Genome-wide association study identifies the common variants in CYP3A4 and CYP3A5 responsible for variation in tacrolimus trough concentration in Caucasian kidney transplant recipients.

The immunosuppressant tacrolimus (TAC) is metabolized by both cytochrome P450 3A4 (CYP3A4) and CYP3A5 enzymes. It is common for European Americans (EA) to carry two CYP3A5 loss-of-function (LoF) variants that profoundly reduces TAC metabolism. Despite having two LoF alleles, there is still considerable variability in TAC troughs and identifying additional variants in genes outside of the CYP3A5 gene could provide insight into this variability. We analyzed TAC trough concentrations in 1345 adult EA recipients with two CYP3A5 LoF alleles in a genome-wide association study. Only CYP3A4*22 was identified and no additional variants were genome-wide significant. Additional high allele frequency genetic variants with strong genetic effects associated with TAC trough variability are unlikely to be associated with TAC variation in the EA population. These data suggest that low allele frequency variants, identified by DNA sequencing, should be evaluated and may identify additional variants that contribute to TAC pharmacokinetic variability.

Genotype-guided tacrolimus dosing in African-American kidney transplant recipients.

Tacrolimus is dependent on CYP3A5 enzyme for metabolism. Expression of the CYP3A5 enzyme is controlled by several alleles including CYP3A5*1, CYP3A5*3, CYP3A5*6 and CYP3A5*7. African Americans (AAs) have on average higher tacrolimus dose requirements than Caucasians; however, some have requirements similar to Caucasians. Studies in AAs have primarily evaluated the CYP3A5*3 variant; however, there are other common nonfunctional variants in AAs (CYP3A5*6 and CYP3A5*7) that do not occur in Caucasians. These variants are associated with lower dose requirements and may explain why some AAs are metabolically similar to Caucasians. We created a tacrolimus clearance model in 354 AAs using a development and validation cohort. Time after transplant, steroid and antiviral use, age and CYP3A5*1, *3, *6 and *7 alleles were significant toward clearance. This study is the first to develop an AA-specific genotype-guided tacrolimus dosing model to personalize therapy.

Genomewide Association Study of Tacrolimus Concentrations in African American Kidney Transplant Recipients Identifies Multiple CYP3A5 Alleles.

We previously reported that tacrolimus (TAC) trough blood concentrations for African American (AA) kidney allograft recipients were lower than those observed in white patients. Subtherapeutic TAC troughs may be associated with acute rejection (AR) and AR-associated allograft failure. This variation in TAC troughs is due, in part, to differences in the frequency of the cytochrome P450 CYP3A5*3 allele (rs776746, expresses nonfunctional enzyme) between white and AA recipients; however, even after accounting for this variant, variability in AA-associated troughs is significant. We conducted a genomewide association study of TAC troughs in AA kidney allograft recipients to search for additional genetic variation. We identified two additional CYP3A5 variants in AA recipients independently associated with TAC troughs: CYP3A5*6 (rs10264272) and CYP3A5*7 (rs41303343). All three variants and clinical factors account for 53.9% of the observed variance in troughs, with 19.8% of the variance coming from demographic and clinical factors including recipient age, glomerular filtration rate, anticytomegalovirus drug use, simultaneous pancreas-kidney transplant and antibody induction. There was no evidence of common genetic variants in AA recipients significantly influencing TAC troughs aside from the CYP3A gene. These results reveal that additional and possibly rare functional variants exist that account for the additional variation.

Studies on induction of lamotrigine metabolism in transgenic UGT1 mice.

