rates in Ghanaians than both Caucasians and Chinese (P < 0.02). In addition, five of the 21 Ghanaian individuals had dextromethorphan metabolic ratios which were unaffected by quinidine. These individuals also had differences in urinary recovery of dextromethorphan and its metabolites when compared to the other Ghanaian individuals. These results confirm the large ethnic differences in probe drug metabolism and quinidine sensitivity among these ethnic groups. They also suggest that the Ghanaians have an additional unidentified allele(s) with altered substrate specificity and quinidine sensitivity which is currently genotyped as CYP2D6*1.

Interactions of amphetamine analogs with human liver CYP2D6.

The interaction of fifteen amphetamine analogs with the genetically polymorphic enzyme CYP2D6 was examined. All fourteen phenylisopropylamines tested were competitive inhibitors of CYP2D6 in human liver microsomes. The presence of a methylenedioxy group in the 3,4-positions of both amphetamine (Ki = 26.5 microM) and methamphetamine (Ki = 25 microM) increased the affinity for CYP2D6 to 1.8 and 0.6 microM, respectively. Addition of a methoxy group to amphetamine in the 2-position also increased the affinity for CYP2D6 (Ki = 11.5 microM). The compound with the highest affinity for CYP2D6 was an amphetamine analog (MMDA-2) having both a methoxy group in the 2-position and a methylenedioxy group (Ki = 0.17 microM). Mescaline did not interact with CYP2D6. O-Demethylation of p-methoxyamphetamine (PMA) by CYP2D6 was characterized (Km = 59.2 +/- 22.4 microM, and Vmax = 29.3 +/- 16.6 nmol/mg/hr, N = 6 livers). This reaction was negligible in CYP2D6-deficient liver microsomes, was inhibited stereoselectively by the quinidine/quinine enantiomer pair, and was cosegregated with dextromethorphan O-demethylation (r = 0.975). The inhibitory effect of methylenedioxymethamphetamine (MDMA) was enhanced by preincubation with microsomes, suggesting that MDMA may produce a metabolite complex with CYP2D6. These findings suggest that phenylisopropylamines as a class interact with CYP2D6 as substrates and/or inhibitors. Their use may cause metabolic interactions with other drugs that are CYP2D6 substrates, and the potential for polymorphic oxidation via CYP2D6 may be a source of interindividual variation in their abuse liability and toxicity.

Effect of CYP2D1 inhibition on the behavioural effects of d-amphetamine.

In rats, amphetamine (AMP) conversion to 4-OH-AMP is metabolized by CYP2D1, the rat equivalent of the human enzyme CYP2D6. To determine the impact of impaired AMP metabolism on its behavioural effects, AMP-induced hyperactivity, AMP discrimination and AMP self-administration were examined in male Wistar rats with or without pretreatment with the CYP2D1 inhibitors quinine and budipine. In vivo, quinine (20 mg/kg) and budipine (10 mg/kg) increased the plasma area under the curve of AMP 4-fold and 3.6-fold respectively, and decreased the plasma levels of 4-OH-AMP, 3-fold and 8.6-fold, confirming that the doses used suppressed CYP2D1 activity. Both inhibitors prolonged AMP-induced hyperactivity (0.3 mg/kg) and prolonged the duration of AMP-appropriate responding for periods of up to 90 min post-AMP administration in a drug discrimination procedure. In rats given a preload dose of AMP (0.8 mg/kg) 3 h prior to the self-administration test session, CYP2D1 inhibition resulted in fewer AMP infusions being taken compared with rats receiving the AMP preload dose alone. These studies indicate that AMP is responsible for the behavioural effects seen in rats and that a rat phenocopy model of the human CYP2D6 deficiency state can be produced by CYP2D1 inhibitors.

Inhibition of cytochrome P450 2D6 metabolism of hydrocodone to hydromorphone does not importantly affect abuse liability.

Enzymatic conversion of hydrocodone to hydromorphone is catalyzed by cytochrome P450 2D6, which is inactive in about 7% of Caucasians [poor metabolizers (PMs)] and can be inhibited by quinidine pretreatment in the remainder [extensive metabolizers (EMs)]. If hydromorphone, having a substantially higher mu-receptor affinity than hydrocodone, contributes importantly to the physiological and subjective effects of oral hydrocodone, then PMs should be less responsive to the same doses, and quinidine pretreatment should cause EMs to temporarily respond as PMs. Seventeen EMs and 8 PMs who previously responded positively to hydromorphone s.c. received placebo and hydrocodone (10 mg, 15 mg and 22.5 mg p.o.) and were retested with their favorite dose after placebo or quinidine (100 mg) pretreatment; physiological and subjective measures were collected at base line and four times after drug administration, and urine was collected for 8 hr. EMs and PMs were equally responsive to oral hydrocodone, and quinidine had no consistent effect on their responses, even though quinidine abolished the pre-existing metabolic differences in hydromorphone production, as measured in urine. These data suggest only a small role of hydromorphone in eliciting abuse-related responses to oral hydrocodone.

