CYP3A Probe Research Articles

Lack of a pharmacokinetic interaction between steady-state roflumilast and single-dose midazolam in healthy subjects.

The aim of this study was to investigate the effects of roflumilast, an investigational PDE4 inhibitor for the treatment of COPD and asthma, on the pharmacokinetics of the CYP3A probe drug midazolam and its major metabolites. In an open, randomized (for midazolam treatment sequence) study, 18 healthy male subjects received single doses of midazolam (2 mg oral and 1 mg i.v., 1 day apart) alone, repeated doses of roflumilast (500 microg once daily for 14 days) alone, and repeated doses of roflumilast together with single doses of midazolam (2 mg oral and 1 mg i.v., 1 day apart). A comparison of clearance and peak and systemic exposure to midazolam following administration of roflumilast indicated no effect of roflumilast dosed to steady state on the pharmacokinetics of midazolam. Point estimates (90% CI) were 0.97 (0.84, 1.13) for the AUC of i.v. midazolam and 0.98 (0.82, 1.17) for that of oral midazolam with and without roflumilast. Therapeutic steady state concentrations of roflumilast and its N-oxide do not alter the disposition of the CYP3A substrate midazolam in healthy subjects. This finding suggests that roflumilast is unlikely to alter the clearance of drugs that are metabolized by CYP3A4.

Metabolism of Endosulfan-α by Human Liver Microsomes and Its Utility as a Simultaneous in Vitro Probe for CYP2B6 and CYP3A4

Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.

Use of omeprazole sulfone in a single plasma sample as a probe for CYP3A4

The hydroxylation of omeprazole, measured as the ratio of omeprazole/5-hydroxyomeprazole in a plasma sample taken 3 h after an oral dose, is an established method to determine CYP2C19 activity, and the ratio of omeprazole AUC/omeprazole sulfone AUC has been used for assessing CYP3A4 activity. The aim of this study was to determine whether the latter ratio from a single 3-h sample can also be used for CYP3A4 phenotyping. Plasma levels of omeprazole and omeprazole sulfone were analyzed by reversed-phase high-performance liquid chromatography in a blood sample drawn 3 h after intake of a single oral 20-mg dose of omeprazole by 22 healthy subjects and five patients with newly diagnosed epilepsy. The procedure was repeated on the 4th day of 200 mg of ketoconazole intake (10 subjects), after 3 weeks of 150-200 mg twice-daily carbamazepine (five patients), and on the 6th day of 4 mg twice-daily tolterodine (12 subjects). Five subjects also took 100 mg and 50 mg of ketoconazole for 3 days before concomitant intake with omeprazole. The mean log10(omeprazole/omeprazole sulfone) ratio was 0.18 3 h after intake of omeprazole alone. After concomitant intake of ketoconazole, the corresponding value was 1.38 (p<0.001); after intake of carbamazepine it was -0.42 (p<0.05); and after tolterodine it was 0.29 (not significant). In the five subjects taking increasing doses of ketoconazole, the ratio was 0.11, 0.79, 1.2, and 1.5 after 0, 50, 100, and 200 mg of ketoconazole, respectively. The correlation between the metabolic ratios from the AUC((0-6h)) and from the single 3-h samples was very good, with a correlation coefficient of 0.92 (p<0.001). A single blood sample taken 3 h after intake of 20 mg of omeprazole can be reliably used to phenotype for both CYP2C19 and CYP3A4 activity.

Effect of tofisopam on the single-oral-dose pharmacokinetics and pharmacodynamics of the cyp3a4 probe drug alprazolam

Effect of tofisopam on the single-oral-dose pharmacokinetics and pharmacodynamics of the cyp3a4 probe drug alprazolam

Predicting Vinorelbine Disposition and Toxicity: Does BSA Provide More Than a “Bad Statistical Association”?

