Hyperthermia-enhanced targeted drug delivery using magnetic resonance-guided focussed ultrasound: a pre-clinical study in a genetic model of pancreatic cancer.

PURPOSE: The lack of effective treatment options for pancreatic cancer has led to a 5-year survival rate of just 8%. Here, we evaluate the ability to enhance targeted drug delivery using mild hyperthermia in combination with the systemic administration of a low-temperature sensitive liposomal formulation of doxorubicin (LTSL-Dox) using a relevant model for pancreas cancer. MATERIALS AND METHODS: Experiments were performed in a genetically engineered mouse model of pancreatic cancer (KPC mice: LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre). LTSL-Dox or free doxorubicin (Dox) was administered via a tail vein catheter. A clinical magnetic resonance-guided high intensity focussed ultrasound (MR-HIFU) system was used to plan treatment, apply the HIFU-induce hyperthermia and monitor therapy. Post-therapy, total Dox concentration in tumour tissue was determined by HPLC and confirmed with fluorescence microscopy. RESULTS: Localized hyperthermia was successfully applied and monitored with a clinical MR-HIFU system. The mild hyperthermia heating algorithm administered by the MR-HIFU system resulted in homogenous heating within the region of interest. MR-HIFU, in combination with LTSL-Dox, resulted in a 23-fold increase in the localised drug concentration and nuclear uptake of doxorubicin within the tumour tissue of KPC mice compared to LTSL-Dox alone. Hyperthermia, in combination with free Dox, resulted in a 2-fold increase compared to Dox alone. CONCLUSION: This study demonstrates that HIFU-induced hyperthermia in combination with LTSL-Dox can be a non-invasive and effective method in enhancing the localised delivery and penetration of doxorubicin into pancreatic tumours.

P-gp/ABCB1 exerts differential impacts on brain and fetal exposure to norbuprenorphine.

Norbuprenorphine is the major active metabolite of buprenorphine which is commonly used to treat opiate addiction during pregnancy. Norbuprenorphine produces marked respiratory depression and was 10 times more potent than buprenorphine. Therefore, it is important to understand the mechanism that controls fetal exposure to norbuprenorphine, as exposure to this compound may pose a significant risk to the developing fetus. P-gp/ABCB1 and BCRP/ABCG2 are two major efflux transporters regulating tissue distribution of drugs. Previous studies have shown that norbuprenorphine, but not buprenorphine, is a P-gp substrate. In this study, we systematically examined and compared the roles of P-gp and BCRP in determining maternal brain and fetal distribution of norbuprenorphine using transporter knockout mouse models. We administered 1mg/kg norbuprenorphine by retro-orbital injection to pregnant FVB wild-type, Abcb1a-/-/1b-/-, and Abcb1a-/-/1b-/-/Abcg2-/- mice on gestation day 15. The fetal AUC of norbuprenorphine was ∼64% of the maternal plasma AUC in wild-type mice, suggesting substantial fetal exposure to norbuprenorphine. The maternal plasma AUCs of norbuprenorphine in Abcb1a-/-/1b-/- and Abcb1a-/-/1b-/-/Abcg2-/- mice were ∼2 times greater than that in wild-type mice. Fetal AUCs in Abcb1a-/-/1b-/- and Abcb1a-/-/1b-/-/Abcg2-/- mice were also increased compared to wild-type mice; however, the fetal-to-maternal plasma AUC ratio remained relatively unchanged by the knockout of Abcb1a/1b or Abcb1a/1b/Abcg2. In contrast, the maternal brain-to-maternal plasma AUC ratio in Abcb1a-/-/1b-/- or Abcb1a-/-/1b-/-/Abcg2-/- mice was increased ∼30-fold compared to wild-type mice. Protein quantification by LC-MS/MS proteomics revealed significantly higher amounts of P-gp protein in the wild-type mice brain than that in the placenta. These results indicate that fetal exposure to norbuprenorphine is substantial and that P-gp has a minor impact on fetal exposure to norbuprenorphine, but plays a significant role in restricting its brain distribution. The differential impacts of P-gp on norbuprenorphine distribution into the brain and fetus are likely, at least in part, due to the differences in amounts of P-gp protein expressed in the blood-brain and blood-placental barriers. BCRP is not as important as P-gp in determining both the systemic and tissue exposure to norbuprenorphine. Finally, fetal AUCs of the metabolite norbuprenorphine-ß-d-glucuronide were 3-7 times greater than maternal plasma AUCs, while the maternal brain AUCs were <50% of maternal plasma AUCs, suggesting that a reversible pool of conjugated metabolite in the fetus may contribute to the high fetal exposure to norbuprenorphine.