A transgenic 'knock-in' mouse model expressing a human UGT1 locus (Tg-UGT1) was recently developed and validated. Although these animals express mouse UGT1A proteins, UGT1A4 is a pseudo-gene in mice. Therefore, Tg-UGT1 mice serve as a 'humanized' UGT1A4 animal model. Lamotrigine (LTG) is primarily metabolized to its N-glucuronide (LTGG) by hUGT1A4. This investigation aimed at examining the impact of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPAR) activators on LTG glucuronidation in vivo and in vitro. Tg-UGT1 mice were administered the inducers phenobarbital (CAR), pregnenolone-16alpha-carbonitrile (PXR), WY-14643 (PPAR-alpha), ciglitazone (PPAR-gamma), or L-165041 (PPAR-beta), once daily for 3 or 4 days. Thereafter, LTG was administered orally and blood samples were collected over 24 h. LTG was measured in blood and formation of LTGG was measured in pooled microsomes made from the livers of treated animals. A three-fold increase in in vivo LTG clearance was seen after phenobarbital administration. In microsomes prepared from phenobarbital-treated Tg-UGT1 animals, 13-fold higher CL(int) (Vmax/K(m)) value was observed as compared with the untreated transgenic mice. A trend toward induction of catalytic activity in vitro and in vivo was also observed following pregnenolone-16alpha-carbonitrile and WY-14643 treatment. This study demonstrates the successful application of Tg-UGT1 mice as a novel tool to study the impact of induction and regulation on metabolism of UGT1A4 substrates.

Variation in glucuronidation of lamotrigine in human liver microsomes.

Lamotrigine (LTG), a diaminotriazine anti-epileptic, is principally metabolized at the 2-position of the triazine ring to form a quaternary ammonium glucuronide (LTGG) by uridine glucuronosyl transferease (UGT) 1A3 and UGT1A4. It has been hypothesized that glucuronidation of anti-epileptic drugs is spared with age, despite a known decrease in liver mass, based on older studies with benzodiazepines such as lorazepam. To examine this, the formation rates of LTGG formation were measured by liquid chromatography-mass spectrometry (LC-MS) in a bank of human liver microsomes (HLMs) obtained from younger and elderly donors at therapeutic concentrations. The formation rate of LTGG was not significantly different in HLMs obtained from younger and elderly subjects. A four- to five-fold variation for the formation of LTGG was observed within each microsomal bank obtained from elderly and younger donors, and the range of LTGG formation was observed to be 0.15-0.78 nmoles min(-1) mg(-1) of protein across the entire set of HLMs (n = 36, elderly and younger HLMs). UGT1A4 and UGT1A3 catalysed the formation of LTGG with an intrinsic clearances of 0.28 and 0.02 microl min(-1) mg(-1) protein, respectively. UGT2B7 and UGT2B4 showed no measurable activity. No correlation was observed across the HLM bank for glucuronidation of LTG and valproic acid (a substrate for multiple UGT isoforms including UGT1A4).

A multicenter trial of selegiline transdermal system for HIV-associated cognitive impairment.

BACKGROUND: Cognitive impairment continues to be a significant neurologic complication of HIV infection and has been associated with oxidative stress-induced neuronal injury. Selegiline is an MAO-B inhibitor with antioxidant and neurotrophic properties. This rationale has led to the design and implementation of this Selegiline Transdermal System (STS) study with the primary aims of assessing safety and tolerability of STS as well as improvement in cognitive performance. METHODS: HIV-1 infected individuals with impaired cognitive functioning were enrolled in this placebo-controlled, three-arm study of STS across 17 sites. Cognitive impairment was determined using a standard battery of neuropsychological tests. Subjects were randomized to receive STS 3 mg/24 hours, STS 6 mg/24 hours, or matching placebo patches daily. The primary efficacy endpoint was defined as the change in neuropsychological composite Z-score (NPZ-6) from baseline to week 24. Measures of safety included frequencies of adverse experiences and abnormal results on laboratory tests. RESULTS: A total of 128 subjects (88% men, 51% white) were enrolled, median age 45 years. Most subjects (62%) had mild to moderate AIDS dementia complex. The 24-week NPZ-6 median (interquartile range) changes were 0.22 (-0.28, 0.55) for the selegiline 3 mg/24 hours arm, 0.21 (-0.18, 0.62) for the selegiline 6 mg/24 hours arm, and 0.28 (-0.16, 0.64) for the placebo arm (a positive score indicates improvement from baseline) (p = 0.914). Severe laboratory abnormalities were few and occurred in similar proportion among the three treatment arms. CONCLUSION: Selegiline was safe and well tolerated by HIV-infected individuals with cognitive impairment and mild to moderate immune suppression; however, no cognitive or functional improvement was observed in this phase II study.

Uridine diphosphoglucuronosyltransferase pharmacogenetics and cancer.