The concept that chemotherapeutic agents are administered at a dose to the maximum that a patient can tolerate before the onset of unacceptable toxicity, is still in wide clinical use today. However, the therapeutic range for most cytotoxic anticancer agents is extremely narrow, and in most cases no information is available before therapy on the intrinsic sensitivity of a patient’s tumor to a particular agent or the patient’s tolerability of a given dose. Hence, the dosage of chemotherapeutic agents remains largely empirical and it is typically only normalized to an individual’s body-surface area (BSA). From a pharmacologic perspective, interindividual differences in body size and body composition present a challenging role for clinicians, as their effects on the time course of drug response (ie, toxicity and efficacy) are still poorly understood. BSA does seem to be a biologically plausible size descriptor to consider in drug dose calculation as it is derived from height and weight, and because of the intuitive belief that patients with a larger BSA would require more drug to induce the same drug effects. Moreover, there are some data describing general physiologic characteristics being associated with BSA, including glomerular filtration rate, blood volume, and basal metabolic rate. In contrast, many studies have now convincingly demonstrated that BSA does not account for the marked differences between patients in drug clearance for many widely used agents, which may be due, in part, to large interindividual variability in drug-metabolizing enzyme activity that is known to exist irrespective of body size and body composition. Based on a retrospective analysis involving 33 different drugs, we have previously estimated that BSA-based dosing is statistically significantly associated with a reduction in interindividual pharmacokinetic variability in only approximately 15% of anticancer drugs. This notion has led several investigators to question the routine use of BSA in dosing strategies for anticancer agents. Overall, however, there is a deficit of knowledge on the influence of body size on interindividual pharmacodynamic variability that does not originate from differences between patients in a drug’s pharmacokinetic profile. In an attempt to overcome this information deficit, Wong et al describe the feasibility and pharmacokinetic/pharmacodynamic relationships for vinorelbine in a group of 41 cancer patients treated with a flat-fixed dose of 60 mg in this issue of the Journal of Clinical Oncology. Using rank correlation analyses, Wong et al demonstrate that BSA did not correlate with vinorelbine clearance, suggesting that BSA is not significantly contributing to explaining interindividual pharmacokinetic variability. Interestingly, among the variables tested, they found that BSA and vinorelbine clearance were the only statistically significant and independent predictors of the post-treatment fractional survival of neutrophils, used as a measure of vinorelbine-induced myelosuppression. The notion that BSA may predict toxicity based on an association with interindividual pharmacodynamic—as opposed to pharmacokinetic— variability is intriguing and warrants additional exploration for vinorelbine, as well as other myelosuppressive chemotherapeutic agents. Although the underlying mechanism for the possible link between BSA and hematologic toxicity remains to be elucidated, the previous finding that BSA is also a risk factor for severe leukopenia in patients treated with a vindesine/cisplatin-containing regimen suggests that this effect may not be limited to vinorelbine. Nonetheless, the currently observed association between BSA and vinorelbine-associated neutropenia shows rather wide scatter and hence better tools to individualize chemotherapeutic treatment with this agent are still highly desired. One of such possible tools evaluated by Wong et al is the application of probe drugs to predict the disposition characteristics of vinorelbine by determining the baseline phenotypic activity of two proteins of putative importance for drug distribution and elimination. Using midazolam, a widely used phenotypic probe for the cytochrome P450 isoforms CYP3A4 and CYP3A5, and technetium labeled sestamibi (99mTc-MIBI), which was used to determine the activity of the transporter protein ABCB1 (MDR1; P-glycoprotein), Wong et al found that only the latter was a significant independent predictor of vinorelbine clearance in the 34 assessable patients. Several factors need to be considered to understand the lack of relationship between the observed phenotypic CYP3A4 activity and vinorelbine clearance. Recent studies have indicated that CYP3A4 probably plays only a modest role in the metabolic breakdown of vinorelbine in vivo, and that this pathway is even relatively insensitive to inhibition (by ritonavir) and induction (by rifampin). Furthermore the ability of a probe drug to predict the metabolism of other CYP3A4 substrates will be compromised if differences in transporter affinities are not identified and fully taken into account. It logically follows that only once all factors contributing to the clearance of a particular CYP3A4 substrate drug are known, including nonmetabolic determinants such as reliance on JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 24 NUMBER 16 JUNE 1 2006

Investigation of sarizotan’s impact on the pharmacokinetics of probe drugs for major cytochrome P450 isoenzymes: a combined cocktail trial

