CYP3A Probe Research Articles

Vincristine-Induced Neuropathic Pain In A Cyp3a5 Non-Expresser With Reduced Cyp3a4 Activity

Vincristine is metabolised by CYP3A5 and CYP3A4 isoforms with CYP3A5 contributing to 75% of vincristine intrinsic clearance. Vincristine is a substrate of the P-glycoprotein (P-gp) transporter. An increase in vincristine neurotoxicity in CYP3A5 nonexpressers has been observed. The severity of neuropathy was found to be inversely correlated to vincristine metabolite concentrations. However, a clear correlation between genetic polymorphisms and vincristine toxicity has not been established. We report the case of a 21-year old African male patient who received vincristine 2 mg on 3 occasions (on 14.10, 30.10 and 06.11.14) for the treatment of pre-B acute lymphoblastic leukemia. Six weeks after the last vincristine dose the patient complained of bilateral severe burning pain in the toes and allodynia, suggestive of neuropathic pain. The patient was genotyped for CYP3A5 using a real-time PCR method as well as for ABCB1 (coding for P-gp) G2677T/A and C3435T SNPs. The results showed that the patient presented a CYP3A5*3/*3 polymorphism indicating that he did not express CYP3A5 enzyme. He was a homozygous ‘wild type’ carrier for ABCB1 SNPs. Furthermore, CYP activity was evaluated using the Geneva phenotyping cocktail including midazolam as a probe for CYP3A4. The patient had a decreased CYP3A activity which could not be explained by the concomitant medication. Similarly he had no CYP3A inhibitor in his medication at the time he received vincristine. The lack of CYP3A5 expression together with decreased CYP3A4 activity probably led to a decrease in vincristine clearance and to an increase in its plasma concentrations. It is a likely explanation for the occurrence of neurotoxicity in our patient despite the low doses of vincristine he received. In patients treated with vincristine, CYP phenotyping and genotyping could be crucial in preventing serious side effects.

Decreased elimination clearance of midazolam by doxorubicin through reductions in the metabolic activity of hepatic CYP3A in rats

1. We examined the effects of doxorubicin (DOX) on the expression level and metabolic activity of CYP3A in the liver as well as on the pharmacokinetics of midazolam (MDZ), a probe for CYP3A, in rats. Changes in the hepatic status of DOX-treated rats were confirmed.2. Serum levels of the biomarkers of hepatic impairment were elevated by the DOX treatment, which was consistent with the results obtained from a histopathological evaluation of the liver.3. No significant difference was observed in the expression of proteins for hepatic CYP3A1 and CYP3A2 between the DOX and control groups. The metabolic production of 1′-hydroxylated and 4′-hydroxylated MDZ by hepatic microsomes was significantly lower in DOX-treated rats than in control rats.4. The area under the curve (AUC) and the half-life (t1/2) of intravenously administered MDZ were significantly increased, and the total clearance (CLtot) and the elimination rate constant at the terminal phase (ke) were significantly decreased without significant changes in the volume of distribution at a steady state (Vdss).5. These results indicated that a DOX-induced depression in the metabolic activity, but not expression level of CYP3A contributed to a decrease in the elimination clearance of MDZ, and also that reduced CYP3A function may be associated with the hepatotoxicity of DOX.

Induction of CYP3A by morroniside in rats

Morroniside is one of the most important iridoid glycosides in the herbal drug Cornus officinalis Sieb. et Zucc. The current study was designed to investigate the ex vivo and in vivo effects of morroniside on CYP3A activity in rats after treatment with morroniside for 7 days (at 10, 30, 90 mg/kg, i.g.). Morroniside was found to induce CYP3A. According to the ex vivo experiment, the activity of CYP3A was measured by the quantification of 1-hydroxymidazolam, which was the metabolite from CYP3A probe substrate, midazolam. The concentration of 1-hydroxymidazolam was determined by using a validated liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) method. The levels of messenger RNA (mRNA) and protein of CYP3A were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting analysis, respectively. The pharmacokinetics of midazolam in rats after treatment with morroniside for 7 days (at 10, 30, 90 mg/kg, i.g.) were investigated in vivo. After treatment with morroniside, the activity, mRNA and protein expression of CYP3A were significantly induced and the absorbance and bioavailability of midazolam in rats were reduced. The results indicated that morroniside could induce the activity of CYP3A.

