17-(R/S)-hydroxyeicosatetraenoic acid (HETE) induces cardiac hypertrophy through the CYP1B1 in enantioselective manners.

Cardiac cellular hypertrophy is the increase in the size of individual cardiac cells. Cytochrome P450 1B1 (CYP1B1) is an extrahepatic inducible enzyme that is associated with toxicity, including cardiotoxicity. We previously reported that 19-hydroxyeicosatetraenoic acid (19-HETE) inhibited CYP1B1 and prevented cardiac hypertrophy in enantioselective manner. Therefore, our aim is to investigate the effect of 17-HETE enantiomers on cardiac hypertrophy and CYP1B1. Human adult cardiomyocyte (AC16) cells were treated with 17-HETE enantiomers (20 µM); cellular hypertrophy was evaluated by cell surface area and cardiac hypertrophy markers. In addition, CYP1B1 gene, protein and activity were assessed. Human recombinant CYP1B1 and heart microsomes of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats were incubated with 17-HETE enantiomers (10-80 nM). Our results demonstrated that 17-HETE induced cellular hypertrophy, which is manifested by increase in cell surface area and cardiac hypertrophy markers. 17-HETE enantiomers allosterically activated CYP1B1 and selectively upregulated CYP1B1 gene and protein expression in AC16 cells at uM range. In addition, CYP1B1 was allosterically activated by 17-HETE enantiomers at nM range in recombinant CYP1B1 and heart microsomes. In conclusion, 17-HETE acts as an autocrine mediator, leading to the cardiac hypertrophy through induction of CYP1B1 activity in the heart.

The enantioselective separation and quantitation of the hydroxy-metabolites of arachidonic acid by liquid chromatography - tandem mass spectrometry.

Arachidonic acid (AA) is a polyunsaturated fatty acid with a structure of 20:4(ω-6). Cytochrome P450s (CYPs) metabolize AA to several regioisomers and enantiomers of hydroxyeicosatetraenoic acids (HETEs). The hydroxy-metabolites (HETEs) exist as enantiomers in the biological system. The chiral assays developed for HETEs are so far limited to a few assays reported for midchain HETEs. The developed method is capable of quantitative analysis for midchain, subterminal HETE enantiomers, and terminal HETEs in microsomes. The peak area or height ratios were linear over concentrations ranging (0.01 -0.6 µg/ml) with r2 > 0.99. The intra-run percent error and coefficient of variation (CV) were ≤ ± 12 %. The inter-run percent error and coefficient of variation (CV)were ≤ ± 13 %, and ≤ 15 %, respectively. The matrix effect for the assay was also within the acceptable limit (≤ ± 15 %). The recovery of HETE metabolites ranged from 70 % to 115 %. The method showed a reliable and robust performance for chiral analysis of cytochrome P450-mediated HETE metabolites.

The multifaceted role of cytochrome P450-Derived arachidonic acid metabolites in diabetes and diabetic cardiomyopathy.

Understanding lipid metabolism is a critical key to understanding the pathogenesis of Diabetes Mellitus (DM). It is known that 60-90% of DM patients are obese or used to be obese. The incidence of obesity is rising owing to the modern sedentary lifestyle that leads to insulin resistance and increased levels of free fatty acids, predisposing tissues to utilize more lipids with less glucose uptake. However, the exact mechanism is not yet fully elucidated. Diabetic cardiomyopathy seems to be associated with these alterations in lipid metabolism. Arachidonic acid (AA) is an important fatty acid that is metabolized to several bioactive compounds by cyclooxygenases, lipoxygenases, and the more recently discovered, cytochrome P450 (P450) enzymes. P450 metabolizes AA to either epoxy-AA (EETs) or hydroxy-AA (HETEs). Studies showed that EETs could have cardioprotective effects and beneficial effects in reversing abnormalities in glucose and insulin homeostasis. Conversely, HETEs, most importantly 12-HETE and 20-HETE, were found to interfere with normal glucose and insulin homeostasis and thus, might be involved in diabetic cardiomyopathy. In this review, we highlight the role of P450-derived AA metabolites in the context of DM and diabetic cardiomyopathy and their potential use as a target for developing new treatments for DM and diabetic cardiomyopathy.