CYP2D6 inhibition in patients treated with sertraline.

Sertraline, a selective serotonin reuptake inhibitor used to treat depression, inhibits CYP2D6 in vitro (Ki = 1.2 microM) less potently than fluoxetine (Ki = 0.15 microM). To determine the extent and time course of CYP2D6 inhibition in patients, six males (mean age: 40 years, range: 29-64 years), who were starting treatment for depression with sertraline, were phenotyped on five occasions (once before treatment and approximately 3, 7, 14, and 21 days later). Phenotype status was determined using oral dextromethorphan (30 mg) by calculating the urinary ratio of O-demethylated metabolites to parent drug (i.e., log ODMR). CYP2D6 genotype was determined by leukocyte DNA analysis using polymerase chain reaction amplification. Compliance was confirmed by sertraline plasma levels. Daily sertraline dosages ranged from 50 to 150 mg. Genotype results indicated all subjects were extensive metabolizers (four homozygous wild type [wt], two heterozygous wt/B mutation). Phenotype results showed that CYP2D6 inhibition in patients treated with sertraline appeared to be related to baseline CYP2D6 activity and sertraline dosage. Some patients with high CYP2D6 activity can demonstrate inhibition with sertraline dosages as low as 50 mg.

Effect of cytochrome P450 2D1 inhibition on hydrocodone metabolism and its behavioral consequences in rats.

Humans that lack cytochrome P450 2D6 (CYP2D6) activity may have an altered risk of drug dependence or abuse because this enzyme is important in the metabolism of some drugs of abuse, including hydrocodone. In rats, hydrocodone conversion to hydromorphone is catalyzed by CYP2D1, the rat homolog of the human CYP2D6. To determine the impact of impaired hydromorphone formation on the behavioral effects of the parent compound, hydrocodone-induced analgesia and hyperactivity, hydrocodone discrimination and self-administration were examined in male Wistar rats, with or without pretreatment with CYP2D1 inhibitors (quinine and budipine). In vivo, quinine (20 mg/kg) and budipine (10 mg/kg) produced a marked suppression in brain and plasma hydromorphone levels detected after the peripheral administration of hydrocodone, thus confirming that the doses used suppressed CYP2D1 activity. In contrast, CYP2D1 inhibition had no impact on the analgesic or discriminative stimulus effects of hydrocodone, nor did this type of manipulation alter hydrocodone self-administration. The effects of quinine on the locomotor activating effects of hydrocodone were subtle at best. Because inhibition of CYP2D1 in this rat strain is proposed to be a useful animal counterpart for studying the impact of CYP2D6 polymorphism in humans, these data suggest that differences in CYP2D6 phenotype will have limited influence on the drug response to hydrocodone after nonoral administration. This has recently been verified in a study showing that inhibition of hydrocodone biotransformation to hydromorphone does not affect measures of abuse liability. Therefore, hydrocodone's behavioral effects are most likely attributable to its own intrinsic effects at mu opioid receptors.

Simultaneous gas chromatographic determination of methamphetamine, amphetamine and their p-hydroxylated metabolites in plasma and urine.

We report a method for the simultaneous determination of methamphetamine, amphetamine and their hydroxylated metabolites in plasma and urine samples using a GC-NPD system. The analytical procedures are: (1) adjust the sample to pH 11.5 with bicarbonate buffer, saturate with NaCl and extract with acetate; (2) back-extract the amines in the ethyl acetate fraction with 0.1 M HCl; (3) adjust the pH of the acid fraction to 11.5 and follow by extraction in ethyl acetate; (4) reduce the volume of ethyl acetate under nitrogen and derivatize the concentrate with trifluoroacetic anhydride or heptafluorobutyric anhydride before the GC analysis. The derivatives were separated on a GC-NPD system equipped with a HP-5 column of 25 m x 0.32 m I.D. and a 0.52 micron film of 5% phenylmethylsilicone. The detection limit (taking a signal-to-noise ratio of 2) of heptafluorobutyl derivatives of methamphetamine and its metabolites in plasma and the trifluoroacetyl derivatives in urine was 1 ng/ml (22 pg on column). The limit of quantitation of the heptafluorobutyl derivatives in the plasma was 1 ng/ml (22 pg on column), and that of the trifluoroacetyl derivatives in urine was 20 ng/ml (73 pg on column). The between-day variation was from 0.9 to 17.4% and within-day variation from 0.9 to 8.3%. This method was used successfully in the quantitative determination of methamphetamine and its p-hydroxylated metabolites in the plasma and urine of human subjects.