Pharmacokinetic interaction of intravenous fentanyl with ketoconazole.

Fentanyl is primarily metabolized by CYP3A, but has also been suggested to act as a weak inhibitor of CYP3A. We investigated the influence of CYP3A inhibition by ketoconazole on the pharmacokinetics of intravenously administered fentanyl and the effect of fentanyl on CYP3A activity. A prospective, open-label, randomized, monocentre, crossover study was conducted in 16 healthy volunteers. They received fentanyl alone (5 microgram per kilogram) or fentanyl plus ketoconazole (200 milligram orally B.I.D. over 2 days). Naloxone (2 × 0.2 milligram i.v.) was given simultaneously with fentanyl to mitigate any opioid effect. Midazolam was administered as a CYP3A probe drug. Fentanyl and its metabolites were quantified by LC/MS/MS in blood and urine samples obtained over 24 hour. Exposure of fentanyl (AUC0- ∞ ) was significantly increased to 133% and systemic clearance was reduced to 78% by ketoconazole, norfentanyl formation was significantly delayed and partial metabolic clearance decreased to 18%. Fentanyl had no influence on midazolam exposure and CYP3A activity whereas ketoconazole decreased CYP3A activity to 13%. Although fentanyl N-dealkylation is substantially inhibited by ketoconazole, exposure of fentanyl itself increased by one third only. Clinically fentanyl dosage adjustments may become necessary when ketoconazole or other strong CYP3A inhibitors are given simultaneously. Fentanyl itself does not influence CYP3A activity.

The influences of aconitine, an active/toxic alkaloid from aconitum, on the oral pharmacokinetics of CYP3A probe drug buspirone in rats.

Aconitine (AC), an active/toxic alkaloid from Aconitum species, is commonly present in Traditional Chinese Medicine (TCM) prescriptions because of the great effectiveness of Aconitum for the treatment of rheumatoid arthritis, cardiovascular diseases, and tumors in clinic. Buspirone (BP) is a sensitive CYP3A probe drug that is administered through oral/intravenous routes as recommended by the U.S. Food and Drug Administration. This study aims to investigate the influences of AC (0.125 mg/kg, oral) on first-pass (intestinal and hepatic) CYP3A activity by using oral BP as the probe in rats. The pharmacokinetics of oral buspirone hydrochloride at different doses (12.5, 25, and 50 mg/kg) were conducted. The pharmacokinetics of oral BP in rats pretreated with single dose or multiple doses (7-day) of AC were investigated. The plasma concentrations of BP and its major metabolites [1-(2-pyrimidinyl)piperazine (1-PP) and 6'-hydroxybuspirone (6'-OH-BP)] were determined. The formation ratios of 1-PP and 6'-OH-BP from BP (AUC0-∞ of 1-PP/AUC0-∞ of BP and AUC0-∞ of 6'-OH-BP/AUC0-∞ of BP values) showed no alternation when the dose of BP changed. Single dose of AC decreased the AUC0-∞ of BP by 53% but increased the formation ratio of 6'-OH-BP by 74% (P<0.05). Multiple AC exposure increased the AUC0-∞ of BP by 110%, and the formation ratios of 1-PP and 6'-OH-BP from BP were increased by 229% and decreased by 95%, respectively (P<0.05). Conclusively, single/multiple AC exposure did not alter the first-pass CYP3A activity when using oral BP as probe in rats. Nevertheless, multiple AC exposure had markedly changed the production of BP metabolites.

Change in pharmacokinetic behavior of intravenously administered midazolam due to increased CYP3A2 expression in rats treated with menthol.