Sex, estrous cycle, and hormone regulation of CYP2D in the brain alters oxycodone metabolism and analgesia.

Opioids, and numerous centrally active drugs, are metabolized by cytochrome P450 2D (CYP2D). There are sex and estrous cycle differences in brain oxycodone analgesia. Here we investigated the mechanism examining the selective role of CYP2D in the brain on sex, estrous cycle, and hormonal regulation. Propranolol, CYP2D-specific mechanism-based inhibitor, or vehicle was delivered into cerebral ventricles 24 h before administering oxycodone (or oxymorphone, negative control) orally to male and female (in estrus and diestrus) rats. Ovariectomized and sham-operated females received no treatment, estradiol, progesterone or vehicle. Analgesia was measured using tail-flick latency, and brain drug and metabolite concentrations were measured by microdialysis. Data were analyzed by two-way or mixed ANOVA. Following propranolol (versus vehicle) inhibition and oral oxycodone, there were greater increases in brain oxycodone concentrations and analgesia, and greater decreases in brain oxymorphone/oxycodone ratios (an in vivo phenotype of CYP2D in brain) in males and females in estrus, compared to females in diestrus; with no impact on plasma drug concentrations. There was no impact of propranolol pre-treatment, sex, or cycle after oral oxymorphone (non-CYP2D substrate) on brain oxymorphone concentrations or analgesia. There was no impact of propranolol pre-treatment following ovariectomy on brain oxycodone concentrations or analgesia, which was restored in ovariectomized females following estradiol, but not progesterone, treatment. Sex, cycle, and estradiol regulation of CYP2D in brain in turn altered brain oxycodone concentration and response, which may contribute to the large inter-individual variation in response to the numerous centrally acting CYP2D substrate drugs, including opioids.

Identifying simultaneous matrix metalloproteinases/soluble epoxide hydrolase inhibitors.

Matrix metalloproteinase (MMP) and soluble epoxide hydrolase (sEH) have completely unrelated biological functions; however, their dysregulation produce similar effects on biological systems. Based on the similarity in the reported structural requirements for their inhibition, the current study aimed to identify a simultaneous inhibitor for MMP and sEH. Six compounds were identified as potential simultaneous MMP/sEH inhibitors and tested for their capacity to inhibit MMP and sEH. Inhibition of MMP and sEH activity using their endogenous and exogenous substrates was measured by liquid chromatography/mass spectrometry, spectrophotometry, and zymography. Two compounds, CTK8G1143 and ONO-4817, were identified to inhibit both MMP and sEH activity. CTK8G1143 and ONO-4817 inhibited the recombinant human sEH activity by an average of 67.4% and 55.2%, respectively. The IC50 values for CTK8G1143 and ONO-4817 to inhibit recombinant human sEH were 5.2 and 3.5 µM, respectively, whereas their maximal inhibition values were 71.4% and 42.8%, respectively. Also, MMP and sEH activity of human cardiomyocytes were simultaneously inhibited by CTK8G1143 and ONO-4817. Regarding other compounds, they showed either MMP or sEH inhibitory activity but not both. In conclusion, these two simultaneous inhibitors of MMP and sEH could provide a promising intervention for the prevention and control of several diseases, especially cardiovascular diseases.

The role of CYP2D in rat brain in methamphetamine-induced striatal dopamine and serotonin release and behavioral sensitization.