The uridine diphosphoglucuronosyltransferases (UGTs) belong to a superfamily of enzymes that catalyse the glucuronidation of numerous endobiotics and xenobiotics. Several human hepatic and extrahepatic UGT isozymes have been characterized with respect to their substrate specificity, tissue expression and gene structure. Genetic polymorphisms have been identified for almost all the UGT family members. A wide variety of anticancer drugs, dietary chemopreventives and carcinogens are known to be conjugated by members of both UGT1A and UGT2B subfamilies. This review examines in detail each UGT isozyme known to be associated with cancer and carcinogenesis. The cancer-related substrates for several UGTs are summarized, and the functionally relevant genetic polymorphisms of UGTs are reviewed. A number of genotype-phenotype association studies have been carried out to characterize the role of UGT pharmacogenetics in several types of cancer, and these examples are discussed here. In summary, this review focuses on the role of the human UGT genetic polymorphisms in carcinogenesis, chemoprevention and cancer risk.

Lamotrigine clearance during pregnancy.

OBJECTIVE: To evaluate changes in lamotrigine (LTG) clearance before, during, and after pregnancy. METHODS: Twelve pregnancies that had complete steady-state data before, during, and after pregnancy were evaluated. Data included weight, LTG dose, and LTG blood levels at preconception, during pregnancy, and postpartum, and concomitant use of other antiepileptic drugs and their dosages. Apparent clearance (L/[kg.day]) of LTG was calculated by dose/level/weight for time points at preconception; during the first trimester, second trimester, and third trimester; and postpartum. Apparent clearance was compared between preconception and each of the three trimesters. Statistical analysis was performed using one-way analysis of variance, the Student-Newman-Keuls test, and the paired Student's t-test. RESULTS: An increase in apparent clearance (>65%) was observed between preconception and the second and third trimesters (p < 0.05). Eleven pregnancies required higher doses of LTG to maintain therapeutic levels during pregnancy. There was no significant change in apparent clearance between each trimester. A decrease in apparent clearance was observed between the last two trimesters and postpartum (p < 0.05). In the postpartum period, apparent clearances returned to the preconception baseline, and LTG doses needed to be reduced. CONCLUSION: Pregnancy increases LTG clearance by >50%. This effect occurs early in pregnancy and reverts quickly after delivery. LTG levels should be monitored before, during, and after pregnancy.

Cytochrome P450 isozymes 3A4 and 2B6 are involved in the in vitro human metabolism of thiotepa to TEPA.

PURPOSE: To establish the cytochrome P450 (CYP) isozymes involved in the metabolism of the alkylating agent, thiotepa, to the pharmacologically active metabolite, TEPA. METHODS: In vitro chemical inhibition studies were conducted by incubating thiotepa and pooled human hepatic microsomes in the presence of known inhibitors to CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Studies were also performed with cloned, expressed CYP3A4, CYP2A6, CYP2E1 and CYP2B6 microsomes, and anti-CYP2B6 monoclonal antibody. RESULTS: Known CYP3A4 inhibitors reduced TEPA production. Inhibition with CYP2E1 inhibitors was inconsistent. All other inhibitors produced little or no change in TEPA formation. Cloned, expressed CYP2B6 and CYP3A4 microsomes catalyzed TEPA formation, whereas CYP2A6 and CYP2E1 did not. Incubation of thiotepa with anti-CYP2B6 antibody and cloned, expressed CYP2B6 microsomes resulted in reductions in the formation of TEPA, but no change in TEPA formation occurred in human liver microsomes. CONCLUSIONS: Thiotepa is metabolized in human liver microsomes by CYP3A4 (major) and CYP2B6 (minor). There is a potential for CYP-mediated drug interactions with thiotepa. Pharmacokinetic variability of thiotepa may be related to expression of hepatic CYP isozymes.

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer.

Tissue-like structures of cells organized in vitro have a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1 transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices.

Probing enhanced cytochrome P450 2B1/2 activity in rat hepatocyte spheroids through confocal laser scanning microscopy.

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic substrate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.

Inhibition of phenytoin hydroxylation in human liver microsomes by several selective serotonin re-uptake inhibitors.

