Investigating the association between CYP2J2 inhibitors and QT prolongation: a literature review.

Drug withdrawal post-marketing due to cardiotoxicity is a major concern for drug developers, regulatory agencies, and patients. One common mechanism of cardiotoxicity is through inhibition of cardiac ion channels, leading to prolongation of the QT interval and sometimes fatal arrythmias. Recently, oxylipin signaling compounds have been shown to bind to and alter ion channel function, and disruption in their cardiac levels may contribute to QT prolongation. Cytochrome P450 2J2 (CYP2J2) is the predominant CYP isoform expressed in cardiomyocytes, where it oxidizes arachidonic acid to cardioprotective epoxyeicosatrienoic acids (EETs). In addition to roles in vasodilation and angiogenesis, EETs bind to and activate various ion channels. CYP2J2 inhibition can lower EET levels and decrease their ability to preserve cardiac rhythm. In this review, we investigated the ability of known CYP inhibitors to cause QT prolongation using Certara's Drug Interaction Database. We discovered that among the multiple CYP isozymes, CYP2J2 inhibitors were more likely to also be QT-prolonging drugs (by approximately 2-fold). We explored potential binding interactions between these inhibitors and CYP2J2 using molecular docking and identified four amino acid residues (Phe61, Ala223, Asn231, and Leu402) predicted to interact with QT-prolonging drugs. The four residues are located near the opening of egress channel 2, highlighting the potential importance of this channel in CYP2J2 binding and inhibition. These findings suggest that if a drug inhibits CYP2J2 and interacts with one of these four residues, then it may have a higher risk of QT prolongation and more preclinical studies are warranted to assess cardiovascular safety.

METTL7A (TMT1A) and METTL7B (TMT1B) Are Responsible for Alkyl S-Thiol Methyl Transferase Activity in Liver.

S-methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine (SAM)-dependent membrane-associated enzyme referred to as thiol methyltransferase (TMT). This putative TMT appeared to have a broad substrate specificity and methylated the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine prodrugs, clopidogrel, and prasugrel. Despite TMT's role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol methyltransferase. METTL7B is an endoplasmic reticulum-associated protein with similar biochemical properties and substrate specificity to the putative TMT. Yet, the historic TMT inhibitor 2,3-dichloro-α-methylbenzylamine (DCMB) did not inhibit METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, is also a SAM-dependent thiol methyltransferase. METTL7A exhibits similar biochemical properties to METTL7B and putative TMT, including inhibition by DCMB (IC50 = 1.17 µM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which are now renamed thiol methyltransferase 1A (TMT1A) and thiol methyltransferase 1B (TMT1B), respectively, that are responsible for thiol methylation activity in human liver microsomes. SIGNIFICANCE STATEMENT: We identified methyltransferase-like protein 7A (thiol methyltransferase 1A) and methyltransferase-like protein 7B (thiol methyltransferase 1B) as the enzymes responsible for the microsomal alkyl thiol methyltransferase (TMT) activity. These are the first two enzymes directly associated with microsomal TMT activity. S-methylation of commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible for this activity will improve our understanding of the drug metabolism and pharmacokinetic (DMPK) properties of alkyl- or phenolic thiol-containing therapeutics.

CYP2D6 Activity Is Correlated with Changes in Plasma Concentrations of Taurocholic Acid during Pregnancy and Postpartum in CYP2D6 Extensive Metabolizers.

Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of >20% of marketed drugs. CYP2D6 expression and activity exhibit high interindividual variability and is induced during pregnancy. The farnesoid X receptor (FXR) is a transcriptional regulator of CYP2D6 that is activated by bile acids. In pregnancy, elevated plasma bile acid concentrations are associated with maternal and fetal risks. However, modest changes in bile acid concentrations may occur during healthy pregnancy, thereby altering FXR signaling. A previous study demonstrated that hepatic tissue concentrations of bile acids positively correlated with the hepatic mRNA expression of CYP2D6. This study sought to characterize the plasma bile acid metabolome in healthy women (n = 47) during midpregnancy (25-28 weeks gestation) and ≥3 months postpartum and to determine if plasma bile acids correlate with CYP2D6 activity. It is hypothesized that during pregnancy, plasma bile acids would favor less hydrophobic bile acids (cholic acid vs. chenodeoxycholic acid) and that plasma concentrations of cholic acid and its conjugates would positively correlate with the urinary ratio of dextrorphan/dextromethorphan. At 25-28 weeks gestation, taurine-conjugated bile acids comprised 23% of the quantified serum bile acids compared with 7% ≥3 months postpartum. Taurocholic acid positively associated with the urinary ratio of dextrorphan/dextromethorphan, a biomarker of CYP2D6 activity. Collectively, these results confirm that the bile acid plasma metabolome differs between pregnancy and postpartum and provide evidence that taurocholic acid may impact CYP2D6 activity during pregnancy. SIGNIFICANCE STATEMENT: Bile acid homeostasis is altered in pregnancy, and plasma concentrations of taurocholic acid positively correlate with CYP2D6 activity. Differences between plasma and/or tissue concentrations of farnesoid X receptor ligands such as bile acids may contribute to the high interindividual variability in CYP2D6 expression and activity.

Future of Biotransformation Science in the Pharmaceutical Industry.

Over the past decades, the number of scientists trained in departments dedicated to traditional medicinal chemistry, biotransformation and/or chemical toxicology have seemingly declined. Yet, there remains a strong demand for such specialized skills in the pharmaceutical industry, particularly within drug metabolism/pharmacokinetics (DMPK) departments. In this position paper, the members of the Biotransformation, Mechanisms, and Pathways Focus Group (BMPFG) steering committee reflect on the diverse roles and responsibilities of scientists trained in the biotransformation field in pharmaceutical companies and contract research organizations. The BMPFG is affiliated with the International Society for the Study of Xenobiotics (ISSX) and was specifically created to promote the exchange of ideas pertaining to topics of current and future interest involving the metabolism of xenobiotics (including drugs). The authors also delve into the relevant education and diverse training skills required to successfully nurture the future cohort of industry biotransformation scientists and guide them toward a rewarding career path. The ability of scientists with a background in biotransformation and organic chemistry to creatively solve complex drug metabolism problems encountered during research and development efforts on both small and large molecular modalities is exemplified in five relevant case studies. Finally, the authors stress the importance and continued commitment to training the next generation of biotransformation scientists who are not only experienced in the metabolism of conventional small molecule therapeutics, but are also equipped to tackle emerging challenges associated with new drug discovery modalities including peptides, protein degraders, and antibodies. SIGNIFICANCE STATEMENT: Biotransformation and mechanistic drug metabolism scientists are critical to advancing chemical entities through discovery and development, yet the number of scientists academically trained for this role is on the decline. This position paper highlights the continuing demand for biotransformation scientists and the necessity of nurturing creative ways to train them and guarantee the future growth of this field.

Selective deuteration of bupropion slows epimerization and reduces metabolism.

Strategically replacing hydrogen with deuterium at sites of metabolism in small molecule drugs can significantly alter clearance and potentially enhance clinical safety. Bupropion is an antidepressant and smoking cessation medication with the potential to cause seizures. We hypothesized that incorporating deuterium at specific sites in bupropion may greatly reduce epimerization, potentially slow metabolism, and reduce the formation of toxic metabolites, namely hydroxybupropion which has been associated with bupropion's toxicity. Four deuterated analogues were synthesized incorporating deuterium at sites of metabolism and epimerization with the aim of altering the metabolic profile of bupropion. Spectroscopic binding and metabolism studies with bupropion and R-or S-d4 and R-or S-d10 analogs were performed with recombinant CYP2B6, human liver microsomes, and human hepatocytes. Results demonstrate that deuterated bupropion analogues exhibited 20-25% decrease in racemization and displayed a significant decrease in the formation of CYP2B6-mediated R,R - or S,S-hydroxybupropion with recombinant protein and human liver microsomes. In primary human hepatocytes, metabolism of deuterated analogs to R,R - and S,S-hydroxybupropion and threo- and erythro-hydrobupropion was significantly less than R/S-d0 bupropion. Selective deuterium substitution at metabolic soft spots in bupropion has the potential to provide a drug with a simplified pharmacokinetic profile, reduced toxicity and improved tolerability in patients.