RATIONALE: Cytochrome P450 2D (CYP2D) enzymes metabolize many addictive drugs, including methamphetamine. Variable CYP2D metabolism in the brain may alter CNS drug/metabolite concentrations, consequently affecting addiction liability and neuropsychiatric outcomes; components of these can be modeled by behavioral sensitization in rats. METHODS: To investigate the role of CYP2D in the brain in methamphetamine-induced behavioral sensitization, rats were pretreated centrally with a CYP2D irreversible inhibitor (or vehicle) 20 h prior to each of 7 daily methamphetamine (0.5 mg/kg subcutaneous) injections. In vivo brain microdialysis was used to assess brain drug and metabolite concentrations, and neurotransmitter release. RESULTS: CYP2D inhibitor (versus vehicle) pretreatment enhanced methamphetamine-induced stereotypy response sensitization. CYP2D inhibitor pretreatment increased brain methamphetamine concentrations and decreased the brain p-hydroxylation metabolic ratio. With microdialysis conducted on days 1 and 7, CYP2D inhibitor pretreatment exacerbated stereotypy sensitization and enhanced dopamine and serotonin release in the dorsal striatum. Day 1 brain methamphetamine and amphetamine concentrations correlated with dopamine and serotonin release, which in turn correlated with the stereotypy response slope across sessions (i.e., day 1 through day 7), used as a measure of sensitization. CONCLUSIONS: CYP2D-mediated methamphetamine metabolism in the brain is sufficient to alter behavioral sensitization, brain drug concentrations, and striatal dopamine and serotonin release. Moreover, day 1 methamphetamine-induced neurotransmitter release may be an important predictor of subsequent behavioral sensitization. This suggests the novel contribution of CYP2D in the brain to methamphetamine-induced behavioral sensitization and suggests that the wide variation in human brain CYP2D6 may contribute to differential methamphetamine responses and chronic effects.

Centrally administered CYP2D inhibitors increase oral tramadol analgesia in rats.

Cytochrome P450 2D (CYP2D) mediates the activation and inactivation of several classes of psychoactive drugs, including opioids, which can alter drug response. Tramadol is a synthetic opioid with analgesic activity of its own as well as being metabolically activated by CYP2D to O-desmethyltramadol (ODMST) an opioid receptor agonist. We investigated the impact of brain CYP2D metabolism on central tramadol and ODSMT levels, and resulting analgesic response after oral tramadol administration in rats. CYP2D inhibitors propranolol and propafenone were administered intracerebroventricularly prior to oral tramadol administration and analgesia was measured by tail-flick latency. Drug levels of tramadol and its metabolites, ODSMT and N-desmethyltramadol, were assessed in plasma and in brain by microdialysis using LC-ESI-MS/MS. Inhibiting brain CYP2D with propafenone pretreatment increased analgesia after oral tramadol administration (ANOVA p = 0.02), resulting in a 1.5-fold increase in area under the analgesia-time curve (AUC0-60, p < 0.01). This effect was associated with changes in the brain levels of tramadol and its metabolites consistent with brain CYP2D inhibition. In conclusion, under oral tramadol dosing pretreatment with a central administration of the CYP2D inhibitor propafenone increased analgesia (without altering plasma drug or metabolite levels), indicating that tramadol itself (and activity of CYP2D within the brain) contributed to analgesia.

Addressing the instability issue of dopamine during microdialysis: the determination of dopamine, serotonin, methamphetamine and its metabolites in rat brain.

Dopamine is a catecholamine neurotransmitter that degrades rapidly in aqueous solutions; hence, its analysis following brain microdialysis is challenging. The aim of the current study was to develop and validate a new microdialysis coupled LC-MS/MS system with improved accuracy, precision, simplicity and turnaround time for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine analysis in the brain. Dopamine degradation was studied with different stabilizing agents under different storage conditions. The modified microdialysis system was tested in vitro, and was optimized for best probe recovery, assessed by %gain. LC-MS/MS assay was developed and validated for the targeted compounds. Stabilizing agents (ascorbic acid, EDTA and acetic acid) as well as internal and cold standards were added on-line to the dialysate flow. Assay linearity range was 0.01-100 ng/mL, precision and accuracy passed criteria, and LOQ and LLOQ were 0.2 and 1.0 pg, respectively. The new microdialysis coupled LC-MS/MS system was used in Wistar rats striatum after 4 mg/kg subcutaneous methamphetamine. Methamphetamine rapidly distributed to rat striatum reaching an average ~200 ng/mL maximum, ~82.5 min post-dose. Amphetamine, followed by 4-hydroxymethamphetamine, was the most abundant metabolite. Dopamine was released following methamphetamine injection, while serotonin was not altered. In conclusion, we proposed and tested an innovative and simplified solution to improve stability, accuracy and turnover time to monitor unstable molecules, such as dopamine, by microdialysis.

Identification of 19-(S/R)Hydroxyeicosatetraenoic Acid as the First Endogenous Noncompetitive Inhibitor of Cytochrome P450 1B1 with Enantioselective Activity.