Venlafaxine oxidation in vitro is catalysed by CYP2D6.

1. Several selective 5-HT reuptake inhibitors (SSRIs) are inhibitors of the genetically polymorphic drug metabolizing enzyme, CYP2D6. We studied the interaction of venlafaxine, a new SSRI, with CYP2D6 in human liver microsomes. 2. Venlafaxine was a less potent inhibitor of this enzyme activity in vitro than other SSRIs tested. The average apparent Ki values determined using CYP2D6-dependent dextromethorphan O-demethylation were: 33, 52 and 22 microM for rac-venlafaxine, R(+)-venlafaxine and S(-)-venlafaxine, respectively, vs 0.065 to 1.8 microM for paroxetine, fluoxetine, norfluoxetine, fluvoxamine and sertraline. 3. Microsomes from human livers (n = 3) and from yeast transformed with an expression plasmid containing human CYP2D6 cDNA catalyzed the O-demethylation of venlafaxine, which is the major metabolic pathway in vivo. Intrinsic metabolic clearance values (Vmax/Km) indicated that S(-)-venlafaxine was cleared preferentially via this pathway. 4. In microsomes from CYP2D6-deficient livers (n = 2), Vmax/Km of O-demethylation of venlafaxine was one to two orders of magnitude lower and was similar to the rate of N-demethylation. 5. Studies with chemical probes which preferentially inhibit P450 isoforms suggested that CYP3A3/4 is involved in venlafaxine N-demethylation. 6. These in vitro findings predict phenotypic differences in the kinetics of venlafaxine in vivo, although the clinical importance of this is unclear as O-demethylvenlafaxine is pharmacologically similar to the parent drug. The findings also predict relatively limited pharmacokinetic interaction between venlafaxine and other CYP2D6 substrates.

Effects of route of administration on dextromethorphan pharmacokinetics and behavioral response in the rat.

One of the potential problems of using dextromethorphan as a neuroprotective or anticonvulsant agent is the phencyclidine-like behavioral effects that have been attributed to its major metabolite dextrorphan. Because previous behavioral studies of dextromethorphan have generally failed to consider metabolic conversion to this metabolite, the present studies were conducted to examine the effects of route of administration on dextromethorphan pharmacokinetics and locomotor activity in the rat. The bioavailability of dextromethorphan was 1.3-fold lower and the formation of dextrorphan and other metabolites was 3-fold greater after i.p. injection of 30 mg/kg of dextromethorphan as compared to the s.c. route, indicating substantial effect of first-pass metabolism. Plasma dextromethorphan was correlated with brain dextromethorphan (r = 0.84, P < .001), and the brain/plasma concentration ratio was about 6.5. Plasma-free dextrorphan, but not conjugated dextrorphan, was correlated with brain dextrorphan (r = 0.97, P < .001). Tmax of brain dextrorphan was earlier, and Cmax was higher after i.p. injection of dextromethorphan than s.c. administration (60 min vs. 120 min and 1.0 nmol/g vs. 0.2 nmol/g). Dextromethorphan (60 mg/kg i.p.) increased locomotor activity in the rat 60 min postdose, whereas the same dose of dextromethorphan administered by s.c. injection was without effect. These data demonstrate the route-specific effects on the disposition of dextromethorphan and dextrorphan in rat plasma and brain, as well as the behavioral consequence of the difference.

Pharmacokinetics of dextromethorphan and metabolites in humans: influence of the CYP2D6 phenotype and quinidine inhibition.

