Determination of CYP450 Expression Levels in the Human Small Intestine by Mass Spectrometry-Based Targeted Proteomics.

The human small intestine can be involved in the first-pass metabolism of drugs. Under this condition, members of the CYP450 superfamily are expected to contribute to drug presystemic biotransformation. The aim of this study was to quantify protein expression levels of 16 major CYP450 isoforms in tissue obtained from nine human organ donors in seven subsections of the small intestine, i.e., duodenum (one section, N = 7 tissue samples), jejunum (three subsections (proximal, mid and distal), N = 9 tissue samples) and ileum (three subsections, (proximal, mid and distal), N = 9 tissue samples), using liquid chromatography tandem mass spectrometry (LC-MS/MS) based targeted proteomics. CYP450 absolute protein expression levels were compared to mRNA levels and enzyme activities by using established probe drugs. Proteins corresponding to seven of sixteen potential CYP450 isoforms were detected and quantified in various sections of the small intestine: CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, CYP3A5 and CYP4F2. Wide inter-subject variability was observed, especially for CYP2D6. CYP2C9 (p = 0.004) and CYP2C19 (p = 0.005) expression levels decreased along the small intestine. From the duodenum to the ileum, CYP2J2 (p = 0.001) increased, and a trend was observed for CYP3A5 (p = 0.13). CYP3A4 expression was higher in the jejunum than in the ileum (p = 0.03), while CYP4F2 expression was lower in the duodenum compared to the jejunum and the ileum (p = 0.005). CYP450 protein levels were better correlated with specific isoform activities than with mRNA levels. This study provides new data on absolute CYP450 quantification in human small intestine that could improve physiologically based pharmacokinetic models. These data could better inform drug absorption profiles while considering the regional expression of CYP450 isoforms.

Development and validation of an absolute protein assay for the simultaneous quantification of fourteen CYP450s in human microsomes by HPLC-MS/MS-based targeted proteomics.

The cytochrome P450 (CYP450) superfamily constitutes the major enzymatic system involved in drug metabolism. CYP450s are highly expressed in the liver and other tissues and limited data on absolute characterization of CYP450s in extra hepatic organs, such as the small intestine, are available. Our objective was to develop and validate an absolute quantification assay by HPLC-MS/MS-based targeted proteomics allowing the simultaneous quantification of fourteen major human CYP450 isoforms (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2) in human liver and intestine microsomes. Absolute protein quantification was performed using two proteotypic peptides for each of the fourteen CYP450s. Peptides were obtained after a tryptic digestion of microsomes and samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Chromatographic separation was performed on a Biobasic-8 analytical column (5 µm 100 x 1 mm) with a gradient elution using acetonitrile and water both fortified with 0.1% formic acid (flow rate: 75 µL/min). Calibration curves were linear over a wide range of concentrations (0.1-50 nM) and the assay met all requirements of sensitivity, linearity, precision, accuracy and matrix effect. Strong correlations were observed between the two proteotypic peptides for each isoenzyme, corroborating the strength of this method. Twelve CYP450s were detected in commercially available human liver microsomes while seven CYP450s were detected in human intestine microsomes. To our knowledge, this is the most sensitive (0.1 nM) and the first most extensively validated assay that can be applied to the absolute quantification of CYP450s in human tissues.

Activity and mRNA expression levels of selected cytochromes P450 in various sections of the human small intestine.

