In vitro to in vivo extrapolation of biotransformation rates for assessing bioaccumulation of hydrophobic organic chemicals in mammals.

Incorporating biotransformation in bioaccumulation assessments of hydrophobic chemicals in both aquatic and terrestrial organisms in a simple, rapid, and cost-effective manner is urgently needed to improve bioaccumulation assessments of potentially bioaccumulative substances. One approach to estimate whole-animal biotransformation rate constants is to combine in vitro measurements of hepatic biotransformation kinetics with in vitro to in vivo extrapolation (IVIVE) and bioaccumulation modeling. An established IVIVE modeling approach exists for pharmaceuticals (referred to in the present study as IVIVE-Ph) and has recently been adapted for chemical bioaccumulation assessments in fish. The present study proposes and tests an alternative IVIVE-B technique to support bioaccumulation assessment of hydrophobic chemicals with a log octanol-water partition coefficient (KOW ) ≥ 4 in mammals. The IVIVE-B approach requires fewer physiological and physiochemical parameters than the IVIVE-Ph approach and does not involve interconversions between clearance and rate constants in the extrapolation. Using in vitro depletion rates, the results show that the IVIVE-B and IVIVE-Ph models yield similar estimates of rat whole-organism biotransformation rate constants for hypothetical chemicals with log KOW ≥ 4. The IVIVE-B approach generated in vivo biotransformation rate constants and biomagnification factors (BMFs) for benzo[a]pyrene that are within the range of empirical observations. The proposed IVIVE-B technique may be a useful tool for assessing BMFs of hydrophobic organic chemicals in mammals. Environ Toxicol Chem 2017;36:1934-1946. © 2016 SETAC.

Chemical activity-based environmental risk analysis of the plasticizer di-ethylhexyl phthalate and its main metabolite mono-ethylhexyl phthalate.

The present study applies a chemical activity-based approach to: 1) evaluate environmental concentrations of di-ethylhexyl phthalate (DEHP; n = 23 651) and its metabolite mono-ethylhexyl phthalate (MEHP; n = 1232) in 16 environmental media from 1174 studies in the United States, Canada, Europe, and Asia, and in vivo toxicity data from 934 studies in 20 species, as well as in vitro biological activity data from the US Environmental Protection Agency's Toxicity Forecaster and other sources; and 2) conduct a comprehensive environmental risk analysis. The results show that the mean chemical activities of DEHP and MEHP in abiotic environmental samples from locations around the globe are 0.001 and 10-8 , respectively. This indicates that DEHP has reached on average 0.1% of saturation in the abiotic environment. The mean chemical activity of DEHP in biological samples is on average 100-fold lower than that in abiotic samples, likely because of biotransformation of DEHP in biota. Biological responses in both in vivo and in vitro tests occur at chemical activities between 0.01 to 1 for DEHP and between approximately 10-6 and 10-2 for MEHP, suggesting a greater potency of MEHP compared with DEHP. Chemical activities of both DEHP and MEHP in biota samples were less than those causing biological responses in the in vitro bioassays, without exception. A small fraction of chemical activities of DEHP in abiotic environmental samples (i.e., 4-8%) and none (0%) for MEHP were within the range of chemical activities associated with observed toxicological responses in the in vivo tests. The present study illustrates the chemical activity approach for conducting risk analyses. Environ Toxicol Chem 2017;36:1483-1492. © 2016 SETAC.

Concentration dependence of biotransformation in fish liver S9: Optimizing substrate concentrations to estimate hepatic clearance for bioaccumulation assessment.

In vitro bioassays to estimate biotransformation rate constants of contaminants in fish are currently being investigated to improve bioaccumulation assessments of hydrophobic contaminants. The present study investigates the relationship between chemical substrate concentration and in vitro biotransformation rate of 4 environmental contaminants (9-methylanthracene, pyrene, chrysene, and benzo[a]pyrene) in rainbow trout (Oncorhynchus mykiss) liver S9 fractions and methods to determine maximum first-order biotransformation rate constants. Substrate depletion experiments using a series of initial substrate concentrations showed that in vitro biotransformation rates exhibit strong concentration dependence, consistent with a Michaelis-Menten kinetic model. The results indicate that depletion rate constants measured at initial substrate concentrations of 1 µM (a current convention) could underestimate the in vitro biotransformation potential and may cause bioconcentration factors to be overestimated if in vitro biotransformation rates are used to assess bioconcentration factors in fish. Depletion rate constants measured using thin-film sorbent dosing experiments were not statistically different from the maximum depletion rate constants derived using a series of solvent delivery-based depletion experiments for 3 of the 4 test chemicals. Multiple solvent delivery-based depletion experiments at a range of initial concentrations are recommended for determining the concentration dependence of in vitro biotransformation rates in fish liver fractions, whereas a single sorbent phase dosing experiment may be able to provide reasonable approximations of maximum depletion rates of very hydrophobic substances.