Dextromethorphan is primarily metabolized to dextrorphan by cytochrome P450 2D6 (CYP2D6), a genetically polymorphic enzyme in humans. Dextrorphan is an active metabolite that produces phencyclidine-like behavioral effects in animals and exhibits anticonvulsant and neuroprotective properties in a variety of experimental models. In these studies, we examined the effects of CYP2D6 phenotype and quinidine inhibition on the pharmacokinetics of dextromethorphan and its metabolites in humans. After a single oral dose of dextromethorphan HBr (30 mg), the major metabolites in the plasma of extensive metabolizers (N = 5) were conjugated dextrorphan and conjugated 3-hydroxymorphinan. Free dextrorphan concentrations were about 100-fold less than the conjugated dextrorphan, and dextromethorphan was not detectable. Pretreatment of these subjects with 100 mg of quinidine, a selective inhibitor of CYP2D6, significantly suppressed the formation of dextrorphan and elevated the concentrations of dextromethorphan (t1/2, 16.4 hours). In poor metabolizers (N = 4) given the same dose, dextromethorphan was the major component in the plasma with a t1/2 of 29.5 hours. Present at concentrations 5- to 10-fold less were conjugated dextrorphan and the other two metabolites. Urinary recovery studies indicated that the inhibition by quinidine was reversible and that the elimination of dextromethorphan primarily depends on CYP2D6 activity rather than renal elimination. These data demonstrated that the CYP2D6 phenotype and the concurrent administration of quinidine significantly affect the disposition of dextromethorphan and the formation of the active metabolite dextrorphan and are important factors to be considered in studies of the pharmacologic and behavioral effects of dextromethorphan.

CYP2D6 phenotype determines the metabolic conversion of hydrocodone to hydromorphone.

The contribution of cytochrome P450 2D6 (CYP2D6) to the formation of hydrocodone's active metabolite, hydromorphone, was examined in vitro and in vivo. Human liver microsomes prepared from an individual homozygous for the D6-B mutation of the CYP2D6 gene catalyzed this reaction at a negligible rate. Urinary metabolic ratios of hydrocodone/hydromorphone were highly correlated with O-demethylation ratios for dextromethorphan, an established marker drug of CYP2D6 activity (rs = 0.85; n = 18). The kinetics of hydrocodone after a single oral dose and its partial metabolic clearance to hydromorphone were investigated in five extensive metabolizers of dextromethorphan, six poor metabolizers, and four extensive metabolizers after pretreatment with quinidine, a selective inhibitor of CYP2D6 activity. The mean values for partial metabolic clearance by O-demethylation in the three groups were 28.1 +/- 10.3, 3.4 +/- 2.4, and 5.0 +/- 3.6 ml/hr/kg, respectively. No statistically significant phenotypic differences in physiologic measures were observed. However, over the first hour after dosing, the extensive metabolizers reported more "good opiate effects" and fewer "bad opiate effects" than poor metabolizers and extensive metabolizers in whom CYP2D6 was inhibited by quinidine. These data establish the importance of CYP2D6 in the formation of hydromorphone from hydrocodone and suggest that the activity of this enzyme may limit the abuse liability of hydrocodone.

Human hepatic cytochrome P450 2D6-like activity in nonhuman primates: catalytic characterization in vitro.

Previous studies have identified the monkey as an animal model for the genetic polymorphism affecting human hepatic cytochrome P450 2D6 enzyme. However, contrary to an earlier in vivo observation, the present study failed to find evidence of polymorphism of this enzyme activity in liver preparations of 84 African green (Cercopithecus aethiops) monkeys. The kinetics of dextromethorphan O-demethylation were similar in liver microsomes from African green (n = 21) and Crab eater (Macaca fascicularis, n = 7) monkeys (Km = 1.1 +/- 0.07 and 1.7 +/- 0.27 microM; Vmax = 215 +/- 71.7 and 152 +/- 21.1 nmol/mg/h, respectively). These Km values were lower and less variable than those in liver microsomes from 10 human extensive metabolizers (5.3 +/- 2.43 microM). Furthermore, the Vmax of the reaction in human liver microsomes was significantly lower (32 +/- 15.7 nmol/mg/h; P < .001). Inhibitor constants (Ki values) determined in monkey and human liver microsomes were highly correlated (r = 0.97), but two high-affinity inhibitors of the human enzyme (quinidine and lobeline) were approximately 40-fold less potent in monkey livers than in human livers. These data show that the monkey enzyme is functionally homologous, but is not identical to human hepatic cytochrome P450 2D6. Failure to observe poor metabolizer monkeys does not preclude their potential usefulness in evaluating the role of human hepatic cytochrome P450 2D6 activity in drug addiction and neurotoxicity because of the possibility of producing poor metabolizer phenocopies by potent hepatic cytochrome P450 2D6-like enzyme inhibitors in monkeys.

