Pharmacological profile of ALKS 7119, an investigational compound evaluated for the treatment of neuropsychiatric disorders, in healthy volunteers.

AIMS: ALKS 7119 is a novel compound with in vitro affinity highest for the SERT, and for µ receptor, α1A -adrenoceptor, α1B -adrenoceptor, NMDA receptor and sigma non-opioid intracellular receptor 1. This first-in-human study evaluated safety and PK/PD effects of single ascending doses (SAD) of ALKS 7119 in healthy males and compared effects with neurotransmitter modulators with partially overlapping targets. METHODS: In 10 cohorts (n = 10 subjects each), PK, safety and PD (NeuroCart tests, measuring neurophysiologic effects [pupillometry, pharmaco-EEG (pEEG)], visuomotor coordination, alertness, [sustained] attention [saccadic peak velocity, adaptive tracking], subjective drug effects [VAS Bowdle and VAS Bond and Lader] and postural stability [body sway]) were evaluated. Neuroendocrine effects (cortisol, prolactin, growth hormone) were measured. Data were analysed over the 12-hour post-dose period using mixed-effects model for repeated measure (MMRM) with baseline as covariate. RESULTS: ALKS 7119 demonstrated linear PK and was generally well tolerated. QTcF interval increases of 30-60 ms compared to baseline were observed with ALKS 7119 doses of ≥50 mg without related adverse events. Significant increases in left and right pupil/iris ratio were observed at dose levels ≥50 mg (estimate of difference [95% CI], P-value) (0.04 [0.01; 0.07], P < .01) and (0.06 [0.03; 0.09], P = .01), respectively. From dose levels ≥50 mg significant increases (% change) of serum cortisol (51.7 [8.4; 112.3], P = .02) and prolactin (77.9 [34.2; 135.8], P < .01) were observed. CONCLUSION: In line with ALKS 7119's in vitro pharmacological profile, the clinical profile observed in this study is most comparable to SERT inhibition.

Effect of food on the bioavailability and tolerability of the JAK2-selective inhibitor fedratinib (SAR302503): Results from two phase I studies in healthy volunteers.

Fedratinib (SAR302503/TG101348) is a Janus kinase 2 (JAK2)-selective inhibitor developed for treatment of patients with myelofibrosis. The effect of food intake on the pharmacokinetics (PKs) and tolerability of single-dose fedratinib was investigated in two Phase I studies (FED12258: 100 mg or 500 mg under fasted or fed [high-fat breakfast] conditions; ALI13451: 500 mg under fasted or fed [low- or high-fat breakfast] conditions) in healthy male subjects. At the 500 mg dose the fed:fasted ratio estimate for area under the plasma concentration-time curve extrapolated to infinity was 0.96 (100 mg; high-fat/fasted), 1.19-1.24 (500 mg; high-fat/fasted), and 1.22 (500 mg; low-fat/fasted). Fedratinib 500 mg attained peak plasma concentration 4 hours after a high-fat breakfast and 2-2.5 hours after a low-fat breakfast or under fasted conditions; terminal half-life was 76-88 hours (fasted) and 73-78 hours (fed). The most frequent adverse events were mild gastrointestinal toxicities, the incidence of which decreased following a high-fat breakfast compared with both fasted and low-fat breakfast conditions (17%, 67%, and 59% of subjects, respectively, in ALI13451). In conclusion, food intake had minimal impact on the PKs of fedratinib, and the tolerability of this drug was improved when taken following a high-fat breakfast.

Lack of clinical pharmacodynamic and pharmacokinetic drug-drug interactions between warfarin and the antisense oligonucleotide mipomersen.

Mipomersen is a second-generation antisense oligonucleotide indicated as an adjunct therapy for homozygous familial hypercholesterolemia (HoFH). Warfarin is commonly prescribed for a variety of cardiac disorders in homozygous familial hypercholesterolemia population, and concurrent use of warfarin and mipomersen is likely. This open-label, single-sequence 2-period phase 1 study in healthy subjects evaluated the potential drug-drug interactions between mipomersen and warfarin. The subjects received a single oral 25 mg dose of warfarin alone on day 1, and after a 7-day washout period, received 200 mg mipomersen alone subcutaneously every other day on days 8-12, and received both concurrently on day 14. Coadministration of mipomersen did not change the pharmacodynamics (international normalized ratio, prothrombin time, and activated partial thromboplastin time) and pharmacokinetics (PK) of warfarin. There were no clinically significant changes in the PK of mipomersen with concurrent administration of warfarin. There were no events indicative of an increase in bleeding tendency when warfarin was coadministered with mipomersen, and the adverse event profile of mipomersen did not appear to be altered in combination with warfarin, as compared with that of the respective reference treatment. The combination of these 2 medications appeared to be safe and well tolerated. These results suggest that the dosage adjustment of warfarin or mipomersen is not expected to be necessary with coadministration.