Pharmacokinetic and pharmacodynamic analysis of inosine monophosphate dehydrogenase activity in hematopoietic cell transplantation recipients treated with mycophenolate mofetil.

A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplantation (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNCs) at 5 time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic-dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory maximum effect model with an IC50 of 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, nonrelapse mortality, and overall mortality. In conclusion, a pharmacokinetic-dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker.

Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients.

Maternal-fetal disposition of glyburide in pregnant mice is dependent on gestational age.

Gestational diabetes mellitus is a major complication of human pregnancy. The oral clearance (CL) of glyburide, an oral antidiabetic drug, increases 2-fold in pregnant women during late gestation versus nonpregnant controls. In this study, we examined gestational age-dependent changes in maternal-fetal pharmacokinetics (PK) of glyburide and metabolites in a pregnant mouse model. Nonpregnant and pregnant FVB mice were given glyburide by retro-orbital injection. Maternal plasma was collected over 240 minutes on gestation days (gd) 0, 7.5, 10, 15, and 19; fetuses were collected on gd 15 and 19. Glyburide and metabolites were quantified using high-performance liquid chromatography-mass spectrometry, and PK analyses were performed using a pooled data bootstrap approach. Maternal CL of glyburide increased approximately 2-fold on gd 10, 15, and 19 compared with nonpregnant controls. Intrinsic CL of glyburide in maternal liver microsomes also increased as gestation progressed. Maternal metabolite/glyburide area under the curve ratios were generally unchanged or slightly decreased throughout gestation. Total fetal exposure to glyburide was <5% of maternal plasma exposure, and was doubled on gd 19 versus gd 15. Fetal metabolite concentrations were below the limit of assay detection. This is the first evidence of gestational age-dependent changes in glyburide PK. Increased maternal glyburide clearance during gestation is attributable to increased hepatic metabolism. Metabolite elimination may also increase during pregnancy. In the mouse model, fetal exposure to glyburide is gestational age-dependent and low compared with maternal plasma exposure. These results indicate that maternal glyburide therapeutic strategies may require adjustments in a gestational age-dependent manner if these same changes occur in humans.

The presence of busulfan metabolites and pharmacometabolomics in plasma drawn immediately before allograft infusion in hematopoietic cell transplant recipients.

Busulfan is hepatically metabolized through glutathione (GSH) conjugation; in vitro, this process depletes hepatocyte GSH stores and generates the cytotoxic metabolite γ-glutamyldehydroalanylglycine, which is too unstable to be quantitated in vivo. We sought to evaluate if pre-graft (i.e., immediately before allograft infusion) concentrations of busulfan metabolites' and of endogenous metabolomic compounds (EMCs) representing the glutathione pathway were associated with clinical outcomes in hematopoietic cell transplant (HCT) recipients receiving busulfan. The clinical outcomes evaluated were relapse, acute graft versus host disease (GVHD), chronic GVHD, non-relapse mortality, and neutrophil nadir. In pre-graft samples obtained from patients immediately before allograft infusion, our objectives were to evaluate for: (1) the presence of busulfan and its metabolites tetrahydrothiophenium ion (THT+), tetrahydrothiophene 1-oxide, sulfolane, and 3-hydroxysulfolane (N = 124); (2) EMCs using a global metabolomics assay (N = 77); and (3) the association of the busulfan metabolites and the EMCs with clinical outcomes. In the pre-graft samples, busulfan and THT+ could not be detected. THT 1-oxide, sulfolane, and 3-hydroxysulfolane were quantitated in 9.6%, 26%, and 58% of pre-graft samples; their concentrations were not associated with clinical outcomes. Four pre-graft EMCs were statistically significantly associated with the neutrophil nadir. The pre-graft EMCs were not associated with the other clinical outcomes. In conclusion, busulfan's metabolites are present in patients' plasma immediately before allograft infusion; the neutrophil nadir is associated with pre-graft EMCs. Future research should investigate the association of clinical outcomes with the concentrations of busulfan's metabolites and EMCs in the pre-graft plasma from allogeneic HCT recipients.