Menthol is used widely as a constituent of functional foods and chemical drugs. The present study investigated changes in the pharmacokinetic behavior of intravenously administered midazolam (MDZ), a probe for CYP3A, when rats were treated with menthol. The study also examined which isoforms of CYP3A1 and 3A2 were menthol-inducible and contributed to the altered disposition of midazolam. Menthol was administered intraperitoneally to rats once daily for 3 days at a dose of 10 mg/kg, while the control rats received vehicle alone. The pharmacokinetic examination of i.v. administered midazolam revealed that serum midazolam concentrations at each sampling point were lower in the menthol-treated rats than in the control rats. Regarding the pharmacokinetic parameters of the menthol-treated group, the area under the curve (AUC) was decreased significantly and, correspondingly, the elimination rate constant at terminal phase (ke) was increased significantly without significant changes in the volume of distribution at steady state (Vdss). The metabolic production of the 1'-hydroxylated and 4'-hydroxylated forms of MDZ by hepatic microsomes was significantly greater in the menthol-treated rats than in the control rats. The expression levels of mRNA and protein for hepatic CYP3A2 were more than 2.5-fold higher than the control levels when the rats were treated with menthol, whereas no changes were observed in the expression levels of CYP3A1. These results indicate that menthol enhanced the elimination clearance of midazolam by inducing hepatic CYP3A2 and that careful attention should be paid when menthol is ingested in combination with drugs that act as substrates for CYP3A.

Pharmacokinetics interaction of dapoxetine with different doses of green tea extract in male healthy volunteers using midazolam as CYP3A4 enzyme probe

This study aimed to investigate the pharmacokinetics interaction of dapoxetine with different doses of green tea extract in healthy volunteers using midazolam (CYP3A4 probe). Method and materials: Twelve healthy males were included in a random three-way crossover study. Each volunteer received dapoxetine 60 mg and midazolam 7.5 mg concurrently after drinking 250 ml of water, 250 ml of fresh extract of 2 gram of green tea or 250 ml of fresh extract of 4 gram of green tea with one week washout period. Plasma samples were analyzed for dapoxetine and midazolam using HPLC. Results: The coadministration of dapoxetine with 4 gm grean tea extract significantly increased dapoxetine AUC∞ (from 3218.74 μg.hr/L to 4207.65 μg.hr/L, P

Gender, but Not CYP2C19 Genotypes and CYP3A Phenotypes, is a Major Determinant of Ilaprazole Pharmacokinetic

The purpose of the study was to assess the impact of CYP2C19 genotypes, CYP3A phenotypes and gender-related difference on the pharmacokinetics of new proton pump inhibitor ilaprazole. Twenty-four healthy Chinese volunteers (age 24.0  1.9 years) were enrolled in an open-label study stratified for gender (12 males and 12 females) and their CYP2C19 genotype (12 of CYP2C19*1/*1 and 12 of CYP2C19*1/*2 or *1/*3). After a single 10-mg dose of ilaprazole was administrated, blood samples were collected at time 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 24, 36h from all subjects. Ilaprazole and its metabolite sulfone-ilaprazole plasma concentrations were measured using the well-validated HPLC/MS/MS method. CYP3A phenotype was determined by the classic CYP3A probe drug midazolam one week after the clinical trial. The kinetics characteristics of ilaprazole and sulfone-ilaprazole were significantly influenced by gender. The clearance/systemic bioavailability (CL/F) of ilaprazole was much lower in female than in male (2.5  1.0 versus 3.7  1.6 h-1, P = 0.029), difference became more significant even after corrected by body weight (P = 0.008). However, the differences on half-life, AUC0-36 and AUC0→∞ of ilaprazole between genders were not significantly after normalized by body weight. As for sulfone ilaprazole, larger AUC0→36 and AUC0→∞ were detected in female when compared with male (406.8  126.3 vs. 246.7  70.0 ng • h/ml, P = 0.007, and 606.7  224.5 vs. 332.0  117.1 ng • h/ml, P = 0.001), discrepancies were still significant after corrected by total body weight, P value were 0.017 and 0.010 respectively. The pharmacokinetics parameters of ilaprazole and ilaprazole sulfone were neither different across CYP2C19 genotype groups nor related to CYP3A phenotype. CL/F of ilaprazole were much smaller in women than in men even after adjusted by body weight, indicating great effect of gender on the pharmacokinetics of ilaprazole. CYP2C19 genotypes and CYP3A phenotypes did not affect the pharmacokinetics of ilaprazole or sulfone-ilaprazole.