AIMS: To characterize mRNA expression levels (17 cytochromes P450) and activity (9 isoforms) of major cytochromes P450 expressed throughout the human small intestine. METHODS: Tissue samples were obtained from 9 deceased subjects and intestinal sections (n = 10) were isolated for each subject. Relative mRNA expression levels were determined using quantitative real-time PCR. Intestinal microsomes were prepared from 5 subsections: duodenum, jejunum (proximal and mid-jejunum) and ileum (proximal and mid-ileum) regions. In vitro incubations were performed with various cytochrome P450 probe substrates: bupropion (CYP2B6), repaglinide (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), chlorzoxazone (CYP2E1), ebastine (CYP2J2), midazolam (CYP3A4/5) and lauric acid (CYP4A11). Metabolite formation was assessed using validated liquid chromatography-tandem mass spectrometry assays. RESULTS: Cytochrome P450 mRNA levels ranked as follows: CYP3A4 > CYP2C9 > CYP2C19 > CYP2J2 > CYP4F2. Cytochrome P450 mRNA transcripts showed different patterns in their relative expression from 1 region to the other but CYP3A4, CYP2C9, CYP2C19 and CYP2J2 displayed the highest levels of mRNA expression (>5%) in all intestinal sections. Cytochrome P450 activities were greater in proximal part of the small intestine with the jejunum showing the greatest drug-metabolism activity. Spearman's correlation analyses indicated that cytochrome P450 mRNA expressions and corresponding cytochrome P450 activities in the human intestine were moderately associated for CYP2C19, CYP2D6 and CYP4A11 (rs  = 0.44-0.56). CONCLUSIONS: Our study provides new and additional information on the expression and activities of selected cytochromes P450 in various sections of the human small intestine.

Epidemiology and outcome of antimicrobial resistance to gram-negative pathogens in bacteriuric kidney transplant recipients.

BACKGROUND: In kidney transplant recipients, episodes of bacteriuria are often treated regardless of the presence of symptoms because of the lack of clear treatment guidelines suggesting otherwise. This practice may lead to the development of antimicrobial resistance. Our aim was to determine the incidence, determinants, and impact of antimicrobial resistance in kidney transplant recipients with gram-negative bacteriuria. METHOD: We conducted a single-center, retrospective cohort study in patients who underwent kidney transplantation between January 2008 and June 2013. To identify risk factors for the development of resistance, we used a logistic regression model with generalized estimating equations to account for within-subject correlation. RESULTS: Among the 318 patients who underwent kidney transplantation during the study period, 147 patients developed 555 gram-negative episodes of bacteriuria. Resistance to trimethoprim-sulfamethoxazole and quinolones, and production of extended-spectrum ß-lactamase (ESBL) occurred in 52%, 21%, and 5% of isolated microorganisms, respectively. An increased risk of resistance to quinolones and production of ESBL were associated with concomitant diabetes (odds ratio [OR]: 2.29, 95% confidence interval [CI]: 1.11-4.74), the first year post transplantation (OR: 2.88, 95% CI: 1.36-6.09), and antibiotic treatment in the previous 6 months (OR: 3.36, 95% CI: 1.66-6.81). This resistance profile was also associated with the presence of symptoms, a longer duration of antibiotic treatment, and a higher rate of hospitalization. CONCLUSION: Antimicrobial resistance to quinolones and production of ESBL were commonly seen, and were shown to demonstrate an adverse impact on outcomes in kidney transplant recipients with gram-negative bacteriuria. The decision on treatment for asymptomatic bacteriuria should be made with caution, given the potential for the selection of resistant strains.

Influence of SLCO1B3 genetic variations on tacrolimus pharmacokinetics in renal transplant recipients.

The immunosuppressive drug tacrolimus requires strict therapeutic monitoring due to its narrow therapeutic index and high interindividual variability. Organic anion transporting polypeptide 1B3 (OATP1B3) is a human hepatocyte transporter involved in the hepatobiliary elimination of diverse endogenous and exogenous substances. Genetic variations within the solute carrier (SLCO) 1B3 gene that encodes OATP1B3 may contribute to interindividual differences in tacrolimus disposition. The purpose of the present study is to investigate the association between SLCO1B3 polymorphisms and tacrolimus pharmacokinetics in renal transplant recipients. We found significant correlations between two linked coding nonsynomymous polymorphisms, T334G and G699A, and mean dose-adjusted tacrolimus trough blood concentrations during the first week post-transplantation (p = 0.04) and when the target dose (10-12 ng/ml) was obtained (p = 0.01). Patients carrying the homozygous mutant haplotype had 14.3-fold higher risk (95% confidence interval: 1.43-100; p = 0.02) of having blood tacrolimus concentrations above the median level, and thus being classified as poor OATP1B3 transporters, than carriers of one or two copies of the wild-type haplotype. This study shows, for the first time, that SLCO1B3 polymorphism is associated with tacrolimus exposure in the early post-transplant period.

Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3: genetic variability and haplotype analysis in white Canadians.

Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are human hepatocyte transporters that mediate the uptake of various endogenous and exogenous substances. Genetic variations in solute carrier transporter 1B1 (SLCO1B1) and SLCO1B3 genes, which encode OATP1B1 and OATP1B3 proteins, could affect the pharmacokinetics of drugs leading to interindividual differences in drug responses. The full extent of SLCO1B1 and SLCO1B3 polymorphisms in white Canadians was analyzed using DNA sequencing procedures. We identified 49 and 41 nucleotide sequence variants leading to 10 and 9 major haplotypes in SLCO1B1 and SLCO1B3 genes, respectively. We report several novel mutations within regulatory and coding regions that could affect gene transcription, translation and function. Comparison with other studies revealed that the distribution of SLCO1B1 and SLCO1B3 polymorphisms and haplotypes differs widely across populations. Data from this survey will ultimately contribute to the design of pharmacogenetic studies in the Canadian population.

The CLEAR study: a 5-day, 3-g loading dose of mycophenolate mofetil versus standard 2-g dosing in renal transplantation.

BACKGROUND AND OBJECTIVES: Adequate early mycophenolic acid (MPA) exposure is associated with lower rates of acute rejection in renal transplantation. The aim of this randomized controlled trial was to determine if higher initial mycophenolate mofetil (MMF) doses increased the proportion of patients reaching therapeutic MPA levels (30 to 60 mg.h/L) by day 5. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: De novo renal transplant patients were randomized to receive intensified dosing of MMF (1.5 g twice daily on days 1 to 5, then 1.0 g twice daily) or standard dosing (1.0 g twice daily). All recipients received tacrolimus and prednisone. Full MPA areas under the curve (AUCs) were completed on days 3 and 5, whereas a limited sampling strategy was utilized at four subsequent time points. RESULTS: At day 5, 47.5% of the MMF 3-g arm achieved the MPA therapeutic window versus 54.4% of the MMF 2-g arm. However, MPA AUC levels were significantly higher in the 3-g arm at day 3 and 5. This resulted in a trend for fewer treated acute rejections at 6 months. Significantly more acute rejections (treated, biopsy-proven including and excluding borderline) occurred in patients with MPA AUC levels<30 mg.h/L compared with those >or=30 mg.h/L at day 5. No significant differences were seen in common adverse events. CONCLUSIONS: A limited intensified dose of MMF increased early MPA exposure and was well tolerated. Further studies are required to determine whether limited intensified MMF dosing can reduce acute rejection.

Mechanisms and management of proteinuria in kidney transplant patients.

Proteinuria is a common complication occurring after kidney transplantation. It is associated with an increased risk of renal failure and patient death. Treatment with ACE inhibitors or angiotensin receptor antagonists (blockers) has been shown to reduce proteinuria after kidney transplantation, as well as improve both graft and patient survival. An increase in proteinuria has been observed in some patients after initiation of sirolimus therapy. Although the mechanism of this remains unclear, high proteinuria at baseline and poor renal function at baseline have been identified as potential risk factors for the development of proteinuria after conversion to sirolimus. Initiation of sirolimus therapy is not recommended in patients with early histological indicators of glomerular damage; however, in patients with healthy grafts, sirolimus may prevent future glomerulosclerosis. Early treatment with an ACE inhibitor and sirolimus, prior to the appearance of glomerular changes, may result in better outcomes.

Cyp3A4, Cyp3A5, and MDR-1 genetic influences on tacrolimus pharmacokinetics in renal transplant recipients.