In vitro biotransformation rates in fish liver S9: effect of dosing techniques.

In vitro biotransformation assays are currently being explored to improve estimates of bioconcentration factors of potentially bioaccumulative organic chemicals in fish. The present study compares thin-film and solvent-delivery dosing techniques as well as single versus multiple chemical dosing for measuring biotransformation rates of selected polycyclic aromatic hydrocarbons in rainbow trout (Oncorhynchus mykiss) liver S9. The findings show that biotransformation rates of very hydrophobic substances can be accurately measured in thin-film sorbent-dosing assays from concentration-time profiles in the incubation medium but not from those in the sorbent phase because of low chemical film-to-incubation-medium mass-transfer rates at the incubation temperature of 13.5 °C required for trout liver assays. Biotransformation rates determined by thin-film dosing were greater than those determined by solvent-delivery dosing for chrysene (octanol-water partition coefficient [KOW ] =10(5.60) ) and benzo[a]pyrene (KOW =10(6.04) ), whereas there were no statistical differences in pyrene (KOW =10(5.18) ) biotransformation rates between the 2 methods. In sorbent delivery-based assays, simultaneous multiple-chemical dosing produced biotransformation rates that were not statistically different from those measured in single-chemical dosing experiments for pyrene and benzo[a]pyrene but not for chrysene. In solvent-delivery experiments, multiple-chemical dosing produced biotransformation rates that were much smaller than those in single-chemical dosing experiments for all test chemicals. While thin-film sorbent-phase and solvent delivery-based dosing methods are both suitable methods for measuring biotransformation rates of substances of intermediate hydrophobicity, thin-film sorbent-phase dosing may be more suitable for superhydrophobic chemicals.

Relationship between biodegradation and sorption of phthalate esters and their metabolites in natural sediments.

Regulatory evaluations of commercial chemicals in Canada, the United States, the European Union, and other countries aim to identify biodegradation rates of chemicals in natural soils and sediments. However, commonly used biodegradation testing methods are limited in their capacity to determine biodegradation rates under natural environmental conditions. As a result, widely varying biodegradation rates have been reported for many very hydrophobic substances. This variability causes difficulties in regulatory evaluations, potentially leading to chemical misclassification. In the present study, the authors developed a model of the relationship between biodegradation, sorption, and hydrophobicity, and tested the model in experiments that measured the biodegradation rates of a range of di-phthalate esters (DPEs) and mono-phthalate esters (MPEs) in natural sediments. The results indicate that DPEs and MPEs have the inherent capacity to be quickly degraded by microbes in sediments at a common rate, but that DPE biodegradation rates in natural sediments decrease with increasing phthalate ester sorption to sediments. The results show that inherently biodegradable substances that are subject to a high degree of sorption can be expected to exhibit long half-lives in natural sediments. The model provides a potential methodology for assessing biodegradation rates in natural sediments from inherent biodegradation rates measured in screening tests by accounting for chemical sorption. The present study indicates that a reduced rate of biodegradation is due to a reduced fraction of freely dissolved chemical concentration in the interstitial water, and that the environmental significance of sorption-reduced biodegradation rates needs to be viewed in the context of risk in chemical evaluations.

Measuring in vitro biotransformation rates of super hydrophobic chemicals in rat liver s9 fractions using thin-film sorbent-phase dosing.