The 5HT(1A) receptor agonist sarizotan is in clinical development for the treatment of dyskinesia, a potentially disabling complication in Parkinson's disease. We investigated the effect of sarizotan on the clinical pharmacokinetics of probe drugs for cytochrome P450 (CYP) to evaluate the risk of CYP-related drug-drug interactions. This was a double-blind, randomised, two-period cross-over interaction study with repeated administration of 5 mg sarizotan HCl or placebo b.i.d. for 8 days in 18 healthy volunteers. On day 4, a single dose of 100 mg metoprolol (CYP2D6 probe) was administered. On day 8, single doses of 100 mg caffeine (CYP1A2 probe), 50 mg diclofenac (CYP2C9 probe), 100 mg mephenytoin (CYP2C19 probe) and 7.5 mg midazolam (CYP3A4 probe) were simultaneously applied. Pharmacokinetic parameters for probe drugs and their metabolites in plasma and urinary recovery were determined. Concentration-time profiles and pharmacokinetic parameters of all probes and their metabolites remained unchanged after co-administration of sarizotan, compared with placebo. Analysis of variance of the area under the plasma concentration-time curve for probe drugs/metabolites, metabolic ratios and urinary excretion resulted in 90% confidence intervals within the acceptance range (0.8-1.25), indicating the absence of drug-drug interactions. At a dose higher than that intended for clinical use (1 mg b.i.d.), sarizotan had no effect on the metabolism and pharmacokinetics of specific probe drugs for CYP isoenzymes 1A2, 2C19, 2C9, 2D6 and 3A4. Pharmacokinetic interactions with co-administered drugs metabolised by these CYP isoforms are not expected, and dose adjustment of co-administered CYP substrates is not necessary.

Use of Omeprazole as a CYP3A Probe Drug: Effect of Sex and Menstrual Cycle Phase on CYP3A Activity in Healthy Caucasian Adults

To determine the effects of sex and menstrual cycle phase on CYP3A activity and to characterize the intraindividual variability of CYP3A, 24 Caucasian adults were given a single dose of omeprazole every 14th day for 3 months (men) or during the mid-follicular and mid-luteal phases over 3 full menstrual cycles (women). The 2-hour plasma metabolic ratio (MR) (omeprazole/omeprazole sulphone) was used as a measure of CYP3A activity. Overall mean MRs for men (2.4 +/- 1.2) and women (2.3 +/- 0.93) were not significantly different. In women, no difference in mean omeprazole MR was observed between the mid-follicular phase (2.2 +/- 0.85) and mid-luteal phase (2.4 +/- 1.0). When all data in the mid-luteal phase were stratified into 3 groups based on the progesterone concentrations (>50 nM [A], 20-50 nM [B], and <20 nM [C]), the MR of group A was significantly lower than that of B and C. The MRs of women during the mid-luteal phase were weakly correlated with progesterone concentrations (r = -0.35; P = .03). Individual coefficients of variation (CV%) in MR ranged from 8.7% to 60.2%, with a median 30.0%. No sex or menstrual cycle differences in CYP3A activity as measured by the probe drug omeprazole were seen. Higher CYP3A activity in women may be associated with higher endogenous progesterone concentrations.

Exploring the Relationship Between Expression of Cytochrome P450??Enzymes and Gefitinib Pharmacokinetics

Exposure to gefitinib (IRESSA, ZD1839), an epidermal growth factor receptor-tyrosine kinase inhibitor, is highly variable between subjects. In an attempt to explain this variability, three pharmacokinetic studies were carried out in healthy volunteers to investigate the relationship between exposure to gefitinib and cytochrome P450 (CYP) 3A phenotype (study 1), CYP3A5 genotype (study 2) and CYP2D6 genotype (study 3). In study 1 all 15 healthy volunteers received single oral doses of midazolam (7.5 mg), as a CYP3A probe, and gefitinib (500 mg), separated by an appropriate washout period. Plasma concentrations of midazolam and gefitinib were measured. In study 2, 73 healthy volunteers with previously defined single-dose gefitinib pharmacokinetic profiles were genotyped for CYP3A5. In study 3 a single oral dose of gefitinib (250 mg) was administered to poor and extensive CYP2D6 metabolisers (n = 15 in each group). Plasma concentrations of gefitinib and its major metabolite, M523595, were measured. Plasma concentrations of gefitinib, M523595 and midazolam were measured using high-performance liquid chromatography with tandem mass spectrometric detection, and appropriate pharmacokinetic parameters were determined by non-compartmental methods. Genetic analysis of CYP3A5 (study 2) and CYP2D6 (study 3) alleles was carried out using standard methodology. In study 1 there was some indication of a correlation between the area under the plasma concentration-time curve from time zero to infinity (AUCinfinity) values of midazolam and gefitinib, although this did not reach statistical significance (p = 0.062, regression analysis). In study 2 eight of 73 volunteers (11%) were identified as CYP3A5 expressers. No apparent relationship was observed between the occurrence of the CYP3A5 expresser genotype and gefitinib plasma clearance or terminal elimination halflife. In study 3 M523595 was not detected in any plasma samples collected from poor CYP2D6 metabolisers. Gefitinib geometric mean AUCinfinity and peak plasma drug concentration were higher in poor CYP2D6 metabolisers compared with extensive metabolisers (AUCinfinity 3060 vs 1430 ng . h/mL, p < 0.05, ANOVA), although the range of values was wide with considerable overlap between the groups. Gefitinib was well tolerated in both groups. Individual differences in CYP3A expression do not explain all the interindividual variability in gefitinib exposure. There is no apparent relationship between CYP3A5 genotype and gefitinib clearance. The lack of measurable levels of M523595 in poor CYP2D6 metabolisers confirms that production of this metabolite is mediated by CYP2D6. Although higher exposure to gefitinib occurs in individuals who are poor CYP2D6 metabolisers, genotyping prior to initiation of therapy and dosage adjustment are not warranted.