Pharmacokinetics, safety and tolerability of mipomersen in healthy Japanese volunteers and comparison with Western subjects.

OBJECTIVES: To characterize the safety, tolerability, pharmacokinetics (PK) and dose proportionality of mipomersen after single subcutaneous (SC) administration to Japanese healthy subjects; and to compare the PK profiles of Japanese and Western subjects. METHODS: 20 healthy first-generation Japanese male subjects were enrolled into one of three treatment cohorts (50, 100 and 200 mg SC) in a dose-escalation design. Within each cohort, subjects were randomized in a 4 : 1 ratio to receive mipomersen or placebo. RESULTS: Mipomersen was absorbed rapidly after SC administration; median tmax varied between 2 and 3 hours. After reaching peak levels, plasma concentrations of mipomersen decayed multiphasically with an initial distribution t1/2 in several hours and a terminal t1/2 of 261 - 393 hours. Mean Cmax increased in a dose-linear manner while all mean AUC from time 0 to different cut points increased slightly more than dose proportionally. Although mean terminal t1/2 varied in the dose range tested, it did not show dose-dependence. The PK profiles of mipomersen in Japanese subjects are similar to those observed in Western subjects. A single SC dose of 50 mg, 100 mg and 200 mg mipomersen was well tolerated by male Japanese subjects. CONCLUSION: Single SC doses of 50 - 200 mg were safe and well tolerated when administered to Japanese subjects. Comparison of PK between Japanese and Western subjects does not support any need for dose adjustment in Japanese population in future clinical development.

A randomized, placebo-controlled study of the pharmacokinetics, pharmacodynamics, and tolerability of the oral JAK2 inhibitor fedratinib (SAR302503) in healthy volunteers.

Fedratinib (SAR302503/TG101348) is a Janus kinase 2 (JAK2)-selective inhibitor in clinical development for the treatment of myelofibrosis. In this randomized, placebo-controlled, Phase 1 study, the pharmacokinetics, pharmacodynamics and tolerability of ascending single doses of fedratinib (10-680 mg) were assessed in healthy male subjects. Fedratinib was rapidly absorbed, with peak plasma concentration observed approximately 3 hours after dosing. The mean terminal half-life of fedratinib was approximately 67 hours, which was unaffected by dose. Fedratinib exposure increased in a greater than dose-proportional manner. Suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation, indicative of JAK2 inhibition, was observed at 3 hours post-dose for subjects in the 300, 500, and 680 mg groups, with the level of suppression increasing with dose. The relationship between fedratinib exposure and suppression of STAT3 phosphorylation was described using an inhibitory effect sigmoid Emax model, with an EC50 of 1,210 ng/mL in healthy subjects. The most common adverse events were mild gastrointestinal toxicities.

Mechanism of cytochrome P450-3A inhibition by ketoconazole.

OBJECTIVES: Ketoconazole is extensively used as an index inhibitor of cytochrome P450-3A (CYP3A) activity in vitro and in vivo, but the mechanism of ketoconazole inhibition of CYP3A still is not clearly established. METHODS: Inhibition of metabolite formation by ketoconazole (seven concentrations from 0.01 to 1.0 µm) was studied in human liver microsomes (n = 4) at six to seven substrate concentrations for triazolam, midazolam, and testosterone, and at two substrate concentrations for nifedipine. KEY FINDINGS: Analysis of multiple data points per liver sample based on a mixed competitive-noncompetitive model yielded mean inhibition constant K(i) values in the range of 0.011 to 0.045 µm. Ketoconazole IC50 increased at higher substrate concentrations, thereby excluding pure noncompetitive inhibition. For triazolam, testosterone, and midazolam α-hydroxylation, mean values of α (indicating the 'mix' of competitive and noncompetitive inhibition) ranged from 2.1 to 6.3. However, inhibition of midazolam 4-hydroxylation was consistent with a competitive process. Determination of K(i) and α based on the relation between 50% inhibitory concentration values and substrate concentration yielded similar values. Pre-incubation of ketoconazole with microsomes before addition of substrate did not enhance inhibition, whereas inhibition by troleandomycin was significantly enhanced by pre-incubation. CONCLUSIONS: Ketoconazole inhibition of triazolam α- and 4-hydroxylation, midazolam α-hydroxylation, testosterone 6ß-hydroxylation, and nifedipine oxidation appeared to be a mixed competitive-noncompetitive process, with the noncompetitive component being dominant but not exclusive. Quantitative estimates of K(i) were in the low nanomolar range for all four substrates.