Effect of CKD and Dialysis Modality on Exposure to Drugs Cleared by Nonrenal Mechanisms

Patients with kidney disease frequently experience adverse effects from medication exposure, even when drugs are cleared by nonrenal pathways. Although many studies suggest that nonrenal drug clearance is decreased in chronic kidney disease (CKD), there remains a paucity of invivo studies in patients with varying degrees of decreased kidney function and those comparing the impact of dialysis modality (eg, hemodialysis [HD] and peritoneal dialysis [PD]). We performed invivo clinical pharmacokinetic studies of midazolam, a nonrenally cleared specific probe for CYP3A4, and fexofenadine, a nonspecific probe for hepatic and intestinal transporters. Healthy controls (n=8), patients with non-dialysis-dependent (NDD)-CKD (n=8), and patients receiving HD (n=10) or PD (n=8). Exposure to midazolam and fexofenadine were quantified using area under the curve (AUC). Comprehensive pharmacokinetic parameters also were calculated for both probes. Midazolam AUC was significantly higher in the HD group (382.8 h·ng/mL) than in the healthy-control (63.0 h·ng/mL; P<0.001), NDD-CKD (84.5 h·ng/mL; P=0.002), and PD (47.4 h·ng/mL; P<0.001) groups.Fexofenadine AUC was significantly higher in each of the NDD-CKD (2,950 h·ng/mL; P=0.003), HD (2,327 h·ng/mL; P=0.01), and PD (2,095 h·ng/mL; P=0.04) groups compared with healthy controls (1,008h·ng/mL). Small study groups had different proportions of diabetic patients, early stages of CKD not available. Our data suggest that selection of dialysis modality is a major determinant of exposure to the CYP3A4 probe midazolam. Exposure to the intestinal and hepatic transporter probe fexofenadine is altered in patients with NDD-CKD and PD and HD patients. Thus, drug development and licensing of nonrenally cleared drugs should include evaluation in these 3 patient groups, with these results included in approved product information labeling. This reinforces the critical need for more invivo studies of humans that evaluate the exposure to drugs cleared by these pathways.

Population pharmacokinetic modelling to assess the impact of CYP2D6 and CYP3A metabolic phenotypes on the pharmacokinetics of tamoxifen and endoxifen

Tamoxifen is considered a pro-drug of its active metabolite endoxifen. The major metabolic enzymes involved in endoxifen formation are CYP2D6 and CYP3A. There is considerable evidence that variability in activity of these enzymes influences endoxifen exposure and thereby may influence the clinical outcome of tamoxifen treatment. We aimed to quantify the impact of metabolic phenotype on the pharmacokinetics of tamoxifen and endoxifen. We assessed the CYP2D6 and CYP3A metabolic phenotypes in 40 breast cancer patients on tamoxifen treatment with a single dose of dextromethorphan as a dual phenotypic probe for CYP2D6 and CYP3A. The pharmacokinetics of dextromethorphan, tamoxifen and their relevant metabolites were analyzed using non-linear mixed effects modelling. Population pharmacokinetic models were developed for dextromethorphan, tamoxifen and their metabolites. In the final model for tamoxifen, the dextromethorphan derived metabolic phenotypes for CYP2D6 as well as CYP3A significantly (P < 0.0001) explained 54% of the observed variability in endoxifen formation (inter-individual variability reduced from 55% to 25%). We have shown that not only CYP2D6, but also CYP3A enzyme activity influences the tamoxifen to endoxifen conversion in breast cancer patients. Our developed model may be used to assess separately the impact of CYP2D6 and CYP3A mediated drug-drug interactions with tamoxifen without the necessity of administering this anti-oestrogenic drug and to support Bayesian guided therapeutic drug monitoring of tamoxifen in routine clinical practice.