Prediction of Busulfan Clearance by Predose Plasma Metabolomic Profiling.

Intravenous busulfan doses are often personalized to a target plasma exposure (targeted busulfan) using an individual's busulfan clearance (BuCL). We evaluated whether BuCL could be predicted by a predose plasma panel of 841 endogenous metabolomic compounds (EMCs). In this prospective cohort of 132 hematopoietic cell transplantation (HCT) patients, all had samples collected immediately before busulfan administration (preBU) and 96 had samples collected 2 weeks before busulfan (2-week-preBU). BuCL was significantly associated with 37 EMCs after univariate linear regression analysis and controlling for false discovery (< 0.05) in the 132 preBU samples. In parallel, with preBU samples, we included all 841 EMCs in a least absolute shrinkage and selection operator-penalized regression which selected 13 EMCs as predominantly associated with BuCL. Then, we constructed a prediction model by estimating coefficients for these 13 EMCs, along with sex, using ordinary least-squares. When the resulting linear prediction model was applied to the 2-week-preBU samples, it explained 40% of the variation in BuCL (adjusted R2  = 0.40). Pathway enrichment analysis revealed 18 pathways associated with BuCL. Lysine degradation followed by steroid biosynthesis, which aligned with the univariate analysis, were the top two pathways. BuCL can be predicted before busulfan administration with a linear regression model of 13 EMCs. This pharmacometabolomics method should be prioritized over use of a busulfan test dose or pharmacogenomics to guide busulfan dosing. These results highlight the potential of pharmacometabolomics as a precision medicine tool to improve or replace pharmacokinetics to personalize busulfan doses.

CYP2D6 mediates 4-hydroxylation of clonidine in vitro: implication for pregnancy-induced changes in clonidine clearance.

Clonidine is a centrally acting, alpha-2 adrenergic agonist used for the treatment of hypertension during pregnancy. The metabolic pathways of clonidine are poorly understood, and the quantitative contribution of specific human cytochrome P450 (P450) isoforms has not been systematically assessed. In this study, 17 cDNA-expressed P450 enzymes, in addition to pooled human liver microsomes, were evaluated for clonidine 4-hydroxylation activity in vitro. Five P450 enzymes-CYP2D6, 1A2, 3A4, 1A1, and 3A5-catalyzed measurable formation of 4-hydroxyclonidine. Selective inhibition studies in human liver microsomes confirmed that these isoforms are jointly responsible for 4-hydroxylation of clonidine in vitro, and CYP2D6 accounted for approximately two-thirds of the activity. The major role of CYP2D6 in clonidine metabolism might explain the increase in its nonrenal clearance during pregnancy.

Effect of N-Acetyltransferase 2 Genotype on the Pharmacokinetics of Hydralazine During Pregnancy.

Hydralazine, an antihypertensive agent used during pregnancy, undergoes N-acetylation primarily via N-acetyltransferase 2 (NAT2) to form 3-methyl-1,2,4-triazolo[3,4-a]phthalazine (MTP). To characterize the steady-state pharmacokinetics (PK) of hydralazine during pregnancy and evaluate the effects of NAT2 genotype on hydralazine and MTP PK during pregnancy, 12 pregnant subjects received oral hydralazine (5-25 mg every 6 hours) in mid- (n = 5) and/or late pregnancy (n = 8). Serial blood samples were collected over 1 dosing interval, and steady-state noncompartmental PK parameters were estimated. Subjects were classified as either (rapid acetylators, n = 6) or slow acetylators (SAs, n = 6) based on NAT2 genotype. During pregnancy, when compared with the SA group, the RA group had faster weight-adjusted hydralazine apparent oral clearance (70.0 ± 13.6 vs 20.1 ± 6.9 L/h, P < .05), lower dose-normalized area under the concentration-time curve (AUC; 1.5 ± 0.8 vs 5.9 ± 3.7 ng·h/mL, P < .05), lower dose-normalized peak concentrations (0.77 ± 0.51 vs 4.04 ± 3.18 ng/mL, P < .05), and larger weight-adjusted apparent oral volume of distribution (302 ± 112 vs 116 ± 45 L/kg, P < .05). Furthermore, the MTP/hydralazine AUC ratio was ∼10-fold higher in the RA group (78 ± 30 vs 8 ± 3, P < .05) than in the SA group. No gestational age or dose-dependent effects were observed, possibly because of the small sample size. This study describes for the first time, the PK of oral hydralazine and its metabolite, MTP, during pregnancy, and confirmed that the PK of oral hydralazine is NAT2 genotype dependent during pregnancy.