Alfentanil‐Induced Miosis Clearance as a Liver CYP3A4 and 3A5 Activity Measure in Healthy Volunteers: Improvement of Experimental Conditions

The aims of this study were to demonstrate the correlation between alfentanil-induced miosis evaluation and alfentanil pharmacokinetics (PK) as a CYP3A4 and 3A5 activity probe in volunteers and to explain the variability in pupilar response and in alfentanil PK. In ambient light, the miosis kinetic parameters were significantly correlated with PK (CLs: r = 0.9, P = .00; AUCs: r = 0.8, P = .01). In dark, a similar correlation was observed between miosis and alfentanil clearances (r = 0.85, P = .03). In 6 volunteers, the sigmoid E(max) model was applicable (average E(max) = 2.5 +/- 0.7 mm, gamma = 2.5 +/- 1.6 and EC(50) = 76.8 +/- 22.3 ng/mL), and in 3, the simple E(max) model was applicable (average E(max) = 2.8 +/- 0.3 mm and EC(50) = 19.9 +/- 8.5 ng/mL). There was a large interindividual variability in PK parameters (coefficient of variation = 19.7%-31.2%). Free drug fraction concentrations were negatively correlated with plasma alpha(1)-AGP (r = -0.9, P = .04) and albumin levels (r = -0.94, P = .02). Alfentanil-induced miosis clearance as a noninvasive CYP3A4 and 3A5 activity measure can be done in both ambient and dark conditions. Drug free fraction may be responsible for large intersubject variability in alfentanil PK.

Sensitivity of Intravenous and Oral Alfentanil and Pupillary Miosis as Minimally Invasive and Noninvasive Probes for Hepatic and First‐Pass CYP3A Activity

This investigation determined the ability of alfentanil miosis and single-point concentrations to detect various degrees of CYP3A inhibition. Results were compared with those for midazolam, an alternative CYP3A probe. Twelve volunteers were studied in a randomized 4-way crossover, targeting 12%, 25%, and 50% inhibition of hepatic CYP3A. They received 0, 100, 200, or 400 mg oral fluconazole, followed 1 hour later by 1 mg intravenous midazolam and then 15 microg/kg intravenous alfentanil 1 hour later. The next day, they received fluconazole, followed by 3 mg oral midazolam and 40 microg/kg oral alfentanil. Dark-adapted pupil diameters were measured coincident with blood sampling. Area under the plasma concentration-time curve (AUC) ratios (fluconazole/control) after 100, 200, and 400 mg fluconazole were (geometric mean) 1.3*, 1.4*, and 2.0* for intravenous midazolam and 1.2*, 1.6*, and 2.2* for intravenous alfentanil (*significantly different from control), indicating 16% to 21%, 31% to 36%, and 43% to 53% inhibition of hepatic CYP3A. Single-point concentration ratios were 1.5*, 1.8*, and 2.4* for intravenous midazolam (at 5 hours) and 1.2*, 1.6*, and 2.2* for intravenous alfentanil (at 4 hours). Pupil miosis AUC ratios were 0.9, 1.0, and 1.2*. After oral dosing, plasma AUC ratios were 2.3*, 3.6*, and 5.3* for midazolam and 1.8*, 2.9*, and 4.9* for alfentanil; plasma single-point ratios were 2.4*, 4.5*, and 6.9* for midazolam and 1.8*, 2.9*, and 4.9* for alfentanil, and alfentanil miosis ratios were 1.1, 1.9*, and 2.7*. Plasma concentration AUC ratios of alfentanil and midazolam were equivalent for detecting hepatic and first-pass CYP3A inhibition. Single-point concentrations were an acceptable surrogate for formal AUC determinations and as sensitive as AUCs for detecting CYP3A inhibition. Alfentanil miosis could detect 50% to 70% inhibition of CYP3A activity, but was less sensitive than plasma AUCs. Further refinements are needed to increase the sensitivity of alfentanil miosis for detecting small CYP3A changes.

CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95 ± 0.24 μM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 ± 0.21 μM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p = 0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75 μmol/mg protein/min) with lower P450 content (0.3 nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC 50 ≈ 21.6 μM) compared to DM induced (IC 50 ≈ 32.3 μM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10–30 μM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1000 μM) a nearly similar inhibition (76%) was observed than that at 50 μM concentration (68.2%).

CYP3A Probes Can Quantitatively Predict the In Vivo Kinetics of Other CYP3A Substrates and Can Accurately Assess CYP3A Induction and Inhibition

Because the rates at which therapeutics are cleared from the body can affect their effectiveness, knowing and accounting for the variables that contribute to drug clearance is of utmost importance when designing a drug dosage regimen for patients. The activities of the manifold cytochrome P450 enzymes [(CYPs), the most clinically important of which is often CYP3A] must be considered as they are essential for modification and metabolism of compounds (i.e., therapeutic, xenobiotic, etc.) prior to their excretion. To this end, much research has been expended on trying to identify and develop drug probes that accurately predict the metabolism of CYP3A substrates in individuals. Recently, Benet has written on the futility of such an enterprise; however, other researchers believe the identification of valuable predictive probes is not only possible but crucial.

Alprazolam as a probe for CYP3A using a single blood sample: pharmacokinetics of parent drug, and of α- and 4-hydroxy metabolites in healthy subjects

(1) To determine the pharmacokinetic parameters of alprazolam and its two metabolites in plasma from healthy volunteers; (2) to identify a suitable single time point to take a plasma sample for CYP3A phenotyping. Twelve healthy Swedish volunteers received a single oral dose of 1 mg alprazolam. Blood samples were collected before drug intake and frequently up to 72 h thereafter. A liquid-chromatography/mass-spectrometry (LC/MS) method was used for the quantification of alprazolam, and 4- and alpha-hydroxyalprazolam. The interindividual variation in the area under the concentration-time curve (AUC) was two, three and fourfold for alprazolam, 4-hydroxyalprazolam and alpha-hydroxyalprazolam, respectively. Plasma concentration ratios collected between 1 h and 48 h for both alprazolam/4-hydroxyalprazolam and alprazolam/alpha-hydroxyalprazolam correlated significantly to the corresponding AUC0-infinity ratios. The metabolic ratios of alprazolam to respective metabolite in a single plasma sample at 3-24 h are suggested to reflect the alprazolam 4- and alpha-hydroxylation activities. In future, it will be important to study these activities in populations where CYP3A5, in addition to CYP3A4, is expressed at a high frequency and to clarify the relative importance of the two enzymatic pathways for in vivo clearance of alprazolam.

CYP3A4 Substrate Selection and Substitution in the Prediction of Potential Drug-Drug Interactions

The complexity of in vitro kinetic phenomena observed for CYP3A4 substrates (homo- or heterotropic cooperativity) confounds the prediction of drug-drug interactions, and an evaluation of alternative and/or pragmatic approaches and substrates is needed. The current study focused on the utility of the three most commonly used CYP3A4 in vitro probes for the prediction of 26 reported in vivo interactions with azole inhibitors (increase in area under the curve ranged from 1.2 to 24, 50% in the range of potent inhibition). In addition to midazolam, testosterone, and nifedipine, quinidine was explored as a more "pragmatic" substrate due to its kinetic properties and specificity toward CYP3A4 in comparison with CYP3A5. Ki estimates obtained in human liver microsomes under standardized in vitro conditions for each of the four probes were used to determine the validity of substrate substitution in CYP3A4 drug-drug interaction prediction. Detailed inhibitor-related (microsomal binding, depletion over incubation time) and substrate-related factors (cooperativity, contribution of other metabolic pathways, or renal excretion) were incorporated in the assessment of the interaction potential. All four CYP3A4 probes predicted 69 to 81% of the interactions with azoles within 2-fold of the mean in vivo value. Comparison of simple and multisite mechanistic models and interaction prediction accuracy for each of the in vitro probes indicated that midazolam and quinidine in vitro data provided the best assessment of a potential interaction, with the lowest bias and the highest precision of the prediction. Further investigations with a wider range of inhibitors are required to substantiate these findings.