Predictive Value of CYP3A and ABCB1 Phenotyping Probes for the Pharmacokinetics of Sunitinib: the ClearSun Study

The wide inter-patient variability in drug exposure partly explains the toxicity and efficacy profile of sunitinib treatment. In this prospective study cytochrome P450 (CYP) 3A and adenosine triphosphate binding cassette (ABC) B1 phenotypes were correlated to the pharmacokinetics of sunitinib and its active metabolite N-desethylsunitinib. A correlation analysis was performed between sunitinib pharmacokinetics and 1'OH-midazolam/midazolam ratio and parameters derived from technetium-99m-2-methoxy isobutyl isonitrile ((99m)Tc-MIBI) scans, respectively. A population pharmacokinetic model using non-linear mixed-effects modeling software NONMEM was built, which included the phenotype tests as covariate. In 52 patients, the mean trough concentration of sunitinib plus metabolite increased from 21.4 ng/mL at day 1 of a cycle to 88.1 ng/mL in the fourth week of treatment. A trend for a correlation was observed between (99m)Tc-MIBI elimination constant and trough concentrations of N-desethylsunitinib; however, this was not significant. Correlations were found between 1'OH-midazolam/midazolam ratio and sunitinib clearance (P = 0.008) and day 1 N-desethylsunitinib trough concentrations (P = 0.005), respectively. Moreover, patients suffering from grade 3 toxicities had significant lower clearance of sunitinib than patients without grade 3 toxicities (34.4 vs. 41.4 L/h; P = 0.025). Phenotype tests for ABCB1 and CYP3A4 did not explain inter-individual variability of sunitinib exposure sufficiently. However, the correlation between sunitinib clearance and the occurrence of severe toxicity suggests a direct exposure-toxicity relationship.

Contribution of baicalin on the plasma protein binding displacement and CYP3A activity inhibition to the pharmacokinetic changes of nifedipine in rats in vivo and in vitro.

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), Vd increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism.

CYP3A activity in severe liver cirrhosis correlates with Child–Pugh and model for end‐stage liver disease (MELD) scores

Impaired liver function often necessitates drug dose adjustment to avoid excessive drug accumulation and adverse events, but a marker for the extent of the required adjustment is lacking. The aim of this study was to investigate whether Child-Pugh (CP) and model for end-stage liver disease (MELD) scores correlate with drug clearance. Midazolam was used as a CYP3A probe and its pharmacokinetics were analyzed in 24 patients with mild to severe liver cirrhosis (n = 4, 10 and 10 with CP class A, B and C, respectively) and six patients without liver disease. Both scores correlated well with unbound midazolam clearance (CLu ), unbound midazolam fraction and half-life (all P < 0.01), whereas the unbound steady-state volume of distribution was not significantly changed. In patients with severe liver cirrhosis unbound midazolam clearance was only 14% of controls (CP C: CLu = 843 ± 346 l h(-1), MELD ≥ 15: CLu = 805 ± 474 l h(-1), controls: CLu = 5815 ± 2649 l h(-1), P < 0.01). The correlation with unbound midazolam clearance suggests that either score predicts the metabolic capacity of CYP3A, the most relevant drug metabolizing enzyme subfamily in humans.

Assessment of interaction potential of AZD2066 using in vitro metabolism tools, physiologically based pharmacokinetic modelling and in vivo cocktail data

Static and dynamic (PBPK) prediction models were applied to estimate the drug-drug interaction (DDI) risk of AZD2066. The predictions were compared to the results of an in vivo cocktail study. Various in vivo measures for tolbutamide as a probe agent for cytochrome P450 2C9 (CYP2C9) were also compared. In vitro inhibition data for AZD2066 were obtained using human liver microsomes and CYP-specific probe substrates. DDI prediction was performed using PBPK modelling with the SimCYP simulator™ or static model. The cocktail study was an open label, baseline, controlled interaction study with 15 healthy volunteers receiving multiple doses of AD2066 for 12days. A cocktail of single doses of 100mg caffeine (CYP1A2 probe), 500mg tolbutamide (CYP2C9 probe), 20mg omeprazole (CYP2C19 probe) and 7.5mg midazolam (CYP3A probe) was simultaneously applied at baseline and during the administration of AZD2066. Bupropion as a CYP2B6 probe (150mg) and 100mg metoprolol (CYP2D6 probe) were administered on separate days. The pharmacokinetic parameters for the probe drugs and their metabolites in plasma and urinary recovery were determined. In vitro AZD2066 inhibited CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6. The static model predicted in vivo interaction with predicted AUC ratio values of >1.1 for all CYP (except CYP3A4). The PBPK simulations predicted no risk for clinical relevant interactions. The cocktail study showed no interaction for the CYP2B6 and CYP2C19 enzymes, a possible weak inhibition of CYP1A2, CYP2C9 and CYP3A4 activities and a slight inhibition (29%) of CYP2D6 activity. The tolbutamide phenotyping metrics indicated that there were significant correlations between CLform and AUCTOL, CL, Aemet and LnTOL24h. The MRAe in urine showed no correlation to CLform. DDI prediction using the static approach based on total concentration indicated that AZD20066 has a potential risk for inhibition. However, no DDI risk could be predicted when a more in vivo-like dynamic prediction method with the PBPK with SimCYP™ software based on early human PK data was used and more parameters (i.e. free fraction in plasma, no DDI risk) were taken into account. The clinical cocktail study showed no or low risks for clinical relevant DDI interactions. Our findings are in line with the hypothesis that the dynamic prediction method predicts DDI in vivo in humans better than the static model based on total plasma concentrations.