The overexpression of cytochrome P450 1B1 (CYP1B1) is a common characteristic of several diseases and conditions, such as inflammation, cancer, and cardiac hypertrophy. CYP1B1 is believed to contribute to pathogenesis of these diseases by mediating the formation of toxic compounds, either from exogenous or endogenous origin. We recently reported that an arachidonic acid metabolite, 19(S/R-)hydroxyeicosatetraenoic (HETE) acid, protects from cardiac hypertrophy by inhibiting the formation of toxic compounds, midchain HETEs, known to be formed by CYP1B1. This raised the question whether 19(S/R)-HETE can directly inhibit CYP1B1. In the current study, we report that 19(S/R)-HETE enantioselectively inhibits human recombinant CYP1B1 activity measured by 7-ethoxyresorufin O-deethylation assay. 19(S)-HETE is more potent than the R enantiomer (K i = 37.3 and 89.1 nM, respectively). Noncompetitive inhibition was identified as the mechanism of CYP1B1 inhibition, which underlines the potentially important physiologic role of 19(S/R)-HETE as an endogenous CYP1B1 inhibitor; to our knowledge, 19(S/R)-HETE is the first inhibitor of its kind to be reported.

Subterminal hydroxyeicosatetraenoic acids: Crucial lipid mediators in normal physiology and disease states.

Cytochrome P450 (P450) enzymes are superfamily of monooxygenases that hold the utmost diversity of substrate structures and catalytic reaction forms amongst all other enzymes. P450 enzymes metabolize arachidonic acid (AA) to a wide array of biologically active lipid mediators. P450-mediated AA metabolites have a significant role in normal physiological and pathophysiological conditions, hence they could be promising therapeutic targets in different disease states. P450 monooxygenases mediate the (ω-n)-hydroxylation reactions, which involve the introduction of a hydroxyl group to the carbon skeleton of AA, forming subterminal hydroxyeicosatetraenoic acids (HETEs). In the current review, we specified different P450 isozymes implicated in the formation of subterminal HETEs in varied tissues. In addition, we focused on the role of subterminal HETEs namely 19-HETE, 16-HETE, 17-HETE and 18-HETE in different organs, importantly the kidneys, heart, liver and brain. Furthermore, we highlighted their role in hypertension, acute coronary syndrome, diabetic retinopathy, non-alcoholic fatty liver disease, ischemic stroke as well as inflammatory diseases. Since each member of subterminal HETEs exist as R and S enantiomer, we addressed the issue of stereoselectivity related to the formation and differential effects of these enantiomers. In conclusion, elucidation of different roles of subterminal HETEs in normal and disease states leads to identification of novel therapeutic targets and development of new therapeutic modalities in different disease states.

DHA and 19,20-EDP induce lysosomal-proteolytic-dependent cytotoxicity through de novo ceramide production in H9c2 cells with a glycolytic profile.

Docosahexaenoic acid (DHA) and their CYP-derived metabolites, epoxydocosapentaenoic acids (EDPs), are important fatty acids obtained from dietary sources. While it is known that they have significant biological effects, which can differ between cell type and disease state, our understanding of how they work remains limited. Previously, we demonstrated that DHA and 19,20-EDP triggered pronounced cytotoxicity in H9c2 cells correlating with increased ceramide production. In this study, we examine whether DHA- and 19,20-EDP-induced cell death depends on the type of metabolism (glycolysis or OXPHOS). We cultivated H9c2 cells in distinct conditions that result in either glycolytic or oxidative metabolism. Our major findings suggest that DHA and its epoxy metabolite, 19,20-EDP, trigger cytotoxic effects toward H9c2 cells with a glycolytic metabolic profile. Cell death occurred through a mechanism involving activation of a lysosomal-proteolytic degradation pathway. Importantly, accumulation of ceramide played a critical role in the susceptibility of glycolytic H9c2 cells to cytotoxicity. Furthermore, our data suggest that an alteration in the cellular metabolic profile is a major factor determining the type and magnitude of cellular toxic response. Together, the novelty of this study demonstrates that DHA and 19,20-EDP induce cell death in H9c2 cells with a glycolytic metabolicwct 2 profile through a lysosomal-proteolytic mechanism.