Inhibition by fluoxetine of cytochrome P450 2D6 activity.

Potent inhibition of cytochrome P450 2D6 (CYP2D6) in human liver microsomes by fluoxetine and its major metabolite norfluoxetine was confirmed (apparent inhibition constant values, 0.2 mumol/L). Several other serotonergic agents were also found to be competitive inhibitors of this genetically polymorphic enzyme. The O-demethylation ratio of dextromethorphan that expressed CYP2D6 activity in 19 patients receiving fluoxetine fell in the region of the antimode separating the O-demethylation ratio values observed in 208 extensive metabolizers from 15 poor metabolizers of a control group of healthy subjects. Inhibition of CYP2D6 activity in patients undergoing treatment with fluoxetine or other serotonin uptake inhibitors could contribute to toxicity or attenuated response from concurrent medications that are substrates of this enzyme. Other in vitro studies indicated that CYP2D6 catalyzes the O-demethylation of oxycodone to form oxymorphone. This reaction was inhibited by fluoxetine and its normetabolite in liver microsomes from both extensive and poor metabolizer individuals, indicating that these compounds are not selective inhibitors of CYP2D6 activity.

Inhibition of human cytochrome P450 2D6 (CYP2D6) by methadone.

1. In microsomes prepared from three human livers, methadone competitively inhibited the O-demethylation of dextromethorphan, a marker substrate for CYP2D6. The apparent Ki value of methadone ranged from 2.5 to 5 microM. 2. Two hundred and fifty-two (252) white Caucasians, including 210 unrelated healthy volunteers and 42 opiate abusers undergoing treatment with methadone were phenotyped using dextromethorphan as the marker drug. Although the frequency of poor metabolizers was similar in both groups, the extensive metabolizers among the opiate abusers tended to have higher O-demethylation metabolic ratios and to excrete less of the dose as dextromethorphan metabolites than control extensive metabolizer subjects. These data suggest inhibition of CYP2D6 by methadone in vivo as well. 3. Because methadone is widely used in the treatment of opiate abuse, inhibition of CYP2D6 activity in these patients might contribute to exaggerated response or unexpected toxicity from drugs that are substrates of this enzyme.

Catalytic and immunologic similarities between monkey and human liver cytochrome P-450db1 (human cytochrome P-450 2D6).

In vivo pharmacogenetic studies have suggested that the monkey may be an animal model for the human polymorphism of cytochrome P-450 2D6 (also called cytochrome P-450db1). In the present study, the catalytic, immunologic, and electrophoretic properties of cytochrome P-450db1 in liver microsomes from African green monkeys (Cercopithecus aethiops) were examined and compared with P-450db1 in human liver microsomes. Using sparteine as the substrate, the activity of microsomal P-450db1 from the two sources was indistinguishable in terms of the pattern of sparteine metabolites produced, the apparent Ki values of 8 competitive inhibitors (r = 0.94, p less than 0.001), and the extent of immunoinhibition by anti-rat P-450db1 antibody. Kinetic analyses demonstrated that the apparent KM values of the high affinity component of sparteine oxidation in monkey liver microsomes fell within the range observed in human livers; the Vmax of this component was as much as six times greater than the highest value reported for human liver. Western immunoblots showed a protein band in monkey liver microsomes that co-migrated with P-450db1 in human liver. The high degree of similarity observed here between P-450db1 of monkey and human liver microsomes suggests that the monkey will be a good animal model for P-450db1 enzyme studies, and possibly for studies of the role of this enzyme in drug abuse and dependence.

Drug metabolism and interactions in abuse liability assessment.

The interpretation of acute abuse liability studies and drug interaction studies would be importantly strengthened by the routine inclusion of drug concentration measurements at appropriate sampling times. Reliance on mean kinetic data misrepresents the variation in drug kinetics and fails to take experimental advantage of the natural differences in the population which may represent the extremes of abuse risk. Pharmacokinetic-pharmacodynamic studies to better understand the relationship of plasma drug concentration, drug concentration in the receptor biophase and specific drug reinforced behaviour will ensure proper study design and yield useful theoretic information. Multi- and poly-drug abuse (including heavy smoking and heavy ethanol use) are very common. Such patterns of use can have quite large effects on drug kinetics. Because of the potentially large qualitative and quantitative differences in drug metabolism and kinetics between pre-clinical species and the human, data should be gathered at the earliest possible time with respect to human metabolic rates, patterns, and identification of inhibitors. The availability of human liver microsomes facilitates such studies.