Deoxyschizandrin, a Naturally Occurring Lignan, Is a Specific Probe Substrate of Human Cytochrome P450 3A

To accurately predict the modifications done during metabolic processes by cytochrome P450 (P450) 3A enzyme, selecting substrates that best represent a broad range of substrate substitutions and that follow the Michaelis-Menten kinetic properties is highly necessary. In the present study, the oxidative pathways of deoxyschizandrin (DS), the most abundant lignan in Fructus Schisandrae fruit extract, were characterized with liver microsomes from human (HLM) and rat (RLM). Only one monohydroxylated metabolite 7(S)-hydroxylated metabolite (isoschizandrin, ISZ), was identified using liquid chromatography-mass spectrometry and nuclear magnetic resonance techniques. CYP3A4 and CYP3A5 were found to be the major isoforms involved in the monohydroxylation of DS. Also, the kinetic studies showed that DS hydroxylation obeyed Michaelis-Menten kinetics both in HLM and in RLM. However, the subsequent metabolism of ISZ was nearly nonexistent when DS was present. More importantly, the interactions between DS and three well characterized CYP3A probe substrates, testosterone (TST), midazolam (MDZ), and nifedipine (NIF), were studied. TST and MDZ were shown to compete with DS for the mutual binding site, causing Km to be increased. The presence of DS also lowered the binding affinities for MDZ and TST. However, DS showed only slight inhibitory effects on nifedipine (NIF) oxidation even though NIF was able to inhibit DS hydroxylation in a noncompetitive fashion. These results show that DS is a good representative substrate of MDZ and TST primarily due to their shared, large binding regions on CYP3A. Therefore, DS is an attractive candidate as a novel CYP3A probe substrate for predicting the metabolic modifications in CYP3A activity.

A Systematic Approach to Evaluate Herb-Drug Interaction Mechanisms: Investigation of Milk Thistle Extracts and Eight Isolated Constituents as CYP3A Inhibitors

Despite increasing recognition of potential untoward interactions between herbal products and conventional medications, a standard system for prospective assessment of these interactions remains elusive. This information gap was addressed by evaluating the drug interaction liability of the model herbal product milk thistle (Silybum marianum) with the CYP3A probe substrate midazolam. The inhibitory effects of commercially available milk thistle extracts and isolated constituents on midazolam 1'-hydroxylation were screened using human liver and intestinal microsomes. Relative to vehicle, the extract silymarin and constituents silybin A, isosilybin A, isosilybin B, and silychristin at 100 μM demonstrated >50% inhibition of CYP3A activity with at least one microsomal preparation, prompting IC50 determination. The IC50s for isosilybin B and silychristin were ∼60 and 90 μM, respectively, whereas those for the remaining constituents were >100 μM. Extracts and constituents that contained the 1,4-dioxane moiety demonstrated a >1.5-fold shift in IC50 when tested as potential mechanism-based inhibitors. The semipurified extract, silibinin, and the two associated constituents (silybin A and silybin B) demonstrated mechanism-based inhibition of recombinant CYP3A4 (KI, ∼100 μM; kinact, ∼0.20 min(-1)) but not microsomal CYP3A activity. The maximum predicted increases in midazolam area under the curve using the static mechanistic equation and recombinant CYP3A4 data were 1.75-fold, which may necessitate clinical assessment. Evaluation of the interaction liability of single herbal product constituents, in addition to commercially available extracts, will enable elucidation of mechanisms underlying potential clinically significant herb-drug interactions. Application of this framework to other herbal products would permit predictions of herb-drug interactions and assist in prioritizing clinical evaluation.

