Cyclophosphamide and 4-hydroxycyclophosphamide pharmacokinetics in patients with glomerulonephritis secondary to lupus and small vessel vasculitis.

AIMS: Cyclophosphamide, the precursor to the active 4-hydroxycyclophosphamide, is used in active glomerulonephritis despite limited pharmacokinetics data. The pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide were evaluated. The influence of laboratory and pharmacogenomic covariates on pharmacokinetics was evaluated as a secondary aim. METHODS: Glomerulonephritis patients (n = 23) participated in a pharmacokinetic evaluation. Blood was serially collected and assayed for cyclophosphamide and 4-hydroxycyclophosphamide by LC/MS methods. Kidney function, serum albumin and polymorphisms in drug metabolism or transport genes were evaluated. Analyses included non-compartmental pharmacokinetics and parametric and non-parametric statistics. RESULTS: The mean area under the plasma concentration-time curve (AUC(0,∞)) data were 110,100 ± 42,900 ng ml(-1) h and 5388 ± 2841 ng ml(-1) h for cyclophosphamide and 4-hydroxycyclophosphamide, respectively. The mean metabolic ratio was 0.06 ± 0.04. A statistically significant relationship was found between increased serum albumin and increased half-life (0.584, P = 0.007, 95% CI 0.176, 0.820) and a borderline relationship with AUC(0,∞) (0.402, P = 0.079, 95% CI -0.064, 0.724) for 4-hydroxycyclophosphamide. Covariate relationships that trended toward significance for cyclophosphamide included decreased serum albumin and increased elimination rate constant (-0.427, P = 0.061, 95% CI 0.738, 0.034), increased urinary protein excretion and increased AUC(0,∞) (-0.392, P = 0.064, 95% CI -0.699 to 0.037), decreased C(max) (0.367, P = 0.085, 95% CI -0.067, 0.684) and decreased plasma clearance (-0.392, P = 0.064, 95% CI -0.699, 0.037). CYP2B6*9 variants vs. wildtype were found to have decreased elimination rate constant (P = 0.0005, 95% CI 0.033, 0.103), increased V(d) (P = 0.0271, 95% CI -57.5, -4.2) and decreased C(max) (P = 0.0176, 95% CI 0.696, 6179) for cyclophosphamide. ABCB1 C3435T variants had a borderline decrease in cyclophosphamide elimination rate constant (P = 0.0858; 95% CI -0.005, 0.102). CONCLUSIONS: Pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in patients with lupus nephritis and small vessel vasculitis are similar. Clinical and pharmacogenetic covariates alter disposition of cyclophosphamide and 4-hydroxycyclophosphamide. Clinical findings of worsened glomerulonephritis lead to increased exposure to cyclophosphamide vs. the active 4-hydroxycyclophosphamide, which could have relevance in terms of clinical efficacy. The CYP2B6*9 and ABCB1 C3435T polymorphisms alter the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis.

CYP2C9 genotype and pharmacodynamic responses to losartan in patients with primary and secondary kidney diseases.

BACKGROUND: Losartan is used for anti-proteinuric as well as blood pressure effects in chronic kidney disease (CKD). It is metabolized by cytochrome P450 (CYP) 2C9 to active E-3174. Single nucleotide polymorphisms in CYP2C9 that reduce catalytic activity could reduce clinical benefits. AIM: The study aims were to determine whether CYP2C9 variant alleles (*2 and *3) altered urinary protein excretion, glomerular filtration rate, and blood pressure in Caucasian patients prescribed losartan. METHODS: Differences between baseline and 6-month follow-up outcomes were compared by CYP2C9 genotypes in 59 patients using unpaired t test or Mann-Whitney U test. RESULTS: Primary renal disease patients had a trend toward less favorable antiproteinuric response (-31.7 +/- 156 vs. -125 +/- 323%; p = 0.123) when carrying variant alleles. Patients with secondary renal diseases had less favorable diastolic blood pressure (9.8 +/- 16.0 vs. -3.2 +/- 10.6 mmHg; p = 0.043) and systolic blood pressure (16.2 +/- 27.1 vs. -5.5 +/- 17.5 mmHg; p = 0.044) with CYP2C9 variants. CONCLUSION: These preliminary results suggest a possible influence of CYP2C9 genotype on proteinuria and blood pressure in Caucasian CKD patients treated with losartan.

Enzyme-mediated protein haptenation of dapsone and sulfamethoxazole in human keratinocytes: I. Expression and role of cytochromes P450.