Reductions in Hydrogen Sulfide and Changes in Mitochondrial Quality Control Proteins Are Evident in the Early Phases of the Corneally Kindled Mouse Model of Epilepsy.

Epilepsy is a heterogenous neurological disorder characterized by recurrent unprovoked seizures, mitochondrial stress, and neurodegeneration. Hydrogen sulfide (H2S) is a gasotransmitter that promotes mitochondrial function and biogenesis, elicits neuromodulation and neuroprotection, and may acutely suppress seizures. A major gap in knowledge remains in understanding the role of mitochondrial dysfunction and progressive changes in H2S levels following acute seizures or during epileptogenesis. We thus sought to quantify changes in H2S and its methylated metabolite (MeSH) via LC-MS/MS following acute maximal electroshock and 6 Hz 44 mA seizures in mice, as well as in the early phases of the corneally kindled mouse model of chronic seizures. Plasma H2S was acutely reduced after a maximal electroshock seizure. H2S or MeSH levels and expressions of related genes in whole brain homogenates from corneally kindled mice were not altered. However, plasma H2S levels were significantly lower during kindling, but not after established kindling. Moreover, we demonstrated a time-dependent increase in expression of mitochondrial membrane integrity-related proteins, OPA1, MFN2, Drp1, and Mff during kindling, which did not correlate with changes in gene expression. Taken together, short-term reductions in plasma H2S could be a novel biomarker for seizures. Future studies should further define the role of H2S and mitochondrial stress in epilepsy.

Cardiac Disease Alters Myocardial Tissue Levels of Epoxyeicosatrienoic Acids and Key Proteins Involved in Their Biosynthesis and Degradation.

CYP2J2 is the main epoxygenase in the heart that is responsible for oxidizing arachidonic acid to cis-epoxyeicosatrienoic acids (EETs). Once formed, EETs can then be hydrolyzed by soluble epoxide hydrolase (sEH, encoded by EPHX2) or re-esterified back to the membrane. EETs have several cardioprotective properties and higher levels are usually associated with better cardiac outcomes/prognosis. This study investigates how cardiovascular disease (CVD) can influence total EET levels by altering protein expression and activity of enzymes involved in their biosynthesis and degradation. Diseased ventricular cardiac tissues were collected from patients receiving Left Ventricular Assist Device (LVAD) or heart transplants and compared to ventricular tissue from controls free of CVD. EETs, and enzymes involved in EETs biosynthesis and degradation, were measured using mass spectrometric assays. Terfenadine hydroxylation was used to probe CYP2J2 activity. Significantly higher cis- and trans-EET levels were observed in control cardiac tissue (n = 17) relative to diseased tissue (n = 24). Control cardiac tissue had higher CYP2J2 protein levels, which resulted in higher rate of terfenadine hydroxylation, compared to diseased cardiac tissues. In addition, levels of both NADPH-Cytochrome P450 oxidoreductase (POR) and sEH proteins were significantly higher in control versus diseased cardiac tissue. Overall, alterations in protein and activity of enzymes involved in the biosynthesis and degradation of EETs provide a mechanistic understanding for decreased EET levels in diseased tissues.

Characterization of Differential Tissue Abundance of Major Non-CYP Enzymes in Human.