Should Midazolam Drug–Drug Interactions be of Concern to Palliative Care Physicians?

I read the article Drug Interactions in Dying Patients: ARetrospective Analysis of Hospice Inpatients in Germany,written by Frechen et al. [1] with great interest. In thepalliative care setting, drug–drug interactions (DDIs) mustbe of special concern, especially in end-of-life care whenthe patient’s condition and organ functions deteriorate, thenumber of drugs taken often exceeds ten, and monitoring ofthe treatment becomes difficult. In the article, the authorsreviewed the main drug combinations that may give rise toDDIs in patients undergoing palliative care. However, Iwould like to also draw special attention to the risk of DDIswith benzodiazepines, and particularly midazolam, whichis classified by the Federal Union of German Associationsof Pharmacists (ABDA) database as a substance with a lowpotential for DDIs.It is well recognized that benzodiazepines carry a risk ofserious pharmacodynamic interactions with many drugsthat exert CNS depressant effects. Some of them are sub-strates of cytochrome P450 (CYP)3A and other CYPenzymes that raise additional risks of pharmacokineticinteractions through that system. These include diazepam,alprazolam, clonazepam, flunitrazepam, clorazepate andmidazolam. The latter is a short-acting benzodiazepine anda CYP3A4 probe substrate. It is available for oral andparenteral administration mostly subcutaneously andintravenously and used more commonly in the palliativecare setting in patients with difficulty swallowing and whendeath approaches, in the treatment of pain, dyspnea, anxi-ety, agitation and, more importantly, for brief and contin-uous palliative sedation in the management of refractorysymptoms and intolerable suffering. The review of theliterature shows that CYP3A inhibitors and inducers suchas antifungal azoles (ketokonazole, itraconazole, fluco-nazole, voriconazole), erythromycin, ritonavir, rifampicinand carbamazepine, among others, significantly change thedisposition of midazolam [2–8]. In one of the earlierstudies of intravenous midazolam, the area under theplasma concentration-time curve (AUC) increased as muchas fivefold after ketokonazole (strong CYP3A4 inhibitor)pretreatment, and intrinsic clearance decreased by 84 %[3]. Surely, the inhibition of midazolam metabolism wasgreater with oral administration (increase of total bio-availability from 25 to 80 % and 16-fold increase in AUCof midazolam) [3]. Such extensive alterations in midazo-lam pharmacokinetics should be expected to be of clinicalrelevance, which is supported by studies. The magnitude ofthe alterations in midazolam pharmacokinetics was welldemonstrated in a study by Backman et al. [9], in whichvolunteers subsequently received itraconazole for 4 daysand, after 2 weeks, rifampin (for a further 5 days).Midazolam was ingested before the first treatment, duringand 4 days after itraconazole as well as 1 and 4 days afterrifampin. During itraconazole treatment, the AUC

Effect of a herbal extract containing curcumin and piperine on midazolam, flurbiprofen and paracetamol (acetaminophen) pharmacokinetics in healthy volunteers.

Turmeric extract derived curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) are currently being evaluated for the treatment of cancer and Alzheimer's dementia. Previous in vitro studies indicate that curcuminoids and piperine (a black pepper derivative that enhances curcuminoid bioavailability) could inhibit human CYP3A, CYP2C9, UGT and SULT dependent drug metabolism. The aim of this study was to determine whether a commercially available curcuminoid/piperine extract alters the pharmacokinetic disposition of probe drugs for these enzymes in human volunteers. A randomized placebo-controlled six way crossover study was conducted in eight healthy volunteers. A standardized curcuminoid/piperine preparation (4 g curcuminoids plus 24 mg piperine) or matched placebo was given orally four times over 2 days before oral administration of midazolam (CYP3A probe), flurbiprofen (CYP2C9 probe) or paracetamol (acetaminophen) (dual UGT and SULT probe). Plasma and urine concentrations of drugs, metabolites and herbals were measured by HPLC. Subject sedation and electroencephalograph effects were also measured following midazolam dosing. Compared with placebo, the curcuminoid/piperine treatment produced no meaningful changes in plasma C(max), AUC, clearance, elimination half-life or metabolite levels of midazolam, flurbiprofen or paracetamol (α = 0.05, paired t-tests). There was also no effect of curcuminoid/piperine treatment on the pharmacodynamics of midazolam. Although curcuminoid and piperine concentrations were readily measured in plasma following glucuronidase/sulfatase treatment, unconjugated concentrations were consistently below the assay thresholds (0.05-0.08 μM and 0.6 μM, respectively). The results indicate that short term use of this piperine-enhanced curcuminoid preparation is unlikely to result in a clinically significant interaction involving CYP3A, CYP2C9 or the paracetamol conjugation enzymes.