Pharmacokinetic and pharmacodynamic study of two doses of bortezomib in patients with relapsed multiple myeloma.

PURPOSE: Characterize bortezomib pharmacokinetics/pharmacodynamics in relapsed myeloma patients after single and repeat intravenous administration at two doses. METHODS: Forty-two patients were randomized to receive bortezomib 1.0 or 1.3 mg/m(2), days 1, 4, 8, 11, for up to eight 21-day treatment cycles (n = 21, each dose group). Serial blood samples for pharmacokinetic/pharmacodynamic analysis were taken on days 1 and 11, cycles 1 and 3. Observational efficacy and safety data were collected. RESULTS: Twelve patients in each dose group were evaluable for pharmacokinetics/pharmacodynamics. Plasma clearance decreased with repeat dosing (102-112 L/h for first dose; 15-32 L/h following repeat dosing), with associated increases in systemic exposure and terminal half-life. Systemic exposures of bortezomib were similar between dose groups considering the relatively narrow dose range and the observed pharmacokinetic variability, although there was no readily apparent deviation from dose-proportionality. Blood 20S proteasome inhibition profiles were similar between groups with mean maximum inhibition ranging from 70 to 84% and decreasing toward baseline over the dosing interval. Response rate (all 42 patients) was 50%, including 7% complete responses. The safety profile was consistent with the predictable and manageable profile previously established; data suggested milder toxicity in the 1.0 mg/m(2) group. CONCLUSIONS: Bortezomib pharmacokinetics change with repeat dose administration, characterized by a reduction in plasma clearance and associated increase in systemic exposure. Bortezomib is pharmacodynamically active and tolerable at 1.0 and 1.3 mg/m(2) doses, with recovery toward baseline blood proteasome activity over the dosing interval following repeat dose administration, supporting the current clinical dosing regimen.

A quantitative framework and strategies for management and evaluation of metabolic drug-drug interactions in oncology drug development: new molecular entities as object drugs.

This article outlines general strategies for the management and evaluation of pharmacokinetic drug-drug interactions (DDIs) resulting from perturbation of clearance of investigational anticancer drug candidates by concomitantly administered agents in a drug development setting, with a focus on drug candidates that cannot be evaluated in first-in-human studies in healthy subjects. A risk level classification is proposed, based on quantitative integration of knowledge derived from preclinical drug-metabolism studies evaluating the projected percentage contribution [f(i)(%)] of individual molecular determinants (e.g. cytochrome P450 isoenzymes) to the overall human clearance of the investigational agent. The following classification is proposed with respect to susceptibility to DDIs with metabolic inhibitors: a projected maximum DDI expected to result in a ≤1.33-fold increase in exposure, representing a low level of risk; a projected maximum DDI expected to result in a >1.33-fold but <2-fold increase in exposure, representing a moderate level of risk; and a projected maximum DDI expected to result in a ≥2-fold increase in exposure, representing a potentially high level of risk. For DDIs with metabolic inducers, the following operational classification is proposed, based on the sum of the percentage contributions of enzymes that are inducible via a common mechanism to the overall clearance of the investigational drug: <<25%, representing a low level of risk; <50%, representing a moderate level of risk; and ≥50%, representing a potentially high level of risk. To ensure patient safety and to minimize bias in determination of the recommended phase II dose (RP2D), it is recommended that strong and moderate inhibitors and inducers of the major contributing enzyme are excluded in phase I dose-escalation studies of high-risk compounds, whereas exclusion of strong inhibitors and inducers of the contributing enzyme(s) is recommended as being sufficient for moderate-risk compounds. For drugs that will be investigated in diseases such as glioblastoma, where there may be relatively frequent use of enzyme-inducing antiepileptic agents (EIAEDs), a separate dose-escalation study in this subpopulation is recommended to define the RP2D. For compounds in the high-risk category, if genetic deficiencies in the activity of the major drug-metabolizing enzyme are known, it is recommended that poor metabolizers be studied separately to define the RP2D for this subpopulation. Whereas concomitant medication exclusion criteria that are utilized in the phase I dose-escalation studies will probably also need to be maintained for high-risk compounds in phase II studies unless the results of a clinical DDI study indicate the absence of a clinically relevant interaction, these exclusion criteria can potentially be relaxed beyond phase I for moderate-risk compounds, if supported by the nature of clinical toxicities and the understanding of the therapeutic index in phase I. Adequately designed clinical DDI studies will not only inform potential relaxation of concomitant medication exclusion criteria in later-phase studies but, importantly, will also inform the development of pharmacokinetically derived dose-modification guidelines for use in clinical practice when coupled with adequate safety monitoring, as illustrated in the prescribing guidance for many recently approved oncology therapeutics.