The availability of assays that predict the contribution of cytochrome P450 (CYP) metabolism allows for the design of new chemical entities (NCEs) with minimal oxidative metabolism. These NCEs are often substrates of non-CYP drug-metabolizing enzymes (DMEs), such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), carboxylesterases (CESs), and aldehyde oxidase (AO). Nearly 30% of clinically approved drugs are metabolized by non-CYP enzymes. However, knowledge about the differential hepatic versus extrahepatic abundance of non-CYP DMEs is limited. In this study, we detected and quantified the protein abundance of eighteen non-CYP DMEs (AO, CES1 and 2, ten UGTs, and five SULTs) across five different human tissues. AO was most abundantly expressed in the liver and to a lesser extent in the kidney; however, it was not detected in the intestine, heart, or lung. CESs were ubiquitously expressed with CES1 being predominant in the liver, while CES2 was enriched in the small intestine. Consistent with the literature, UGT1A4, UGT2B4, and UGT2B15 demonstrated liver-specific expression, whereas UGT1A10 expression was specific to the intestine. UGT1A1 and UGT1A3 were expressed in both the liver and intestine; UGT1A9 was expressed in the liver and kidney; and UGT2B17 levels were significantly higher in the intestine than in the liver. All five SULTs were detected in the liver and intestine, and SULT1A1 and 1A3 were detected in the lung. Kidney abundance was the most variable among the studied tissues, and overall, high interindividual variability (>15-fold) was observed for UGT2B17, CES2 (intestine), SULT1A1 (liver), UGT1A9, UGT2B7, and CES1 (kidney). These differential tissue abundance data can be integrated into physiologically based pharmacokinetic (PBPK) models for the prediction of non-CYP drug metabolism and toxicity in hepatic and extrahepatic tissues.

Critical Differences between Enzyme Sources in Sensitivity to Detect Time-Dependent Inactivation of CYP2C8.

Clopidogrel acyl-ß-d-glucuronide is a mechanism-based inhibitor of cytochrome P450 2C8 in human liver microsomes (HLMs). However, time-dependent inactivation (TDI) of CYP2C8 could not be detected in an earlier study in human recombinant CYP2C8 (Supersomes). Here, we investigate whether different enzyme sources exhibit differences in detection of CYP2C8 TDI under identical experimental conditions. Inactivation of CYP2C8 by amiodarone (100 µM), clopidogrel acyl-ß-d-glucuronide (100 µM), gemfibrozil 1-O-ß-glucuronide (100 µM), and phenelzine (100 µM) was investigated in HLMs and three recombinant human CYP2C8 preparations (Supersomes, Bactosomes, and EasyCYP Bactosomes) using amodiaquine N-deethylation as the marker reaction. Furthermore, the inactivation kinetics of CYP2C8 by clopidogrel glucuronide (5-250 µM) was determined in Supersomes and Bactosomes. Amiodarone caused weak TDI in all enzyme preparations tested, while the extent of inactivation by clopidogrel glucuronide, gemfibrozil glucuronide, and phenelzine varied markedly between preparations, and even different Supersome lots. Both glucuronides caused strong inactivation of CYP2C8 in HLMs, Bactosomes and in one Supersome lot (>50% inhibition), but significant inactivation could not be reliably detected in other Supersome lots or EasyCYP Bactosomes. In Bactosomes, the concentration producing half of kinact (KI) and maximal inactivation rate (kinact) of clopidogrel glucuronide (14 µM and 0.054 minute-1) were similar to those determined previously in HLMs. Phenelzine caused strong inactivation of CYP2C8 in one Supersome lot (91% inhibition) but not in HLMs or other recombinant CYP2C8 preparations. In conclusion, different enzyme sources and different lots of the same recombinant enzyme preparation are not equally sensitive to detect inactivation of CYP2C8, suggesting that recombinant CYPs should be avoided when identifying mechanism-based inhibitors.

CYP2J2 Expression in Adult Ventricular Myocytes Protects Against Reactive Oxygen Species Toxicity.