1678P - Predictive Value of Two Phenotyping Probes for the Pharmacokinetics of Sunitinib in Cancer Patients

ABSTRACT Background Cancer patients treated with sunitinib show a wide inter-patient variability in drug exposure. This may at least partly explain the variation in toxicity and efficacy related to sunitinib treatment. The primary aim of this study was to investigate whether the clearance of 2 phenotyping probes are related to the pharmacokinetics (PK) of sunitinib. Patients and methods A prospective multi-center study was performed in cancer patients treated with single agent sunitinib. PK of sunitinib and its active metabolite N-desethyl-sunitinib were measured at start of sunitinib intake and within 4 weeks of continuous daily dosing. A correlation analysis was performed between these PK data and midazolam clearance (as a CYP3A phenotyping probe) and hepatic 99mTc-MIBI scans (as a probe for ABCB1 [P-glycoprotein] activity). Results A total of 52 GIST and renal cell cancer patients were included in this study, of whom 46 were evaluable for analysis of sunitinib PK. Mean trough level of sunitinib, adjusted for dose, at day 1 of a new course was 14.7 ng/mL (range 7.2-28.9 ng/mL), and 56.2 ng/mL (range 19.1-151.1 ng/mL) in the 4th week of a treatment cycle. A significant correlation between N-desethyl-sunitinib trough levels and plasma clearance of midazolam (r = 0.58, P = 0.006) within 24 hours after the first intake of sunitinib was found. In addition, a trend was seen for the correlation between midazolam clearance and sunitinib trough levels at steady state (r = 0.26, P = 0.09). No correlation between sunitinib trough levels and hepatic 99mTc-MIBI clearance was seen in an interim analysis after 27 patients. Conclusions These observations show a correlation between the midazolam clearance test that serves as a probe for CYP3A and the exposure to sunitinib. Meanwhile, a lack of correlation was seen between sunitinib PK and the studied ABCB1 probe. These data could ultimately lead to a further personalization of sunitinib dosing. Additional investigations are in progress, including population PK modeling of sunitinib, and incorporation of pharmacogenetic parameters to further unravel the mechanisms behind these findings. Disclosure All authors have declared no conflicts of interest.

An Investigation of the Bioactivation Potential and Metabolism Profile of Zebrafish versus Human

The zebrafish model has been increasingly explored as an alternative model for toxicity screening of pharmaceutical drugs. However, little is understood about the bioactivation of drug to reactive metabolite and phase I and II metabolism of chemical in zebrafish as compared with human. The primary aim of our study was to establish the bioactivation potential of zebrafish using acetaminophen as a probe substrate. Our secondary aim was to perform metabolite profiling experiments on testosterone, a CYP3A probe substrate, in zebrafish and compare the metabolite profiles with that of human. The glutathione trapping assay of N-acetyl-p-benzoquinone imine demonstrated that zebrafish generates the same reactive metabolite as humans from the bioactivation of acetaminophen. Zebrafish possesses functional CYP3A4/5-like and UDP-glucuronosyltransferase metabolic activities on testosterone. Differential testosterone metabolism was observed among the two species. In silico docking studies suggested that the zebrafish CYP3A65 was responsible for the bioactivation of acetaminophen and phase I hydroxylation of testosterone. Our findings reinforce the need to further characterize the drug metabolism phenotype of zebrafish before the model can fully achieve its potential as an alternative toxicity screening model in drug research.