Cutaneous drug reactions (CDRs) are among the most common adverse drug reactions and are responsible for numerous minor to life-threatening complications. Several arylamine drugs, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation, resulting in adduction to cellular proteins. These adducted proteins may initiate the immune response that ultimately results in a CDR. Recent studies have demonstrated that normal human epidermal keratinocytes (NHEKs) can bioactivate these drugs, resulting in protein haptenation. We sought to identify the enzyme(s) responsible for this bioactivation in NHEKs. Using immunofluorescence confocal microscopy and an adduct-specific enzyme-linked immunosorbent assay (ELISA), we found that N-acetylation of the primary amine of SMX and DDS markedly reduced the level of protein haptenation in NHEKs. Detection of mRNA and/or protein confirmed the presence of CYP3A4, CYP3A5, and CYP2E1 in NHEKs. In contrast, although a faint band suggestive of CYP2C9 protein was detected in one NHEK sample, a CYP2C9 message was not detectable. We also examined the ability of chemical inhibitors of cytochromes P450 (aminobenzotriazole and 1-dichloroethylene) and cyclooxygenase (indomethacin) to reduce protein haptenation when NHEKs were incubated with SMX or DDS by either confocal microscopy or ELISA. These inhibitors did not significantly attenuate protein adduction with either SMX or DDS, indicating that cytochromes P450 and cyclooxygenase do not play important roles in the bioactivation of these xenobiotics in NHEKs and thus suggesting the importance of other enzymes in these cells.

Discovery of new potentially defective alleles of human CYP2C9.

CYP2C9 is a clinically important enzyme, responsible for the metabolism of numerous clinically important therapeutic drugs. In the present study, we discovered 38 single nucleotide polymorphisms in CYP2C9 by resequencing of genomic DNA from 92 individuals from three different racial groups. Haplotype analysis predicted that there are at least 21 alleles of CYP2C9 in this group of individuals. Six new alleles were identified that contained coding changes: L19I (CYP2C9*7), R150H (CYP2C9*8), H251R (CYP2C9*9), E272G (CYP2C9*10), R335W(CYP2C9*11) and P489S (CYP2C9*12). When expressed in a bacterial cDNA expression system, several alleles exhibited altered catalytic activity. CYP2C9*11 appeared to be a putative poor metabolizer allele, exhibiting a three-fold increase in the Km and more than a two-fold decrease in the intrinsic clearance for tolbutamide. Examination of the crystal structure of human CYP2C9 reveals that R335 is located in the turn between the J and J' helices and forms a hydrogen-bonding ion pair with D341 from the J' helix. Abolishing this interaction in CYP2C9*11 individuals could destabilize the secondary structure and alter the substrate affinity. This new putative poor metabolizer (PM) allele was found in Africans. A second potentially PM allele CYP2C9*12 found in a racially unidentified sample also exhibited a modest decrease in the Vmax and the intrinsic clearance for tolbutamide in a recombinant system. Further clinical studies are needed to determine the effect of these new polymorphisms on the metabolism of CYP2C9 substrates.

Cloning, expression, and characterization of three new mouse cytochrome p450 enzymes and partial characterization of their fatty acid oxidation activities.

The mammalian CYP2C subfamily is one of the largest and most complicated in the cytochrome P450 superfamily. In this report, we describe the organization of the mouse Cyp2c locus, which contains 15 genes and four pseudogenes, all of which are located in a 5.5-megabase region on chromosome 19. We cloned three novel mouse CYP2C cDNAs (designated CYP2C50, CYP2C54, and CYP2C55) from mouse heart, liver, and colon, respectively. All three cDNAs contain open reading frames that encode 490 amino acid polypeptides that are 57 to 95% identical to other CYP2Cs. The recombinant CYP2C proteins were expressed in Escherichia coli after N-terminal modification, partially purified, and shown to be active in the metabolism of both arachidonic acid (AA) and linoleic acid, albeit with different catalytic efficiencies and profiles. CYP2C50 and CYP2C54 metabolize AA to epoxyeicosatrienoic acids (EETs) primarily, and linoleic acid to epoxyoctadecenoic acids (EOAs) primarily, whereas CYP2C55 metabolizes AA to EETs and hydroxyeicosatetraenoic acids and linoleic acid to EOAs and hydroxyoctadecadienoic acids. Northern blotting and reverse transcription-polymerase chain reaction analysis reveal that CYP2C50 transcripts are abundant in liver and heart; CYP2C54 transcripts are present in liver, kidney, and stomach; and CYP2C55 transcripts are abundant in liver, colon, and kidney. Immunoblotting studies demonstrate that CYP2C50 protein is expressed in liver and heart, CYP2C54 protein is detected primarily in liver, and CYP2C55 protein is present primarily in colon. Immunohistochemistry reveals that CYP2C55 is most abundant in surface columnar epithelium in the cecum. We conclude that these new CYP2C enzymes are probably involved in AA and linoleic acid metabolism in mouse hepatic and extrahepatic tissues.

Losartan and E3174 pharmacokinetics in cytochrome P450 2C9*1/*1, *1/*2, and *1/*3 individuals.