The Role of Soluble Epoxide Hydrolase Enzyme on Daunorubicin-Mediated Cardiotoxicity.

Several studies have demonstrated the role of cytochrome P450 (CYP) and its associated arachidonic acid (AA) metabolites in the anthracyclines-induced cardiac toxicity. However, the ability of daunorubicin (DNR) to induce cardiotoxicity through the modulation of CYP and its associated AA metabolites has not been investigated yet. Therefore, we hypothesized that DNR-induced cardiotoxicity is mediated through the induction of cardiotoxic hydroxyeicosatetraenoic acids and/or the inhibition of cardioprotctive epoxyeicosatrienoic acids (EETs). To test our hypothesis, Sprague-Dawley rats were treated with DNR (5 mg/kg i.p.) for 24 h, whereas human ventricular cardiomyocytes RL-14 cells were exposed to DNR in the presence and absence of 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (tAUCB), a soluble epoxide hydrolase (sEH) inhibitor. Thereafter, real-time PCR, Western blot analysis and liquid chromatography-electron spray ionization mass spectroscopy were used to determine the level of gene expression, protein expression and AA metabolites, respectively. Our results showed that DNR-induced cardiotoxicity in vivo and in vitro as evidenced by the induction of hypertrophic and fibrotic markers. Moreover, the DNR-induced cardiotoxicity was associated with a dramatic increase in the formation of cardiac DHET/EET metabolites both in vivo and in RL-14 cells suggesting a sEH enzyme dependent mechanism. Interestingly, inhibition of sEH using tAUCB, a selective sEH inhibitor, significantly protects against DNR-induced cardiotoxicity. Mechanistically, the protective effect tAUCB was mediated through the induction of P50 nuclear factor-κB and the inhibition of phosphorylated p38. In conclusion, our study provides the first evidence that DNR induces cardiotoxicity through a sEH-mediated EETs degradation-dependent mechanism.

Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation.

AIMS: In addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor. METHODS AND RESULTS: Utilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbß3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation. CONCLUSION: These findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.

Inhibition of Mid-chain HETEs Protects Against Angiotensin II-induced Cardiac Hypertrophy.

Recent data demonstrated the role of CYP1B1 in cardiovascular disease. It was, therefore, necessary to examine whether the inhibition of CYP1B1 and hence inhibiting the formation of its metabolites, using 2,4,3',5'-tetramethoxystilbene (TMS), would have a cardioprotective effect against angiotensin II (Ang II)-induced cardiac hypertrophy. For this purpose, male Sprague Dawley rats were treated with Ang II with or without TMS (300 µg/kg every third day i.p.). Thereafter, cardiac hypertrophy and the formation of mid-chain HETEs and arachidonic acid were assessed. In vitro, RL-14 cells were treated with Ang II (10 µM) in the presence and absence of TMS (0.5 µM). Then, reactive oxygen species, mitogen-activated protein kinase phosphorylation levels, and nuclear factor-kappa B-binding activity were determined. Our results demonstrated that TMS protects against Ang II-induced cardiac hypertrophy as indicated by the improvement in cardiac functions shown by the echocardiography as well as by reversing the increase in heart weight to tibial length ratio caused by Ang II. In addition, the cardioprotective effect of TMS was associated with a significant decrease in cardiac mid-chain HETEs levels. Mechanistically, TMS inhibited reactive oxygen species formation, the phosphorylation of ERK1/2, p38 mitogen-activated protein kinase, and the binding of p65 NF-κB.

The role of cytochrome P450 1B1 and its associated mid-chain hydroxyeicosatetraenoic acid metabolites in the development of cardiac hypertrophy induced by isoproterenol.