Several case reports have indicated that the selective serotonin re-uptake inhibitor (SSRI) fluoxetine increases phenytoin blood levels when given concurrently. The mechanism of this drug-drug interaction has been attributed to inhibition of CYP2C9-catalyzed hydroxylation of phenytoin to its major oxidative metabolite in humans, para-hydroxyphenyl phenyl hydantoin (HPPH). With a bank of human liver microsomes (HLM), four SSRIs (fluoxetine, norfluoxetine, sertraline, and paroxetine) were tested for inhibition of HPPH formation. Initially, the K(m) and V(max) values of phenytoin hydroxylation to HPPH were determined in the individual HLM samples. The average K(m) (n=8) was 9.7+/-2.9 microM. The V(max) varied fivefold, with an average value of 113+/-53 pmol HPPH/min/nmol CYP450. All of the SSRIs inhibited HPPH formation; resulting Ki values were 31.1+/-10.1 microM (fluoxetine) (n=5), 51.1+/-9.4 microM (norfluoxetine) (n=3), 52.2+/-21.5 microM (sertraline) (n=3), and 80.0+/-7.2 microM (paroxetine) (n=3). Sulfaphenazole (10 microM), utilized as a positive control for inhibition of HPPH formation, inhibited phenytoin hydroxylation (>95%) in all HLM samples. Diclofenac hydroxylation to 4'-OH diclofenac, a specific marker for CYP2C9 activity, was determined in HLM1-HLM6 and was highly correlated with HPPH formation in HLM1-HLM6, indicating that phenytoin hydroxylation in human liver microsomes is largely due to CYP2C9. This work presents direct evidence that the effect of fluoxetine on phenytoin blood levels may be explained by inhibition of CYP2C9-catalyzed phenytoin hydroxylation. In light of typical SSRI blood levels observed in patients, this study also suggests that the risk of a SSRI-phenytoin interaction is highest with fluoxetine and norfluoxetine, and less likely with sertraline and paroxetine.

Relative bioavailability of lamotrigine chewable dispersible tablets administered rectally.

STUDY OBJECTIVE: To determine the relative bioavailability of lamotrigine (LTG) chewable dispersible tablets after rectal administration. DESIGN: Two-period, crossover study with a 2-week washout between dosing periods. SETTING: Clinical research center. PATIENTS: Twelve healthy adult volunteers. INTERVENTION: One hundred milligrams of a LTG chewable dispersible tablet was administered by oral and rectal routes. MEASUREMENTS AND MAIN RESULTS: Plasma samples were collected before and up to 120 hours after drug administration. The samples were analyzed for LTG by high-performance liquid chromatography, and the relative bioavailability was determined. Drug concentrations were lower after rectal than after oral administration. The relative bioavailability (F = AUC(rectal)/AUC(oral)) was 0.52 +/- 0.23 (SD). CONCLUSION: Drug prepared from LTG chewable dispersible tablets is absorbed rectally, although not to the same extent as when given orally. Rectal administration of suspension of these tablets can be an acceptable route of administration.

Indinavir plasma protein binding in HIV-1-infected adults.

OBJECTIVE: To quantify unbound indinavir concentrations and characterize indinavir plasma protein binding in HIV-infected adults. DESIGN: Pharmacokinetic study in antiretroviral-naive, HIV-infected persons with CD4 T lymphocytes > 100 x 10(6) cells/L and HIV-RNA in plasma >5000 copies/ml at baseline who were participating in an open-label study of zidovudine, lamivudine and indinavir therapy. METHODS: Eight men underwent 8 h intensive pharmacokinetic studies for indinavir on two occasions 6 months apart. Unbound indinavir was separated by ultra-filtration, and unbound and total concentrations were quantified by a validated high-performance liquid chromatography method. RESULTS: Overall indinavir protein binding was 61+/-6%, with a range among the profiles of 54 to 70%. Indinavir binding was higher at the 8 h post-dose concentration compared with the 1 h post-dose concentration (66 versus 57%, P = 0.0006). CONCLUSIONS: The mean 61% protein binding for indinavir in these HIV-infected persons is similar to the in vitro report of 60%. However, the fraction bound was concentration-dependent, and considerable variability in binding was present among patients. Quantification of unbound protease inhibitor concentrations opens new avenues of research to advance our understanding of the pharmacologically-relevant moieties of antiretroviral agents and thereby the pharmacotherapy of HIV infection.