STUDY OBJECTIVE: To determine if differences in the pharmacokinetics of losartan and its pharmacologically active E3174 metabolite exist among individuals expressing the cytochrome P450 (CYP) 2C9*1/*1, *1/*2, and *1/*3 genotypes. DESIGN: Single-dose pharmacokinetic study. SETTING: University general clinical research center. SUBJECTS: Fifteen healthy volunteers, five from each genotype: CYP2C9*1/*1, *1/*2, and *1/*3. INTERVENTION: A single oral dose of losartan 50 mg. MEASUREMENTS AND MAIN RESULTS: Plasma and urine samples were collected for 24 hours, and losartan and E3174 pharmacokinetic data were compared across the three genotypes. Orthostatic blood pressure was measured over 12 hours after dosing. No significant differences were observed among the three groups in losartan or E3174 area under the plasma concentration-time curve, losartan or E3174 elimination half-life, or losartan oral clearance. A significant association between CYP2C9 genotype and losartan to E3174 formation clearance was observed, such that 50% of the variability was accounted for by the genotype. No significant relationship between that genotype and blood pressure was observed at any time. CONCLUSION: Differences in the pharmacokinetics of losartan and its active E3174 metabolite were not observed in healthy subjects with the genotype of CYP2C9*1/*2 and *1/*3 compared with those expressing *1/*1. Alterations in losartan dosing in CYP2C9*1/*2 and *1/*3 individuals does not appear necessary.

Differences in flurbiprofen pharmacokinetics between CYP2C9*1/*1, *1/*2, and *1/*3 genotypes.

OBJECTIVE: This study was conducted to examine differences in flurbiprofen metabolism among individuals with the CYP2C9*1/*1, *1/*2, and *1/*3 genotypes. METHODS: Fifteen individuals with the CYP2C9*1/*1 ( n=5), *1/*2 ( n=5), and *1/*3 ( n=5) genotypes received a single 50-mg oral dose of flurbiprofen. Plasma and urine samples were collected over 24 h, and flurbiprofen and 4'-hydroxyflurbiprofen pharmacokinetic data were compared across genotypes. RESULTS: CYP2C9 genotype was a significant predictor of flurbiprofen metabolism and accounted for 59% of the variability in flurbiprofen AUC(0- infinity ), and approximately 50% of the variability in flurbiprofen oral clearance, formation clearance to 4'-hydroxyflurbiprofen, and the 0 to 24-h urinary metabolic ratio of flurbiprofen to 4'-hydroxyflurbiprofen. Flurbiprofen AUC(0- infinity )was significantly higher and all measures of flurbiprofen clearance were significantly lower in the CYP2C9*1/*3 individuals than in those with *1/*1. Significant differences in these parameters were not detected between *1/*2 subjects and *1/*1 subjects. CONCLUSIONS: CYP2C9 genotype is a significant predictor of flurbiprofen disposition in humans by altering CYP2C9-mediated metabolism and reducing systemic clearance. The effects are most pronounced in individuals carrying the *3 allele.

Tolbutamide, flurbiprofen, and losartan as probes of CYP2C9 activity in humans.

The metabolic activity of CYP2C9 in 16 subjects expressing four different genotypes (CYP2C9*1/*1, *1/*2, *1/*3, and *2/*2) was evaluated. Single oral doses of tolbutamide, flurbiprofen, and losartan were administered in a randomized, crossover design. Plasma and urine were collected over 24 hours. The urinary metabolic ratio and amount of metabolite(s) excreted were correlated with formation clearance. The formation clearance of tolbutamide to its CYP2C9-mediated metabolites demonstrated a stronger association with genotype compared to flurbiprofen and losartan, respectively (r2 = 0.64 vs. 0.53 vs. 0.42). A statistically significant correlation was observed between formation clearance of tolbutamide and the 0- to 12-hour urinary amount of 4'-hydroxytolbutamide and carboxytolbutamide (r = 0.84). Compared to tolbutamide, the correlations observed between the respective measures of flurbiprofen and losartan metabolism were not as strong. Tolbutamide is a better CYP2C9 probe than flurbiprofen and losartan, and the 0- to 12-hour amount of 4'-hydroxytolbutamide and carboxytolbutamide is the best urinary measure of its metabolism.

Identification and functional characterization of new potentially defective alleles of human CYP2C19.