An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers.

OBJECTIVES: The co-primary objectives of this study were to determine the human pharmacokinetics (PK) of oral NR and the effect of NR on whole blood nicotinamide adenine dinucleotide (NAD+) levels. BACKGROUND: Though mitochondrial dysfunction plays a critical role in the development and progression of heart failure, no mitochondria-targeted therapies have been translated into clinical practice. Recent murine studies have reported associations between imbalances in the NADH/NAD+ ratio with mitochondrial dysfunction in multiple tissues, including myocardium. Moreover, an NAD+ precursor, nicotinamide mononucleotide, improved cardiac function, while another NAD+ precursor, nicotinamide riboside (NR), improved mitochondrial function in muscle, liver and brown adipose. Thus, PK studies of NR in humans is critical for future clinical trials. METHODS: In this non-randomized, open-label PK study of 8 healthy volunteers, 250 mg NR was orally administered on Days 1 and 2, then uptitrated to peak dose of 1000 mg twice daily on Days 7 and 8. On the morning of Day 9, subjects completed a 24-hour PK study after receiving 1000 mg NR at t = 0. Whole-blood levels of NR, clinical blood chemistry, and NAD+ levels were analyzed. RESULTS: Oral NR was well tolerated with no adverse events. Significant increases comparing baseline to mean concentrations at steady state (Cave,ss) were observed for both NR (p = 0.03) and NAD+ (p = 0.001); the latter increased by 100%. Absolute changes from baseline to Day 9 in NR and NAD+ levels correlated highly (R2 = 0.72, p = 0.008). CONCLUSIONS: Because NR increases circulating NAD+ in humans, NR may have potential as a therapy in patients with mitochondrial dysfunction due to genetic and/or acquired diseases.

Rapid and sensitive analysis of reduced and oxidized coenzyme Q10 in human plasma by ultra performance liquid chromatography-tandem mass spectrometry and application to studies in healthy human subjects.

BACKGROUND: Coenzyme Q10 is an endogenous antioxidant as well as a popular dietary supplement. In blood circulation, coenzyme Q10 exists predominantly as its reduced ubiquinol-10 form, which readily oxidizes to ubiquinone-10 ex vivo. Plasma concentrations of coenzyme Q10 reflect net overall metabolic demand, and the ratio of ubiquinol-10:ubiquinone-10 has been established as an important biomarker for oxidative stress. However, the lability of ubiquinol-10 makes accurate determination of both forms of coenzyme Q10 difficult. Ex vivo oxidation of ubiquinol-10 to ubiquinone-10 during sample collection, processing and analysis may obfuscate the in vivo ratio. METHODS: We developed a rapid and sensitive method for the determination of ubiquinol-10 and ubiquinone-10 in human plasma, using coenzyme Q9 analogues as internal standards. Single-step protein precipitation in 1-propanol, a lipophilic and water-soluble alcohol, allowed for rapid extraction. RESULTS: Analysis by ultra performance liquid chromatography-tandem mass spectrometry provided rapid run-time and high sensitivity, with lower limits of quantitation for ubiquinol-10 and ubiquinone-10 of 5 µg/L and 10 µg/L, respectively. CONCLUSIONS: This method is suitable for clinical studies with coenzyme Q10 supplementation in various disease states where this lipid-antioxidant may be beneficial. We have applied this method to >300 plasma samples from coenzyme Q10 research studies in chronic haemodialysis patients and postsurgical patients.

Pulsed High-Intensity Focused Ultrasound Enhances Delivery of Doxorubicin in a Preclinical Model of Pancreatic Cancer.