Zidovudine triphosphate and lamivudine triphosphate concentration-response relationships in HIV-infected persons.

OBJECTIVE: To quantitate intracellular concentrations of zidovudine and lamivudine triphosphate and explore relationships with virologic and immunologic responses to antiretroviral therapy. DESIGN: Eight antiretroviral-naive, HIV-infected persons with CD4 T cell counts > 100 x 10(6) cells/l, and HIV RNA in plasma > 5000 copies/ml participating in a prospective, randomized, open-label study of standard dose versus concentration-controlled therapy with zidovudine, lamivudine, and indinavir. METHODS: Peripheral blood mononuclear cells and plasma were collected frequently throughout the study for quantitation of intracellular zidovudine triphosphate and lamivudine triphosphate concentrations, and zidovudine and lamivudine concentrations in plasma. CD4 T cells and HIV RNA in plasma (Roche Amplicor Ultrasensitive Assay) were measured at baseline and every 4 weeks throughout the study. Relationships among intracellular and plasma concentrations, and CD4 T cells and HIV RNA in plasma were investigated with regression analyses. RESULTS: Significant relationships were observed between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate and the baseline level of CD4 cells. Lamivudine triphosphate concentrations were related in a linear manner to the apparent oral clearance of lamivudine from plasma. A direct linear relationship was found between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. The percent change in CD4 cells during therapy and the rate of decline in HIV RNA in plasma were related to the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. CONCLUSION: These studies into the intracellular clinical pharmacology of nucleoside reverse transcriptase inhibitors illustrate potential clinical implications as determinants of therapeutic success. Moreover, these findings provide several leads and a strong impetus for future investigations with nucleoside reverse transcriptase inhibitors particularly when given in combination and sequentially.

Ester and amide derivatives of the nonsteroidal antiinflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors.

Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with ICo5 values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 microM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.

Antiepileptic drug pharmacokinetics and interactions: impact on treatment of epilepsy.

An understanding of epilepsy therapy's pharmacokinetic and drug interaction principles-combined with knowledge of antiepileptic drug (AED) clinical pharmacology-allows more effective use of these drugs. The most desirable pharmacokinetic characteristic is a linear relationship between dose and steady-state concentration, as this determines the ease or difficulty in determining the appropriate dose. Drug-drug interactions affecting AED metabolism are common, clinically important, and, until recently, often unpredictable. Advances in molecular biology have identified specific enzymes responsible for AED metabolism and interactions. Clinicians now can identify potential interactions and avoid or manage them by adjusting drug dosage. Most newer AEDs follow or approximate linear pharmacokinetics, are absorbed extensively and consistently, are not significantly bound to plasma proteins, do not form active metabolites, and have few, if any, drug interactions. In cases where interactions occur between newer AEDs and other drugs, knowledge of these interactions reduces the likelihood of serious adverse events. The pharmacokinetics of the newer AEDs simplify drug dosing and monitoring and should lead to improved patient care.

Evaluation of the effect of culture matrices on induction of CYP3A isoforms in cultured porcine hepatocytes.

Several bioartificial liver devices have been developed as temporary therapy for patients suffering from fulminant hepatic failure. Some of these devices contain porcine hepatocytes entrapped in collagen matrices. In order to improve the function of these BAL devices, there exists a need to optimize metabolic function of cultured hepatocytes. The goal of these investigations was to evaluate the effect of altering culture conditions on rifampin-mediated induction of CYP3A isoforms in cultured porcine hepatocytes. Midazolam metabolism was compared in porcine hepatocytes cultured in a monolayer configuration on collagen gels, in a sandwich configuration between collagen gels and a Matrigel overlay, and in spheroidal cultures. The effect of culture conditions was evaluated, by measuring CYP3A-mediated metabolism of midazolam and by immunoblotting to detect CYP3A proteins, in control cultures and in rifampin-treated cultures. Results obtained by normalizing the metabolism rate data to cell numbers (based on DNA content) present at the end of the culture experiment, showed that there was no difference between the different culture conditions tested. Our results suggest that culturing porcine hepatocytes as spheroids or in a sandwich configuration between collagen and Matrigel, offers no advantage in terms of CYP3A-mediated metabolic function on a per cell basis compared to culturing on collagen gels.