Methods for rapid and cost-effective assessment of the biotransformation potential of very hydrophobic and potentially bioaccumulative chemicals in mammals are urgently needed for the ongoing global evaluation of the environmental behavior of commercial chemicals. We developed and tested a novel solvent-free, thin-film sorbent-phase in vitro dosing system to measure the in vitro biotransformation rates of very hydrophobic chemicals in male Sprague-Dawley rat liver S9 homogenates and compared the rates to those measured by conventional solvent-delivery dosing. The thin-film sorbent-phase dosing system using ethylene vinyl acetate coated vials was developed to eliminate the incomplete dissolution of very hydrophobic substances in largely aqueous liver homogenates, to determine biotransformation rates at low substrate concentrations, to measure the unbound fraction of substrate in solution, and to simplify chemical analysis by avoiding the difficult extraction of test chemicals from complex biological matrices. Biotransformation rates using sorbent-phase dosing were 2-fold greater than those measured using solvent-delivery dosing. Unbound concentrations of very hydrophobic test chemicals were found to decline with increasing S9 and protein concentrations, causing measured biotransformation rates to be independent of S9 or protein concentrations. The results emphasize the importance of specifying both protein content and unbound substrate fraction in the measurement and reporting of in vitro biotransformation rates of very hydrophobic substances, which can be achieved in a thin-film sorbent-phase dosing system.

Assessing exposure of sediment biota to organic contaminants by thin-film solid phase extraction.

Differences in bioavailability among sediments are a source of variability and uncertainty in sediment quality assessment. We present three sets of studies designed to test a thin-film solid phase extraction technique for characterizing the bioavailability of organic chemicals in sediments. Laboratory studies with spiked natural sediments reveal highly reproducible thin-film extractions for chemicals with octanol-water partition coefficients between 10(4.5) and 10(8.5), with 95% equilibration times between 1 and 600 h. Studies with field-collected sediments illustrate that method detection limits are sufficiently low for field application at contaminated sites. Bioaccumulation studies with clams (Macoma balthica) show excellent correlations between thin-film and animal tissue concentrations. We conclude that thin-film extraction provides an ecologically relevant, fugacity-based measure of chemical exposure that can be expected to improve sediment quality assessments.

Biodegradation of mono-alkyl phthalate esters in natural sediments.

Mono-alkyl phthalate esters (MPEs) are primary metabolites of di-alkyl phthalate esters (DPEs), a family of industrial chemicals widely used in the production of soft polyvinyl chloride and a large range of other products. To better understand the long term fate of DPEs in the environment, we measured the biodegradation kinetics of eight MPEs (-ethyl, -n-butyl, -benzyl, -i-hexyl, -2-ethyl-hexyl, -n-octyl, -i-nonyl, and -i-decyl monoesters) in marine and freshwater sediments collected from three locations in the Greater Vancouver area. After a lag period in which no apparent biodegradation occurred, all MPEs tested showed degradation rates in both marine and freshwater sediments at 22 degrees C with half-lives ranging between 16 and 39 h. Half-lives increased approximately 8-fold in incubations performed at 5 degrees C. Biodegradation rates did not differ between marine and freshwater sediments. Half-lives did not show a relationship with increasing alkyl chain length. We conclude that MPEs can be quickly degraded in natural sediments and that the similarity in MPE degradation kinetics among sediment types suggests a wide occurrence of nonspecific esterases in microorganisms from various locations, as has been reported previously.

T cell lymphocytosis associated with polymyositis, myasthenia gravis and thymoma.

Peripheral T cell lymphocytosis is a rare finding in association with malignant thymomas. In the majority of previous cases, the tumours have behaved aggressively with symptoms arising from local invasion. We describe a patient with ocular myasthenia gravis who presented with a rapidly progressive polymyositis and neuropathy and who was subsequently found to have a thymic mass and a mild T cell lymphocytosis. The thymoma did not give rise to local symptoms and showed no evidence of progression over a 14-month period of follow-up. The possibility of an underlying thymic tumour should be considered in any patient with chronic T cell lymphocytosis if the circulating cells show mature morphology and there is no molecular evidence of monoclonality.

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.

Unrelated donor bone marrow transplantation in adults: some current controversies.

The results of unrelated donor bone marrow transplantation are continually improving. These improved results are due to a better understanding of the complications of the procedure and the devising of strategies to avoid them. Nonetheless, many problems remain. This review will address some of the major controversies of the field including the indications for UD-BMT, infection, GVHD prophylaxis and treatment and whether UD-BMT should only be performed in specialist centres. Conclusions will be supported by evidence from the limited published literature and the authors' experience in 3 major transplant centres.

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.

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.

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.