Pancreatic cancer is characterized by extensive stromal desmoplasia, which decreases blood perfusion and impedes chemotherapy delivery. Breaking the stromal barrier could both increase perfusion and permeabilize the tumor, enhancing chemotherapy penetration. Mechanical disruption of the stroma can be achieved using ultrasound-induced bubble activity-cavitation. Cavitation is also known to result in microstreaming and could have the added benefit of actively enhancing diffusion into the tumors. Here, we report the ability to enhance chemotherapeutic drug doxorubicin penetration using ultrasound-induced cavitation in a genetically engineered mouse model (KPC mouse) of pancreatic ductal adenocarcinoma. To induce localized inertial cavitation in pancreatic tumors, pulsed high-intensity focused ultrasound (pHIFU) was used either during or before doxorubicin administration to elucidate the mechanisms of enhanced drug delivery (active vs. passive drug diffusion). For both types, the pHIFU exposures that were associated with high cavitation activity resulted in disruption of the highly fibrotic stromal matrix and enhanced the normalized doxorubicin concentration by up to 4.5-fold compared with controls. Furthermore, normalized doxorubicin concentration was associated with the cavitation metrics (P < 0.01), indicating that high and sustained cavitation results in increased chemotherapy penetration. No significant difference between the outcomes of the two types, that is, doxorubicin infusion during or after pHIFU treatment, was observed, suggesting that passive diffusion into previously permeabilized tissue is the major mechanism for the increase in drug concentration. Together, the data indicate that pHIFU treatment of pancreatic tumors when resulting in high and sustained cavitation can efficiently enhance chemotherapy delivery to pancreatic tumors. .

Maintenance of high-density lipoprotein blood levels prior to spontaneous abortion in pig-tailed macaques (Macaca nemestrina).

Blood was drawn throughout the first half of the pregnancies of 24 pig-tailed macaques (Macaca nemestrina) to evaluate longitudinal high-density lipoprotein (HDL) changes. In all 15 normal pregnancies, HDL decreased at least 50%; the mean value for the group fell from 0.45 gm/liter to 0.17 gm/liter. HDL began to fall after about four weeks of pregnancy. However, no comparable HDL change occurred in nine pregnancies that terminated in spontaneous abortions. This lack of an HDL decrease was unexpected. Subsequent studies showed that the predominant decrease was in the HDL2 subfraction. The data indicate that the normal physiologic metabolism or utilization of HDL is aberrant early in pregnancies ending in spontaneous abortions and may be due to a dysfunctional fetal-placental unit.

Identification of CYP3A7 for glyburide metabolism in human fetal livers.

Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u=37.1, 13.0, and 8.7ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1-M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4'-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers.

Contribution of CYP3A5 to hepatic and renal ifosfamide N-dechloroethylation.

Ifosfamide nephrotoxicity is attributed to the formation of a toxic metabolite, chloroacetaldehyde, via N-dechloroethylation, a reaction that is purportedly catalyzed by CYP3A and CYP2B6. Because allelic variants of CYP3A5 are associated with polymorphic expression of microsomal CYP3A5 in human liver and kidneys, we hypothesized that ifosfamide N-dechloroethylation depends on CYP3A5 genotype. We compared ifosfamide N-dechloroethylation activity in cDNA-expressed CYP3A4 and CYP3A5. Ifosfamide N-dechloroethylation was also assessed in liver (N = 20) and kidney (N = 21) microsomes from human donors with different CYP3A5 genotypes. Ifosfamide N-dechloroethylation was catalyzed by recombinant CYP3A5 at a rate comparable with recombinant CYP3A4. In human liver microsomes matched for CYP3A4 protein content, N-dechloroethylation was more than 2-fold higher in that from donors carrying CYP3A5*1 allele that express CYP3A5 relative to that from donors homozygous for the mutant CYP3A5*3. Correlation analysis revealed that ifosfamide N-dechloroethylation was significantly associated with CYP3A4 and CYP3A5 protein concentration but not with age, sex, or CYP2B6 protein concentration. In hepatic microsomes not expressing CYP3A5 protein, ifosfamide N-dechloroethylation was inhibited 53 to 61% and 0 to 3% by monoclonal antibodies specific for CYP3A4/5 or CYP2B6, respectively. Ifosfamide N-dechloroethylation was not detected in renal microsomes obtained from CYP3A5*3/*3 donors. In contrast, it was readily measurable in microsomes isolated from four kidneys of CYP3A5*1 carriers, which was almost completely inhibited by the CYP3A inhibitor ketoconazole. CYP2B6 protein could not be detected in this panel of human renal microsomes. In conclusion, CYP3A5*1 genotype is associated with higher rates of ifosfamide N-dechloroethylation in human liver and kidneys.