Cytochrome P450 2J2 isoform (CYP2J2) is a drug-metabolizing enzyme that is highly expressed in adult ventricular myocytes. It is responsible for the bioactivation of arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs). EETs are biologically active signaling compounds that protect against disease progression, particularly in cardiovascular diseases. As a drug-metabolizing enzyme, CYP2J2 is susceptible to drug interactions that could lead to cardiotoxicity. CYP2J2 has been shown to be resistant to induction by canonical CYP inducers such as phenytoin and rifampin. It is, however, unknown how cellular stresses augment CYP2J2 expression. Here, we determine the effects of oxidative stress on gene expression in adult ventricular myocytes. Further, we assess the consequences of CYP2J2 inhibition and CYP2J2 silencing on cells when levels of reactive oxygen species (ROS) are elevated. Findings indicate that CYP2J2 expression increases in response to external ROS or when internal ROS levels are elevated. In addition, cell survival decreases with ROS exposure when CYP2J2 is chemically inhibited or when CYP2J2 expression is reduced using small interfering RNA. These effects are mitigated with external addition of EETs to the cells. Finally, we determined the results of external EETs on gene expression and show that only two of the four regioisomers cause an increase in HMOX1 expression. This work is the first to determine the consequence of cellular stress, specifically high ROS levels, on CYP2J2 expression in human ventricular myocytes and discusses how this enzyme may play an important role in response to cardiac oxidative stress.

Regulation of CYP2J2 and EET Levels in Cardiac Disease and Diabetes.

Cytochrome P450 2J2 (CYP2J2) is a known arachidonic acid (AA) epoxygenase that mediates the formation of four bioactive regioisomers of cis-epoxyeicosatrienoic acids (EETs). Although its expression in the liver is low, CYP2J2 is mainly observed in extrahepatic tissues, including the small intestine, pancreas, lung, and heart. Changes in CYP2J2 levels or activity by xenobiotics, disease states, or polymorphisms are proposed to lead to various organ dysfunctions. Several studies have investigated the regulation of CYP2J2 and EET formation in various cell lines and have demonstrated that such regulation is tissue-dependent. In addition, studies linking CYP2J2 polymorphisms to the risk of developing cardiovascular disease (CVD) yielded contradictory results. This review will focus on the mechanisms of regulation of CYP2J2 by inducers, inhibitors, and oxidative stress modeling certain disease states in various cell lines and tissues. The implication of CYP2J2 expression, polymorphisms, activity and, as a result, EET levels in the pathophysiology of diabetes and CVD will also be discussed.

The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides.

BACKGROUND: Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure. METHODS: The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures. RESULTS: Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F-G loop regions and active site cavity volumes for the F87A mutated structures. CONCLUSIONS: Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site. GENERAL SIGNIFICANCE: The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.

Expression and Functional Characterization of Breast Cancer-Associated Cytochrome P450 4Z1 in Saccharomyces cerevisiae.

CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in Saccharomyces cerevisiae and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC50 ∼15 µM). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.

The Methyltransferases METTL7A and METTL7B Confer Resistance to Thiol-Based Histone Deacetylase Inhibitors.