Sources of variability in ketoconazole inhibition of human cytochrome P450 3A in vitro.

Despite the extensive use of ketoconazole as an index inhibitor of human cytochrome P450 3A (CYP3A) isoforms in vitro, literature reports of the quantitative inhibitory potency of ketoconazole are highly variable. In 51 published studies reporting 76 values of ketoconazole inhibition constants (K(i)) versus in vitro clearance of 31 different CYP3A substrates, the K(i) values ranged from 0.001 µM to 25 µM. The geometric mean was 0.1 µM (90% confidence interval: 0.07 to 0.15 µM), and the median was 0.08 µM. Even for one specific substrate metabolized to one specific metabolite (midazolam α-hydroxylation), variability was still extensive (K(i) range: 0.004-0.18 µM). Only about 20% of overall variability in K(i) was explained by a combination of incubation, duration, and microsomal protein concentration. The remaining variation is unexplained, but could be attributable to factors such as: in vitro clearance by non-CYP3A pathways; incorrect assignment of inhibition mechanism; and variable relative content of CYP3A4 and CYP3A5 in different microsomal preparations. However, the role of these factors still is not established. Until sources of variation are more clearly defined, variability can be minimized by use of low microsomal protein concentrations, short incubation periods, and data analysis procedures that use untransformed reaction velocities and inhibition models that allow for mixed competitive-noncompetitive mechanisms.

Quantitative prediction and clinical observation of a CYP3A inhibitor-based drug-drug interactions with MLN3897, a potent C-C chemokine receptor-1 antagonist.

A novel in vitro model was recently developed in our laboratories for the prediction of magnitude of clinical pharmacokinetic drug-drug interactions (DDIs), based on reversible hepatic cytochrome P450 (P450) inhibition. This approach, using inhibition data from human hepatocytes incubated in human plasma, and quantitative P450 phenotyping data from hepatic microsomal incubations, successfully predicted DDIs for 15 marketed drugs with ketoconazole, a strong competitive inhibitor of CYP3A4/5, generally used to demonstrate a "worst-case scenario" for CYP3A inhibition. In addition, this approach was successfully extended to DDI predictions with the moderate competitive CYP3A inhibitor fluconazole for nine marketed drugs. In the current report, the general applicability of the model has been demonstrated by prospectively predicting the degree of inhibition and then conducting DDI studies in the clinic for an investigational CCR1 antagonist MLN3897, which is cleared predominantly by CYP3A. The clinical studies involved treatment of healthy volunteers (n = 17-20), in a crossover design, with ketoconazole (200 mg b.i.d.) or fluconazole (400 mg once a day), while receiving MLN3897. Administration of MLN3897 and ketoconazole led to an average 8.28-fold increase in area under the curve of plasma concentration-time plot (AUC) of MLN3897 at steady state, compared with the 8.33-fold increase predicted from the in vitro data. Similarly for fluconazole, an average increase of 3.93-fold in AUC was observed for MLN3897 in comparison with a predicted value of 3.26-fold. Thus, our model reliably predicted the exposure changes for MLN3897 in interaction studies with competitive CYP3A inhibitors in humans, further strengthening the utility of our in vitro model.