CYP2C19 is a clinically important enzyme responsible for the metabolism of a number of therapeutic drugs, such as mephenytoin, omeprazole, diazepam, proguanil, propranolol and certain antidepressants. Genetic polymorphisms in this enzyme result in poor metabolizers of these drugs. There are racial differences in the incidence of the poor metabolizer trait, which represents 13-23% of Asians but only 3-5% of Caucasians. In this study, single nucleotide polymorphisms (SNPs) in CYP2C19 were identified by direct sequencing of genomic DNA from 92 individuals from three different racial groups of varied ethnic background, including Caucasians, Asians and blacks. Several new alleles were identified containing the coding changes Arg114 His (CYP2C19*9), Pro227 Leu (CYP2C19*10), Arg150 His (CYP2C19*11), stop491 Cys (CYP2C19*12), Arg410 Cys (CYP2C19*13), Leu17 Pro (CYP2C19*14) and Ile19 Leu (CYP2C19*15). When expressed in a bacterial cDNA expression system, CYP2C19*9 exhibited a modest decrease in the V(max) for 4'-hydroxylation of -mephenytoin, and no alteration in its affinity for reductase. CYP2C19*10 exhibited a dramatically higher K(m) and lower V(max) for mephenytoin. CYP2C19*12was unstable and expressed poorly in a bacterial cDNA expression system. Clinical studies will be required to confirm whether this allele is defective in vivo. CYP2C19*9, CYP2C19*10 and CYP2C19*12 all occurred in African-Americans, or individuals of African descent, and represent new potentially defective alleles of CYP2C19 which are predicted to alter risk of these populations to clinically important drugs.

Evaluation of cytochrome P4502C9 metabolic activity with tolbutamide in CYP2C91 heterozygotes.

OBJECTIVES: Multiple single-nucleotide polymorphisms in the gene encoding cytochrome P450 (CYP) 2C9 have been identified, but the functional significance of the various putative defective genotypes in humans merits further study. METHODS: Using tolbutamide as a probe of CYP2C9 activity, we evaluated CYP2C9 phenotype in 15 healthy individuals expressing the CYP2C9(*)1/(*)1, (*)1/(*)2, and (*)1/(*)3 genotypes (n = 5 per group). CYP2C9 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism methods. Subjects received 500 mg of tolbutamide, with plasma and urine collected over a 24-hour period. Plasma tolbutamide and urinary tolbutamide, 4'-hydroxytolbutamide, and carboxytolbutamide concentrations were determined by an HPLC method. RESULTS: Tolbutamide area under the plasma concentration-time curve from time zero to infinity [AUC(0- infinity )] significantly increased by 1.5-fold and 1.9-fold, respectively, in subjects expressing the CYP2C9(*)1/(*)2 and (*)1/(*)3 genotypes compared with (*)1/(*)1 subjects. Statistically significant reductions in tolbutamide oral clearance (29% and 48%) and formation clearance (38% and 56%) were detected in the (*)1/(*)2 and (*)1/(*)3 individuals, respectively, compared with (*)1/(*)1 subjects. The increases in AUC(0- infinity) and decreases in oral clearance observed in the (*)1/(*)3 individuals were also significantly greater than those expressing the (*)1/(*)2 genotype (P <.05). The amount of urinary 4'-hydroxytolbutamide and carboxytolbutamide excreted in the 0- to 12-hour and 6- to 12-hour collection intervals was significantly less in (*)1/(*)2 and (*)1/(*)3 individuals compared with (*)1/(*)1 subjects. With tolbutamide used as a CYP2C9 probe, CYP2C9 genotype was the major determinant of CYP2C9 phenotype (r(2) = 0.77). CONCLUSIONS: CYP2C9 activity was significantly reduced in (*)1 heterozygotes compared with (*)1 homozygotes, and metabolism was more severely impaired in (*)1/(*)3 individuals compared with those expressing (*)1/(*)2.

Analysis of the CYP2C19 polymorphism in a North-eastern Thai population.

CYP2C19 is a polymorphically expressed cytochrome P450 responsible for the metabolism of several clinically used drugs, including some barbiturates, diazepam, proguanil, propranolol and several proton pump inhibitors. Genetic polymorphism of this enzyme shows marked interracial differences, with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 11-23% of Oriental populations. In the present study, CYP2C19 phenotype and genotype were investigated in 107 North-eastern Thai subjects using the omeprazole hydroxylation index (HI) and polymerase chain reaction-restriction fragment length polymorphism technique, respectively. It was found that the distribution of HI in these subjects was bimodal. Seven subjects [6.54%, 95% confidence (CI) 1.86-11.22%] were identified as PM, with an HI > 7. Analysis of CYP2C19 genotypes in these 107 Thai subjects revealed that the allele frequencies for CYP2C19*1, CYP2C19*2 and CYP2C19*3 were 0.71 (95% CI 0.65-0.77), 0.27 (95% CI 0.21-0.33) and 0.02 (95% CI 0.01-0.05), respectively. The PM phenotype and the frequencies of CYP2C19 defective alleles in Thais, particularly CYP2C19*3, were lower than those observed in other Oriental populations. It is noteworthy that there was a case of nonaccordance between phenotype and genotype in one of the PMs. Whether this PM represents a novel defective allele requires further investigation.