Histone deacetylase inhibitors (HDACi) are part of a growing class of epigenetic therapies used for the treatment of cancer. Although HDACis are effective in the treatment of T-cell lymphomas, treatment of solid tumors with this class of drugs has not been successful. Overexpression of the multidrug resistance protein P-glycoprotein (P-gp), encoded by ABCB1, is known to confer resistance to the HDACi romidepsin in vitro, yet increased ABCB1 expression has not been associated with resistance in patients, suggesting that other mechanisms of resistance arise in the clinic. To identify alternative mechanisms of resistance to romidepsin, we selected MCF-7 breast cancer cells with romidepsin in the presence of the P-gp inhibitor verapamil to reduce the likelihood of P-gp-mediated resistance. The resulting cell line, MCF-7 DpVp300, does not express P-gp and was found to be selectively resistant to romidepsin but not to other HDACis such as belinostat, panobinostat, or vorinostat. RNA-sequencing analysis revealed upregulation of the mRNA coding for the putative methyltransferase, METTL7A, whose paralog, METTL7B, was previously shown to methylate thiol groups on hydrogen sulfide and captopril. As romidepsin has a thiol as the zinc-binding moiety, we hypothesized that METTL7A could inactivate romidepsin and other thiol-based HDACis via methylation of the thiol group. We demonstrate that expression of METTL7A or METTL7B confers resistance to thiol-based HDACis and that both enzymes are capable of methylating thiol-containing HDACis. We thus propose that METTL7A and METTL7B confer resistance to thiol-based HDACis by methylating and inactivating the zinc-binding thiol.

OATP1B1-related drug-drug and drug-gene interactions as potential risk factors for cerivastatin-induced rhabdomyolysis.

OBJECTIVE: Genetic variation in drug metabolizing enzymes and membrane transporters as well as concomitant drug therapy can modulate the beneficial and the deleterious effects of drugs. We investigated whether patients exhibiting rhabdomyolysis who were taking cerivastatin possess functional genetic variants in SLCO1B1 and whether they were on concomitant medications that inhibit OATP1B1, resulting in accumulation of cerivastatin. METHODS: This study had three components: (a) resequencing the SLCO1B1 gene in 122 patients who developed rhabdomyolysis while on cerivastatin; (b) functional evaluation of the identified SLCO1B1 nonsynonymous variants and haplotypes in in-vitro HEK293/FRT cells stably transfected with pcDNA5/FRT empty vector, SLCO1B1 reference, variants, and haplotypes; and (c) in-vitro screening of 15 drugs commonly used among the rhabdomyolysis cases for inhibition of OATP1B1-mediated uptake of cerivastatin in HEK293/FRT cells stably transfected with reference SLCO1B1. RESULTS: The resequencing of the SLCO1B1 gene identified 54 variants. In-vitro functional analysis of SLCO1B1 nonsynonymous variants and haplotypes showed that the V174A, R57Q, and P155T variants, a novel frameshift insertion, OATP1B1*14 and OATP1B1*15 haplotype were associated with a significant reduction (P<0.001) in cerivastatin uptake (32, 18, 72, 3.4, 2.1 and 5.7% of reference, respectively). Furthermore, clopidogrel and seven other drugs were shown to inhibit OATP1B1-mediated uptake of cerivastatin. CONCLUSION: Reduced function of OATP1B1 related to genetic variation and drug-drug interactions likely contributed to cerivastatin-induced rhabdomyolysis. Although cerivastatin is no longer in clinical use, these findings may translate to related statins and other substrates of OATP1B1.

Activity, inhibition, and induction of cytochrome P450 2J2 in adult human primary cardiomyocytes.

Cytochrome P450 2J2 plays a significant role in the epoxidation of arachidonic acid to signaling molecules important in cardiovascular events. CYP2J2 also contributes to drug metabolism and is responsible for the intestinal clearance of ebastine. However, the interaction between arachidonic acid metabolism and drug metabolism in cardiac tissue, the main expression site of CYP2J2, has not been examined. Here we investigate an adult-derived human primary cardiac cell line as a suitable model to study metabolic drug interactions (inhibition and induction) of CYP2J2 in cardiac tissue. The primary human cardiomyocyte cell line demonstrated similar mRNA-expression profiles of P450 enzymes to adult human ventricular tissue. CYP2J2 was the dominant isozyme with minor contributions from CYP2D6 and CYP2E1. Both terfenadine and astemizole oxidation were observed in this cell line, whereas midazolam was not metabolized suggesting lack of CYP3A activity. Compared with recombinant CYP2J2, terfenadine was hydroxylated in cardiomyocytes at a similar K(m) value of 1.5 µM. The V(max) of terfenadine hydroxylation in recombinant enzyme was found to be 29.4 pmol/pmol P450 per minute and in the cells 6.0 pmol/pmol P450 per minute. CYP2J2 activity in the cell line was inhibited by danazol, astemizole, and ketoconazole in submicromolar range, but also by xenobiotics known to cause cardiac adverse effects. Of the 14 compounds tested for CYP2J2 induction, only rosiglitazone increased mRNA expression, by 1.8-fold. This cell model can be a useful in vitro model to investigate the role of CYP2J2-mediated drug metabolism, arachidonic acid metabolism, and their association to drug induced cardiotoxicity.