Role of NADPH-cytochrome P450 reductase and cytochrome-b5/NADH-b5 reductase in variability of CYP3A activity in human liver microsomes.

NADPH-cytochrome P450 reductase (CPR) and cytochrome-b(5) (b(5)) together with NADH-b(5) reductase (b(5)R) play important roles in cytochrome P450 3A-mediated drug metabolism via electron transfer. However, it is not clear whether variability in expression of these accessory proteins contributes to the known interindividual variability in CYP3A activity. CPR and b(5) were measured in human liver microsomes (HLMs) by spectrophotometry and immunoblotting. HLMs from elderly (>or=46 years) male donors (n=11) averaged 27% (P=0.034) and 41% (P=0.011) lower CPR levels than young (

Gender has a small but statistically significant effect on clearance of CYP3A substrate drugs.

The role of gender on the disposition of drugs metabolized by cytochrome P4503A (CYP3A) remains controversial. Some sources suggest that CYP3A activity in women exceeds that in men, but evidence to support this position is inconsistent at best. We evaluated 38 data sets in which clearance of CYP3A substrate drugs was studied in healthy young male and young female subjects. None of these drugs was a substrate for transport by P-glycoprotein (P-gp). The overall mean (+/-SE) for the female/male ratio of weight-normalized clearance was 1.26 (+/-0.07) for parenteral dosage and 1.17 (+/-0.07) for oral dosage. Both ratios were significantly different (P < .05) from 1.0. For oral dosage studies, the female/male clearance ratio was unrelated to the drug's absolute oral bioavailability. Thus gender has a small and statistically significant, although most likely clinically unimportant, influence on CYP3A phenotype for substrates not transported by P-gp.

Predicting interactions between conventional medications and botanical products on the basis of in vitro investigations.

The potential for various natural products to perturb the metabolism and disposition of medications has been recognized for decades. There are numerous in vitro and in vivo methods available to screen botanical products for drug interaction potential. Although many normal volunteer botanical-drug interaction studies have been performed, clearly, in vitro studies assessing the potential for drug interactions with various natural products represent the predominant type of published research performed to date. In addition to the recognized limitations of in vitro screening methodologies to assess conventional drug interactions, further difficulties emerge when examining botanical products. Primary challenges include assigning hepatic concentrations and accounting for bioavailability, distribution, first-pass metabolism and active metabolites. Additionally, variability in the chemical composition of commercially available botanical supplements, the lack of analytical standards and the inability to accurately screen the entities as mixtures add to complexities in experimental design. This mini-review is intended to address the particular problems and challenges in evaluating botanical supplements using in vitro methods, and review what can and cannot be learned from such investigations.

Ritonavir greatly impairs CYP3A activity in HIV infection with chronic viral hepatitis.

PURPOSE: Ritonavir is a powerful inhibitor of cytochrome P450 3A (CYP3A) that metabolizes many antiretrovirals. We examined the effect of ritonavir and of chronic viral hepatitis (CVH) status on CYP3A activity. METHODS: Twenty-six HIV-positive men (13 with CVH, 16 on chronic ritonavir-based highly active antiretroviral therapy) received oral and intravenous midazolam, a probe for CYP3A phenotypic activity. RESULTS: CYP3A activity was expressed as oral clearance of the midazolam probe. In HIV-positive subjects not on ritonavir, CYP3A activity (mean +/- SD) did not differ between subjects by CVH (no CVH, controls: 28.5 +/- 9.0 vs. CVH+: 23.2 +/- 6.2 mL/min/kg, not significant). In those on ritonavir (R), CYP3A activity was 7% of controls (R: 2.1 +/- 0.8 vs. no R 28.5 +/- 9.0 mL/min/kg, P < 0.0004). CYP3A activity in subjects on ritonavir and with CVH was further reduced to 4% of controls (no CVH, R+ 2.1 +/- 0.8 vs. R+, CVH+ 1.0 +/- 0.4 mL/min/kg, P < 0.006). CONCLUSIONS: Ritonavir markedly decreases CYP3A activity. In the presence of CVH, ritonavir-based therapy further reduces CYP3A activity by half. Coinfection with CVH impairs CYP3A activity in the presence of the CYP3A inhibitor ritonavir.

Induction of P-glycoprotein expression and activity by ritonavir in bovine brain microvessel endothelial cells.