Oxymorphone Extended Release Does Not Affect CYP2C9 or CYP3A4 Metabolic Pathways

Two 14-day, randomized, open-label, parallel-group studies examined the effects of extended-release (ER) oxymorphone on CYP2C9 or CYP3A4 metabolic activities in healthy subjects. On days -1, 7, and 14, subjects received either a CYP2C9 probe (tolbutamide 500 mg) or CYP3A4 probes (midazolam and [14C N-methyl]-erythromycin for the erythromycin breath test). Subjects were randomized to 5 groups: high-dose oxymorphone ER (3 x 20 mg q12h) + naltrexone (50 mg q24h); low-dose oxymorphone ER (10-20 mg q12h); rifampin (2 x 300 mg q24h), an inducer of CYP2C9 and CYP3A4 activities; naltrexone (50 mg q24h); or CYP probes alone (controls). Probe metabolism was significantly altered by rifampin on days 7 and 14 (P < .05), whereas probe metabolism was not significantly affected by low-dose oxymorphone ER or by high-dose oxymorphone ER plus naltrexone. Oxymorphone ER exhibits a minimal potential for causing metabolic drug-drug interactions mediated by CYP2C9 or CYP3A4.

The ratio of 1-hydroxymidazolam (1-OH MDZ) to midazolam (MDZ) plasma concentrations is not an accurate measurement for CYP3A activity when using oral (PO) MDZ

Background The ratio of 1-OH MDZ to MDZ plasma concentrations has been proposed as an index to reduce sampling when measuring CYP3A activity. The aims of this study were to 1) assess if this ratio can be used to predict MDZ AUC when used as a CYP3A probe and 2) determine an optimal sampling strategy for the use of this ratio. Methods Data from 3 previous studies in 41 healthy adults were used. Subjects abstained from any drugs known to affect CYP3A. Plasma samples were collected at specified time points (Chr) after PO MDZ (0.075mg/kg) administration. 1-OH MDZ and MDZ concentrations were analyzed by LC/MS/MS. MDZ AUC0-∞ was determined by non-compartmental analysis. 20 subjects were randomly selected for the training set and the remaining 21 subjects for the validation set. The ratios of 1-OH MDZ to MDZ C0.5, 1, 2, 4, and 6 and AUC0-∞ were log-transformed. Stepwise multiple linear regression was used to derive equation models to predict MDZ AUC (AUCpred). Results One equation using C2 was statistically significant but had a low regression coefficient (R2=0.32): Log(AUCpred)=4.43–0.75[log(1-OH MDZ C2/MDZ C2)]. This equation did not meet the acceptable limits of bias and precision (Mean prediction error=−9.8%, Root mean square error=52.3%). Conclusions The ratio of 1-OH MDZ to MDZ plasma concentrations does not correlate with MDZ AUC and cannot be used to accurately predict MDZ exposure when PO MDZ is used as a CYP3A biomarker. Clinical Pharmacology & Therapeutics (2005) 77, P32–P32; doi: 10.1016/j.clpt.2004.12.016

Effect of tadalafil on cytochrome P450 3A4?mediated clearance: Studies in vitro and in vivo