Cardioprotective mechanisms of cytochrome P450 derived oxylipins from ω-3 and ω-6 PUFAs.

The seminal discovery that cytochrome P450 enzymes (CYPs) can oxidize polyunsaturated fatty acids (PUFAs) sparked a new area of research aimed at discovering the role of these metabolites in cardiac physiology and pathophysiology. CYPs metabolize arachidonic acid, an ω-6 PUFA, to alcohols and epoxides with the latter providing cardioprotection following myocardial infarction, hypertrophy, and diabetes-induced cardiomyopathy through their anti-inflammatory, vasodilatory and antioxidant properties. Despite their protective properties, the use of EETs as therapeutic agents is hampered mainly by their rapid hydrolysis to less active vicinal diols by soluble epoxide hydrolase (sEH). Several approaches have been investigated to prolong EET signaling effects using small molecule sEH inhibitors, chemically and biologically stable analogs of EETs and more recently, through the development of an sEH vaccine. Alternatively, research investigating the cardioprotective outcomes of ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), mainly focused on dietary intake or supplementation studies. EPA and DHA have overlapping but distinct effects on myocardial function and merit separate studies to fully understand their mechanism of cardiac protection. In contrast to EETs, relatively fewer studies examined the protective mechanisms of EPA and DHA derived epoxides to determine if some protective effects are in part due to the CYP mediated downstream metabolites. The actions of CYPs on PUFAs generate potent oxylipins utilizing diverse cardioprotective mechanisms and the extent of their full potential will be important for the future development of therapeutics to prevent or treat cardiovascular disease.

Plasma hydrogen sulfide, nitric oxide, and thiocyanate levels are lower during pregnancy compared to postpartum in a cohort of women from the Pacific northwest of the United States.

AIMS: Pregnancy alters multiple physiological processes including angiogenesis, vasodilation, inflammation, and cellular redox, which are partially modulated by the gasotransmitters hydrogen sulfide (H2S) and nitric oxide (NO). In this study, we sought to determine how plasma levels of H2S, NO, and the H2S-related metabolites thiocyanate (SCN-), and methanethiol (CH3SH) change during pregnancy progression. MATERIALS AND METHODS: Plasma was collected from 45 women at three points: 25-28 weeks gestation, 28-32 week gestation, and at ≥3 months postpartum. Plasma levels of H2S, SCN-, and CH3SH were measured following derivatization using monobromobimane followed by LC-MS/MS. Plasma NO was measured indirectly using the Griess reagent. KEY FINDINGS: NO and SCN- were significantly lower in women at 25-28 weeks gestation and 28-32 weeks gestation than postpartum while plasma H2S levels were significantly lower at 28-32 weeks gestation than postpartum. No significant differences were observed in CH3SH. SIGNIFICANCE: Previous reports demonstrated that the production of H2S and NO are stimulated during pregnancy, but we observed lower levels during pregnancy compared to postpartum. Previous reports on NO have been mixed, but given the related effects of H2S and NO, it is expected that their levels would be higher during pregnancy vs. postpartum. Future studies determining the mechanism for decreased H2S and NO during pregnancy will elucidate the role of these gasotransmitters during normal and pathological progression of pregnancy.

Effects of Pregnancy on Plasma Sphingolipids Using a Metabolomic and Quantitative Analysis Approach.