Extended treatment with human immunodeficiency virus (HIV) protease inhibitors (HPIs) is standard in HIV/AIDS therapy. While these drugs have helped decrease the overall incidence of AIDS defining illnesses, the relative prevalence of HIV/AIDS dementia has increased. HPIs may cause induction of blood-brain barrier (BBB) drug transporters (P-glycoprotein; P-gp) and thereby limit entry of HPIs into brain tissue, increasing the probability that the brain could become an HIV sanctuary site. Using bovine brain microvessel endothelial cells (BMEC) as an in-vitro model of the BBB, the potential for the HIV protease inhibitor ritonavir to cause induction of P-gp activity and expression was examined. BMEC were isolated from fresh cow brain by enzymatic digest and density centrifugation. Primary culture BMEC were co-incubated with ritonavir or vehicle control for 120 h. Quantitative drug accumulation of rhodamine 123 (Rh123) and fluorescence microscopy were used as measures of P-gp activity. P-gp expression was assessed using quantitative Western blotting. Ritonavir decreased Rh123 cell accumulation and increased P-gp immunoreactive protein in a concentration-dependent manner. Fluorescent microscopy mirrored Rh123 quantitative studies. In BMEC pretreated with 30 microM ritonavir, Rh123 accumulation was decreased 40% and immunoreactive P-gp protein increased 2-fold. Collectively, a strong correlation between decreased Rh123 BMEC accumulation and increased P-gp immunoreactive protein was observed (Spearman r2 = 0.77, P < 0.0001). Thus extended exposure of BMEC to ritonavir caused a concentration-dependent increase in P-gp activity and expression. Similar findings may occur at the clinical level with prolonged HIV protease inhibitor use, giving insight into the central nervous system as an HIV sanctuary site and eventual development of HIV dementia.

Role of CYP3A and CYP2E1 in alcohol-mediated increases in acetaminophen hepatotoxicity: comparison of wild-type and Cyp2e1(-/-) mice.

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.

Human pregnane X receptor: genetic polymorphisms, alternative mRNA splice variants, and cytochrome P450 3A metabolic activity.

Human pregnane X receptor (hPXR) gene polymorphisms (spanning exon 2 to exon 5) and alternative mRNA splicing were investigated as possible contributors to individual variability in CYP3A metabolic activity measured both in vivo and in vitro. None of the 9 variants evaluated, including the 2 most common nonsynonymous variants (Pro27Ser and Gly36Arg), was found to be associated with midazolam 1'-hydroxylation rate measured in a bank of human livers (48 European Americans, 4 African Americans, 2 Hispanics). In contrast, 3 linked hPXR variants (g.252A > G, g.275A > G, and g.4760G > A) were significantly (P < .05) associated with oral midazolam clearance in a mixed race/ethnicity population (n = 26) and the African American subpopulation (n = 14) but not in European Americans (n = 9). Although the amount of hPXR mRNA normally spliced at the exon 4-5 junction correlated well with midazolam 1'-hydroxylation activities (P < .05), none of the 6 hPXR mRNA splice variants identified was associated with midazolam 1'-hydroxylation. In conclusion, several hPXR polymorphisms have been identified that may have predictive value for oral midazolam clearance, particularly in African Americans.

The novel UGT1A9 intronic I399 polymorphism appears as a predictor of 7-ethyl-10-hydroxycamptothecin glucuronidation levels in the liver.