Numerous experimental studies have demonstrated the role of cytochrome P450 1B1 (CYP1B1) and its associated mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) metabolite in the pathogenesis of cardiac hypertrophy. However, the ability of isoproterenol (ISO) to induce cardiac hypertrophy through mid-chain HETEs has not been investigated yet. Therefore, we hypothesized that ISO induces cardiac hypertrophy through the induction of CYP1B1 and its associated mid-chain HETE metabolites. To test our hypothesis, Sprague-Dawley rats were treated with ISO (5 mg/kg i.p.) for 12 and 72 h whereas, human ventricular cardiomyocytes RL-14 cells were exposed to 100 µM ISO in the presence and absence of 0.5 µM tetramethoxystilbene (TMS) a selective CYP1B1 inhibitor, or 25 nM CYP1B1-siRNA. Moreover, RL-14 cells were transiently transfected with the CRISPR-CYP1B1 plasmid. Thereafter, real-time PCR, western blot analysis, and liquid chromatography-electrospray ionization mass spectroscopy were used to determine the level of gene expression, protein expression, and mid-chain HETEs, respectively. Our results showed that ISO induced CYP1B1 protein expression and the level of cardiac mid-chain HETEs in vivo at pre-hypertrophic and hypertrophic stage. In vitro, inhibition of CYP1B1 using TMS or CYP1B1-siRNA significantly attenuates ISO-induced hypertrophy. Furthermore, overexpression of CYP1B1 significantly induced cellular hypertrophy and mid-chain HETEs metabolite. Mechanistically, the protective effect of TMS against cardiac hypertrophy was mediated through the modulation of superoxide anion, mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that CYP1B1 and its associated mid-chain HETE metabolites are directly involved in the ISO-induced cardiac hypertrophy.

Pharmacokinetics of metformin in the rat: assessment of the effect of hyperlipidemia and evidence for its metabolism to guanylurea.

Metformin pharmacokinetics are highly dependent upon organic cationic transporters. There is evidence of a change in its renal clearance in hyperlipidemic obese patients, and no information on its metabolic fate. To study some of these aspects, the influence of poloxamer 407 (P407)-induced hyperlipidemia on metformin pharmacokinetics was assessed. Control and P407-treated adult male rats were administered 30 mg/kg metformin intravenously (i.v.). The pharmacokinetic assessments were performed at 2 time points, 36 and 108 h, following the intraperitoneal dose of P407 (1 g/kg). mRNA and protein expressions of cationic drug transporters were also measured. There was no evidence of a change in metformin pharmacokinetics after i.v. doses as a consequence of short-term hyperlipidemia, and a change in transporter mRNA but not protein expression was observed in the P407- treated rats 108 h after P407 injection. Urinary recovery of unchanged drug was high (>90%) but incomplete. Presumed metabolite peaks were detected in chromatograms of hepatocytes and microsomal protein spiked with metformin. Comparative chromatographic elution times and mass spectra suggested that one of the predominant metabolites was guanylurea. Hyperlipidemia by itself did not affect the pharmacokinetics of metformin. Guanylurea is a putative metabolite of metformin in rats.

Microsomal cytochrome P450 as a target for drug discovery and repurposing.

Cytochrome P450 (P450) enzymes are ancient electron-transfer-chain system of remarkable biological importance. Microsomal P450 enzymes are the P450 attached to endoplasmic reticulum, which, in humans, are critical for body's defenses against xenobiotics by mediating their metabolism, and cell signaling by mediating arachidonic acid (AA) transformation to several potent bioactive molecules. Only recently, modulating P450-mediated AA metabolism has risen as a promising new drug target. This review presents the therapeutic potential of finding effective, selective and safe treatments targeting P450-mediated AA metabolism, and the several approaches that have been used to find these treatments; among which, our focus was on modulators of P450 activities. We detailed the efforts done to develop new molecular entities designed to modulate P450, and the more recent efforts tried to employ our previous knowledge on drug metabolism to repurpose old drugs with the capacity to alter P450-mediated drug metabolism to target AA metabolism. Because of the long recognition of P450 role in xenobiotic metabolism, several clinically approved agents were identified to alter P450 activity. Repurposing old drugs as P450 modulators can facilitate bringing treatments targeting P450-mediated AA metabolism to clinical trials. However, the capacity of the modulation of P450-derived AA metabolites of clinically approved drugs has to be systematically investigated and validated for their new use in humans.

Repurposing Resveratrol and Fluconazole To Modulate Human Cytochrome P450-Mediated Arachidonic Acid Metabolism.

