Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential

Phase II metabolism is prominently governed by UDP-glucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30–34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%–184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential

The utility of pathway selective estrogen receptor ligands that inhibit nuclear factor-κB transcriptional activity in models of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that produces synovial proliferation and joint erosions. The pathologic lesions of RA are driven through the production of inflammatory mediators in the synovium mediated, in part, by the transcription factor NF-κB. We have identified a non-steroidal estrogen receptor ligand, WAY-169916, that selectively inhibits NF-κB transcriptional activity but is devoid of conventional estrogenic activity. The activity of WAY-169916 was monitored in two models of arthritis, the HLA-B27 transgenic rat and the Lewis rat adjuvant-induced model, after daily oral administration. In both models, a near complete reversal in hindpaw scores was observed as well as marked improvements in the histological scores. In the Lewis rat adjuvant model, WAY-169916 markedly suppresses the adjuvant induction of three serum acute phase proteins: haptoglobin, α1-acid glycoprotein (α1-AGP), and C-reactive protein (CRP). Gene expression experiments also demonstrate a global suppression of adjuvant-induced gene expression in the spleen, liver, and popliteal lymph nodes. Finally, WAY-169916 was effective in suppressing tumor necrosis factor-α-mediated inflammatory gene expression in fibroblast-like synoviocytes isolated from patients with RA. Together, these data suggest the utility of WAY-169916, and other compounds in its class, in treating RA through global suppression of inflammation via selective blockade of NF-κB transcriptional activity

Tamoxifen's protection against breast cancer recurrence is not reduced by concurrent use of the SSRI citalopram

Tamoxifen remains an important adjuvant therapy to reduce the rate of breast cancer recurrence among patients with oestrogen-receptor-positive tumours. Cytochrome P-450 2D6 metabolises tamoxifen to metabolites that more readily bind the oestrogen receptor. This enzyme also metabolises selective serotonin reuptake inhibitors (SSRI), so these widely used drugs – when taken concurrently – may reduce tamoxifen's prevention of breast cancer recurrence. We studied citalopram use in 184 cases of breast cancer recurrence and 184 matched controls without recurrence after equivalent follow-up. Cases and controls were nested in a population of female residents of Northern Denmark with stages I–III oestrogen-receptor-positive breast cancer 1985–2001 and who took tamoxifen for 1, 2, or most often for 5 years. We ascertained prescription histories by linking participants' central personal registry numbers to prescription databases from the National Health Service. Seventeen cases (9%) and 21 controls (11%) received at least one prescription for the SSRI citalopram while taking tamoxifen (adjusted conditional odds ratio=0.85, 95% confidence interval=0.42, 1.7). We also observed no reduction of tamoxifen effectiveness among regular citalopram users (⩾30% overlap with tamoxifen use). These results suggest that concurrent use of citalopram does not reduce tamoxifen's prevention of breast cancer recurrence

The role of human CYP2C8 and CYP2C9 variants in pioglitazone metabolism in vitro

The cytochrome P450 enzyme CYP2C8 appears to have a major role in pioglitazone metabolism. The present study was conducted to further clarify the role of individual CYPs and of the CYP2C8/9 polymorphisms in the primary metabolism of pioglitazone in vitro. Pioglitazone (2-400 microM) was incubated with isolated cytochrome P450 enzymes or human liver microsomes, some of them carrying either the CYP2C8*3/*3 genotype (and also the CYP2C9*2/*2 genotype) or the CYP2C8*1/*1 genotype (five samples each). The formation of the primary pioglitazone metabolite M-IV was monitored by HPLC. Enzyme kinetics were estimated assuming a single binding site. Mean intrinsic clearance of pioglitazone to the metabolite M-IV was highest for CYP2C8 and CYP1A2 with 58 pmol M-IV/min/nmol CYP P450/microM pioglitazone each, 53 for CYP2D6*1, 40 for CYP2C19*1, and 34 for CYP2C9*2, respectively. CYP2A6, CYP2B6, CYP2C9*1, CYP2C9*3, CYP2E1, CYP3A4 and CYP3A5 did not form quantifiable amounts of M-IV. CYP2C8*1/*1 microsomes (25 +/- 4 pmol M-IV/min/mg protein/muM pioglitazone) showed lower intrinsic clearance of pioglitazone than CYP2C8*3/*3 microsomes (35 +/- 9, p = 0.04). In all samples, metabolite formation showed substrate inhibition, while pioglitazone did not inhibit CYP2C8-mediated paclitaxel metabolism. CYP2C8, CYP1A2 and CYP2D6 are major CYPs forming M-IV in vitro. The higher activity of CYP2C8*3/CYP2C9*2 microsomes may result from a contribution of CYP2C9*2, or from differences in CYP2C8 expression. The evidence for substrate-specific inhibitory effects of pioglitazone on CYP2C-mediated metabolism needs to be tested in further studies