OBJECTIVE: The immunosuppressive drug tacrolimus requires strict therapeutic monitoring due to its narrow therapeutic index and great inter-individual variability. Cytochrome P450 3A4 (Cyp3A4) and Cyp3A5 are the most important contributors to tacrolimus metabolism while the P-glycoprotein pump (MDR-1) modulates its bioavailability. The objective was to investigate the association between Cyp3A4, Cyp3A5, and MDR-1 polymorphisms and tacrolimus pharmacokinetics in the early period after renal transplantation. METHODS: Forty-four renal transplant recipients were genotyped for 8 Cyp3A4, 7 Cyp3A5, and 5 MDR-1 genetic variants affecting the proteins' expression and/or function. Dose-adjusted tacrolimus though levels were determined during the first week after transplantation and correlated with corresponding genotype. RESULTS: We found no correlation between Cyp3A4 polymorphism and tacrolimus pharmacokinetics. Patients who do not carry both Cyp3A5*3 alleles achieved lower mean dose-adjusted tacrolimus blood concentrations (p<0.001) and needed a longer time to reach the target concentration (10-12 ng/ml; p<0.001) compared to Cyp3A5*3 homozygotes. Patients with less than three copies of MDR-1 (T-129C, C3435T and G2677T) polymorphisms, associated with reduced expression of P-glycoprotein, had also lower dose-adjusted tacrolimus blood concentrations compared to patients having equal to or greater than three copies of MDR-1 genetic variants (P=0.003). There was no difference in the rate of biopsy-confirmed acute rejection among groups during the first 3 months after transplantation. CONCLUSION: The complete absence of Cyp3A5*3 allele and the accumulation of less than three copies of MDR-1 (T-129C, C3435T and G2677T) polymorphisms are associated with lower tacrolimus blood levels identifying these genotypes as markers for patients requiring higher tacrolimus doses.

CYP3A5 genetic polymorphisms in different ethnic populations.

Cyp3A5 activity varies within any given ethnic population, suggesting that genetic variation within the Cyp3A5 gene may be the most important contributor to interindividual and interracial differences in Cyp3A-dependent drug clearance and response. The full extent of Cyp3A5 polymorphism in a white and an indigenous African population was analyzed using DNA direct sequencing procedures. The presence of 10 and 12 single nucleotide polymorphisms was detected in the white and African samples, respectively. Thirteen novel mutations occurring at low frequencies were identified in these populations. Significant differences were observed in the distribution of Cyp3A5*3, Cyp3A5*6, and Cyp3A5*7 alleles among white and African populations. The frequency of Cyp3A5*3 allele in white Canadians (approximately 93%) is higher than in Zimbabweans (77.6%) (p < 0.001). In contrast, Cyp3A5*6 and Cyp3A5*7 alleles are relatively frequent in African subjects (10-22%) but absent in white subjects (p < 0.001). These differences may reflect evolutionary pressures generated by environmental factors in geographically distinct regions. However, the genetic polymorphism of Cyp3A5 alone does not explain the interindividual differences in Cyp3A-mediated metabolism.

Synergistic effects of RAD and Neoral in inhibition of host-vs.-graft and graft-vs.-host immune responses in rat small-bowel transplantation.

The combined effects of RAD and Neoral were tested in a rat orthotopic small-bowel transplantation model. Seven groups (n = 6) were involved in this study, and each one was included in three rejection models for the evaluation of host-vs.-graft disease (HVG) (LBN-F1 to LEW), graft-vs.-host disease (GVH) (LEW to LBN-F1), and combined HVG and GVH immune responses (BN to LEW). Both drugs were administered orally throughout the study. Low doses of RAD (1.0-2.5 mg/kg/day) combined with Neoral (2.0-5.0 mg/kg/day) produced strong synergistic effects in the prolongation of small-bowel graft survival in HVG (combination index, CI = 0.095, 0.1212), GVH (CI = 0.027, 0.020), and combined HVG and GVH immune responses (CI = 0.070, 0.301). The combination therapy of RAD and Neoral produces a strong synergistic effect toward the inhibition of HVG, GVH, and combined HVG and GVH immune responses in a rat small-bowel transplantation model.