Changes in the maternal metabolome, and specifically the maternal lipidome, that occur during pregnancy are relatively unknown. The objective of this investigation was to evaluate the effects of pregnancy on sphingolipid levels using metabolomics analysis followed by confirmational, targeted quantitative analysis. We focused on three subclasses of sphingolipids: ceramides, sphingomyelins, and sphingosines. Forty-seven pregnant women aged 18 to 50 years old participated in this study. Blood samples were collected on two study days for metabolomics analysis. The pregnancy samples were collected between 25 and 28 weeks of gestation and the postpartum study day samples were collected ≥3 months postpartum. Each participant served as their own control. These samples were analyzed using a Ultra-performance liquid chromatography/mass spectroscopy/mass spectroscopy (UPLC/MS/MS) assay that yielded semi-quantitative peak area values that were used to compare sphingolipid levels between pregnancy and postpartum. Following this lipidomic analysis, quantitative LC/MS/MS targeted/confirmatory analysis was performed on the same study samples. In the metabolomic analysis, 43 sphingolipid metabolites were identified and their levels were assessed using relative peak area values. These profiled sphingolipids fell into three categories: ceramides, sphingomyelins, and sphingosines. Of the 43 analytes measured, 35 were significantly different during pregnancy (p < 0.05) (including seven ceramides, 26 sphingomyelins, and two sphingosines) and 32 were significantly higher during pregnancy compared to postpartum. Following metabolomics, a separate quantitative analysis was performed and yielded quantified concentration values for 23 different sphingolipids, four of which were also detected in the metabolomics study. Quantitative analysis supported the metabolomics results with 17 of the 23 analytes measured found to be significantly different during pregnancy including 11 ceramides, four sphingomyelins, and two sphingosines. Fourteen of these were significantly higher during pregnancy. Our data suggest an overall increase in plasma sphingolipid concentrations with possible implications in endothelial function, gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy, and fetal development. This study provides evidence for alterations in maternal sphingolipid metabolism during pregnancy.

The Effects of Pregnancy on Amino Acid Levels and Nitrogen Disposition.

Limited data are available on the effects of pregnancy on the maternal metabolome. Therefore, the objective of this study was to use metabolomics analysis to determine pathways impacted by pregnancy followed by targeted confirmatory analysis to provide more powerful conclusions about metabolic alterations during pregnancy. Forty-seven pregnant women, 18-50 years of age were included in this study, with each subject serving as their own control. Plasma samples were collected between 25 and 28 weeks gestation and again ≥3 months postpartum for metabolomics analysis utilizing an HILIC/UHPLC/MS/MS assay with confirmatory targeted specific concentration analysis for 10 of the significantly altered amino acids utilizing an LC/MS assay. Principle component analysis (PCA) on metabolomics data clearly separated pregnant and postpartum groups and identified outliers in a preliminary assessment. Of the 980 metabolites recorded, 706 were determined to be significantly different between pregnancy and postpartum. Pathway analysis revealed three significantly impacted pathways, arginine biosynthesis (p = 2 × 10-5 and FDR = 1 × 10-3), valine, leucine, and isoleucine metabolism (p = 2 × 10-5 and FDR = 2 × 10-3), and xanthine metabolism (p = 4 × 10-5 and FDR = 4 × 10-3). Of these we focused analysis on arginine biosynthesis and branched-chain amino acid (BCAA) metabolism due to their clinical importance and interconnected roles in amino acid metabolism. In the confirmational analysis, 7 of 10 metabolites were confirmed as significant and all 10 confirmed the direction of change of concentrations observed in the metabolomics analysis. The data support an alteration in urea nitrogen disposition and amino acid metabolism during pregnancy. These changes could also impact endogenous nitric oxide production and contribute to diseases of pregnancy. This study provides evidence for changes in both the ammonia-urea nitrogen and the BCAA metabolism taking place during pregnancy.