Polymorphisms in UGT1A9 were associated with reduced toxicity and increased response to irinotecan in cancer patients. UDP-glucuronosyltransferase (UGT) protein expression, glucuronidation activities for 7-ethyl-10-hydroxycamptothecin (SN-38), and probe substrates of the UGT1A9 and UGT1A1 were measured in 48 human livers to clarify the role of UGT1A9 variants on the in vitro glucuronidation of SN-38. Genotypes were assessed for UGT1A9 (-2152C>T, -275T>A, and -118T(9>10)), three novel UGT1A9 variants (-5366G>T, -4549T>C, and I399C>T), and UGT1A1 (-53TA(6>7), -3156G>A, and -3279T>G). Of all the variants, the UGT1A9 I399C>T was associated with the most dramatic change in SN-38-glucuronide (SN-38G) (2.64-fold; p = 0.0007). Compared with UGT1A9 I399C/C, homozygous I399T/T presented elevated UGT1A1 and UGT1A9 proteins and higher glucuronidation of UGT1A9 and UGT1A1 substrates (p < 0.05). The very low linkage disequilibrium (r(2) < 0.19) between UGT1A9 I399 and all the other UGT1A1 and UGT1A9 variants suggests a direct effect or linkage to unknown functional variant(s) relevant to SN-38 glucuronidation. The UGT1A9 -118T(9/10) was also linked to alteration of SN-38 glucuronidation profiles in the liver (p < 0.05) and was associated with higher UGT1A1 protein (p = 0.03). However, UGT1A9 -118T(10) appears to have low functional impact as a result of the lack of correlation with UGT1A9 protein levels and a modest 1.4-fold higher reporter gene expression associated with the -118T(10) allele in HepG2 cells (p = 0.004). In contrast, the UGT1A9 -5366T, -4549C, -2152T, and -275A, associated with higher UGT1A9 protein (2-fold; p < 0.05), have no influence on SN-38G. Despite limitations resulting from sample size, results indicate that UGT1A9 I399 and -118T(9/10) may represent additional candidates in combination with UGT1A1 promoter haplotypes for the prediction of SN-38 glucuronidation profile in vivo.

Ritonavir has minimal impact on the pharmacokinetic disposition of a single dose of bupropion administered to human volunteers.

A drug-drug interaction study was conducted to determine whether ritonavir (200 mg; 4 doses over 2 days) alters the pharmacokinetic disposition of bupropion (75 mg; once) coadministered to 7 healthy volunteers in a placebo-controlled 2-way crossover study. Serum samples collected from 0 to 24 hours after bupropion administration were assayed for concentrations of bupropion and metabolites (hydroxybupropion, threohydrobupropion, and erythrohydrobupropion). Derived pharmacokinetic parameters were compared between placebo/bupropion and ritonavir/bupropion trials by paired t test. The effect of ritonavir on most pharmacokinetic parameters was minimal (<20% mean change). The only parameters that showed a statistically significant effect were threohydrobupropion area under the blood concentration curve (14% +/- 5% decrease, mean +/- SE; P = .04) and erythrohydrobupropion time-to-maximal serum concentration (161% +/- 92% increase, P = .03), suggesting that ritonavir may inhibit the carbonyl reductase enzyme responsible for formation of these metabolites. These findings indicate that short-term ritonavir dosing has only minimal impact on the pharmacokinetic disposition of a single dose of bupropion in healthy volunteers.

Factors influencing midazolam hydroxylation activity in human liver microsomes.

The cytochrome P450 3A (CYP3A) subfamily (mainly CYP3A4 and CYP3A5) is responsible for metabolizing approximately half of currently marketed drugs, but with considerable interindividual variability in expression and function. To investigate factors contributing to this variability, rates of midazolam (MDZ) 1'-hydroxylation and CYP3A4 and CYP3A5 protein content were determined using a set of 54 human liver microsomes (HLMs). Genetic factors investigated included CYP3A4 and CYP3A5 single nucleotide polymorphisms (SNPs) and haplotypes, CYP3A4 mRNA alternative splicing, and CYP3A4 transcriptional start and polyadenylation sites. Demographic and environmental factors investigated included age, gender, and histories of smoking and alcohol consumption. MDZ 1'-hydroxylation rates varied from 0.025 to 3.106 nmol/min/mg protein, with significant correlation to CYP3A4 protein content (r(s) = 0.92, P < 0.001) but less robust correlation to CYP3A5 protein content (r(s) = 0.60, P < 0.001). We identified eight CYP3A4 SNPs (five novel) and nine CYP3A5 SNPs (one novel), as well as seven CYP3A4 and two CYP3A5 haplotypes (all novel). No influence of genotype or haplotype on MDZ 1'-hydroxylation rate was observed, although CYP3A5*3A (g.6986a>g; g.31611c>t) carriers had lower CYP3A5 protein content compared with noncarriers (P = 0.004). No alternative splicing of CYP3A4 mRNA was found. Likewise, only a single transcriptional start site and polyadenylation site for CYP3A4 mRNA were identified. Subjects with a history of alcohol consumption had 2.2-fold higher median MDZ 1'-hydroxylation (P = 0.017), whereas no influence of age, gender, or smoking was evident. In conclusion, the investigated genetic factors did not contribute substantially to the large interindividual variability in midazolam hydroxylation, although alcohol consumption has a discernable but modest influence.