Tadalafil was examined in vitro and in vivo for its ability to affect human cytochrome P450 (CYP) 3A-mediated metabolism. Reversible and mechanism-based inhibition of CYP3A by tadalafil was examined in human liver microsomes. The ability of tadalafil to influence CYP3A activity was also examined in primary cultures of human hepatocytes. The effect of tadalafil on the pharmacokinetics of CYP3A probe substrates was evaluated in human volunteers before and after coadministration with either a single dose or multiple doses of tadalafil (10 or 20 mg). Negligible competitive inhibition of CYP3A was observed in vitro. Mechanism-based inhibition of CYP3A was detected, albeit with a low potency. In human hepatocytes, exposure to 1 micromol/L or greater of tadalafil resulted in increased CYP3A protein expression; however, as with a combined effect of induction and inhibition, a corresponding increase in CYP3A activity did not occur. The clinical pharmacokinetics of midazolam and lovastatin, probe substrates of CYP3A, were unaffected by up to 14 days of tadalafil administration (90% confidence intervals for the ratio of least squares means for the pharmacokinetic parameters of tadalafil were contained within the no-effect boundaries of 0.7 to 1.43). In vitro results suggested that tadalafil would have little effect on the pharmacokinetics of drugs metabolized by CYP3A. Clinical studies demonstrated that the pharmacokinetics of 2 different CYP3A substrates, midazolam and lovastatin, were virtually unchanged after tadalafil coadministration. Thus therapeutic concentrations of tadalafil do not produce clinically significant changes in the clearance of drugs metabolized by CYP3A.

METABOLISM OF ALFENTANIL BY CYTOCHROME P4503A (CYP3A) ENZYMES

The synthetic opioid alfentanil is an analgesic and an in vivo probe for hepatic and first-pass CYP3A activity. Alfentanil is a particularly useful CYP3A probe because pupil diameter change is a surrogate for plasma concentrations, thereby affording noninvasive assessment of CYP3A. Alfentanil undergoes extensive CYP3A4 metabolism via two major pathways, forming noralfentanil and N-phenylpropionamide. This investigation evaluated alfentanil metabolism in vitro to noralfentanil and N-phenylpropionamide, by expressed CYP3A5 and CYP3A7 in addition to CYP3A4, with and without coexpressed or exogenous cytochrome b(5). Effects of the CYP3A inhibitors troleandomycin and ketoconazole were also determined. Rates of noralfentanil and N-phenylpropionamide formation by CYP3A4 and 3A5 in the absence of b(5) were generally equivalent, although the metabolite formation ratio differed, whereas those by CYP3A7 were substantially less. CYP3A4 and 3A5 were equipotently inhibited by troleandomycin, whereas ketoconazole was an order of magnitude more potent toward CYP3A4. Cytochrome b(5) qualitatively and quantitatively altered alfentanil metabolism, with b(5) coexpression having a greater effect than exogenous addition. Addition or coexpression of b(5) markedly stimulated the formation of both metabolites and changed the formation of noralfentanil but not N-phenylpropionamide from apparent single-site to multisite Michaelis-Menten kinetics. These results demonstrate that alfentanil is a substrate for CYP3A5 in addition to CYP3A4, and the effects of the CYP3A inhibitors troleandomycin and ketoconazole are CYP3A enzyme-selective. Alfentanil is one of the few CYP3A substrates that is metabolized in vitro as avidly by both CYP3A4 and 3A5. Polymorphic CYP3A5 expression may contribute to inter-individual variability in alfentanil metabolism.

Intravenous and oral alfentanil as in vivo probes for hepatic and first-pass cytochrome P450 3A activity: Noninvasive assessment by use of pupillary miosis

Systemic clearance of intravenous (IV) alfentanil (ALF) is an in vivo probe for hepatic cytochrome P450 (CYP) 3A activity, miosis is a surrogate for plasma ALF concentrations, and IV ALF miosis is a noninvasive probe for hepatic CYP3A. This investigation characterized the bioavailability and first-pass metabolism of oral ALF and tested the hypotheses that (1) first-pass ALF clearance reflects first-pass CYP3A activity, (2) miosis after oral ALF will reflect intestinal and hepatic CYP3A activity, and (3) miosis can approximate plasma concentration-based pharmacokinetic measures for IV and oral ALF as a noninvasive in vivo probe for hepatic and first-pass CYP3A activity and drug interactions. Results were compared with those for midazolam (MDZ), an alternative CYP3A probe. Ten volunteers were studied by use of a randomized, 9-way, crossover design after administration of rifampin (INN, rifampicin) (hepatic and intestinal CYP3A induction), troleandomycin (TAO) (hepatic and intestinal CYP3A inhibition), grapefruit juice (selective intestine CYP3A inhibition), or nothing (control). For each condition, they received 1 mg IV MDZ and then 15 microg/kg IV ALF, as well as 3 mg oral MDZ and then oral ALF (23 or 60 microg/kg) on another day. Plasma concentrations were determined by liquid chromatography-mass spectrometry. Dark-adapted pupil diameters were measured coincident with blood sampling. ALF effect was analyzed similarly to concentration to yield an effect "clearance" (Dose/Area under the pupil diameter change versus time curve). Bioavailability (Foral), hepatic extraction (EH), and intestinal availability (FG) were 0.26 +/- 0.08, 0.52 +/- 0.09, and 0.56 +/- 0.20, respectively, for MDZ and 0.42 +/- 0.15, 0.28 +/- 0.09, and 0.56 +/- 0.18, respectively, for ALF. Oral clearance (CL/F) was 34.7 +/- 12.8 and 10.9 +/- 3.5 mL.kg -1.min -1 , respectively, for MDZ and ALF. After rifampin, TAO, and grapefruit juice, ALF F oral was 0.04 +/- 0.02 (P <.05, versus control), 0.99 +/- 0.18 (P <.05, versus control), and 0.62 +/- 0.18 (P <.05, versus control), respectively; E H was 0.69 +/- 0.14 (P < .05, versus control), 0.04 +/- 0.01 (P <.05, versus control), and 0.26 +/- 0.08, respectively; F G was 0.16 +/- 0.10 (P <.05, versus control), 1.0 +/- 0.2 (P <.05, versus control), and 0.85 +/- 0.30 (P <.05, versus control), respectively; CL/F was 339 +/- 233 (P <.05, versus control), 0.62 +/- 0.26 (P <.05, versus control), and 6.7 +/- 2.5 (P <.05, versus control), respectively, and effect clearance was 2.1 +/- 1.1 (P <.05, versus control), 0.087 +/- 0.056 (P <.05, versus control), and 0.54 +/- 0.30 (0.73 +/- 0.43 mg.mm -1.h -1 in controls), respectively. There were significant correlations between ALF and MDZ systemic clearances (r2= 0.92), EH (r2=0.93), and CL/F (r2= 0.97), as well as between oral ALF effect (miosis) clearance and oral clearance (r2=0.59). ALF and MDZ have similar intestinal extraction but low and intermediate hepatic extraction, respectively. Systemic and oral clearances of ALF are excellent in vivo probes for hepatic and first-pass CYP3A activities and drug interactions. Miosis was an acceptable surrogate for plasma ALF. ALF miosis may be a suitable noninvasive in vivo probe for both hepatic and first-pass CYP3A.

Opioids: clinical pharmacology: Oxymorphone does not affect CYP450 enzymes 2C9 or 3A4: Positive implications for pain management

In vitro, oxymorphone does not inhibit or induce CYP450 enzymes at pharmacotherapeutic concentrations and only modestly affects CYP2C9 and CYP3A4 at suprapharmacologic concentrations (300x therapeutic levels). This study was conducted to investigate the in vivo potential for oxymorphone extended-release (ER) tablets to induce or inhibit CYP3A4 and 2C9 isoforms. Two randomized, open-label, parallel-group trials were conducted with 80 healthy adult volunteers per trial. On day -1, the subjects received 1 dose of a CYP2C9 probe (oral tolbutamide 500 mg) or CYP3A4 probes (oral midazolam and intravenous [14CH3 N-methyl]-erythromycin for the erythromycin breath test.) In both studies, subjects were randomized to 5 groups for 14 days of treatment with either high-dose oxymorphone ER (3x20 mg q12h)+oral naltrexone (50 mg q24h to minimize opioid effects); low-dose oxymorphone ER (10-20 mg q12h); oral rifampin (2x300 mg q24h), an inducer of CYP2C9 and CYP3A4 activities; naltrexone (50 mg q24h); or CYP probes alone. Tolbutamide area under the curve (AUC), hydroxytolbutamide:tolbutamide (H/T) urine ratio, midazolam AUC, and change from baseline in expired 14CO2 were determined on days -1, 7, and 14. In the oxymorphone ER and naltrexone groups, the changes from baseline to days 7 and 14 in midazolam and tolbutamide AUCs, H/T urine ratio, and the percentage of expired 14CO2/hour in ERMBT were not significantly different from the controls, whereas the positive control rifampin significantly changed the values of each of these parameters on days 7 and 14. Concomitant use of drugs metabolized by CYP450 enzymes may adversely affect plasma levels of one or both drugs. Oxymorphone ER has no effect on CYP450 enzymes, indicating a reduced risk of interactions with other agents (ie, SSRIs) commonly used by chronic pain patients. Endo Pharmaceuticals Inc. and Penwest Pharmaceuticals Co. provided partial support for this work.