Cytochrome P450 (P450) enzymes metabolize arachidonic acid (AA) to several biologically active epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). Repurposing clinically-approved drugs could provide safe and readily available means to control EETs and HETEs levels in humans. Our aim was to determine how to significantly and selectively modulate P450-AA metabolism in humans by clinically-approved drugs. Liquid chromatography-mass spectrometry was used to determine the formation of 15 AA metabolites by human recombinant P450 enzymes, as well as human liver and kidney microsomes. CYP2C19 showed the highest EET-forming activity, while CYP1B1 and CYP2C8 showed the highest midchain HETE-forming activities. CYP1A1 and CYP4 showed the highest subterminal- and 20-HETE-forming activity, respectively. Resveratrol and fluconazole produced the most selective and significant modulation of hepatic P450-AA metabolism, comparable to investigational agents. Monte Carlo simulations showed that 90% of human population would experience a decrease by 6-22%, 16-39%, and 16-35% in 16-, 18-, and 20-HETE formation, respectively, after 2.5 g daily of resveratrol, and by 22-31% and 14-23% in 8,9- and 14,15-EET formation after 50 mg of fluconazole. In conclusion, clinically-approved drugs can provide selective and effective means to modulate P450-AA metabolism, comparable to investigational drugs. Resveratrol and fluconazole are good candidates to be repurposed as new P450-based treatments.

Ketoconazole Stereoisomers Differentially Induce Cytochrome P450 1A1 Between Human Hepatoma HepG2 and Mouse Hepatoma Hepa1c1c7 Cells.

Ketoconazole (KTZ) has 2 chiral centers with the therapeutically active form being a racemic mixture of 2 cis-enantiomers, namely, (2R,4S)-(+)-KTZ and (2S,4R)-(-)-KTZ. The aims of the present study were to examine the effects of (+)-KTZ, (-)-KTZ, and (±)-KTZ on aryl hydrocarbon receptor activation and subsequently CYP1A1 induction in both human HepG2 and murine Hepa1c1c7 hepatoma cells, and to further test their inhibitory effect using recombinant human and mouse CYP1A1 enzymes. Our results demonstrated that (+)-KTZ induced human CYP1A1 more than (-)-KTZ, whereas on the other hand (-)-KTZ induced murine Cyp1a1 more than (+)-KTZ at the mRNA, and activity levels. Human CYP1A1 showed higher affinity to 7ER compared with murine Cyp1a1 (Km values 13.29 nM for human vs. 168.1 nM for murine). The intrinsic clearance values for human and murine CYP1A1 were 194.1 and 87.6 µL/pmol P450/min, respectively, whereas, Vmax values were 2.58 and 14.73 pmol/pmol P450/min, respectively. (+)-KTZ and (-)-KTZ directly inhibited CYP1A1 activity by noncompetitive mechanism. The affinity of (-)-KTZ to interact with human CYP1A1 and murine Cyp1a1 was significantly different from (+)-KTZ, as the Ki values for human CYP1A1 and murine Cyp1a1 were 199.4 and 413.7 nM, respectively, for (+)-KTZ, and 269.3 and 230.8 nM, respectively, for (-)-KTZ.

19-Hydroxyeicosatetraenoic acid and isoniazid protect against angiotensin II-induced cardiac hypertrophy.

We have recently demonstrated that 19-hydroxyeicosatetraenoic acid (19-HETE) is the major subterminal-HETE formed in the heart tissue, and its formation was decreased during cardiac hypertrophy. In the current study, we examined whether 19-HETE confers cardioprotection against angiotensin II (Ang II)-induced cardiac hypertrophy. The effect of Ang II, with and without 19-HETE (20 µM), on the development of cellular hypertrophy in cardiomyocyte RL-14 cells was assessed by real-time PCR. Also, cardiac hypertrophy was induced in Sprague-Dawley rats by Ang II, and the effect of increasing 19-HETE by isoniazid (INH; 200mg/kg/day) was assessed by heart weight and echocardiography. Also, alterations in cardiac cytochrome P450 (CYP) and their associated arachidonic acid (AA) metabolites were determined by real-time PCR, Western blotting and liquid-chromatography-mass-spectrometry. Our results demonstrated that 19-HETE conferred a cardioprotective effect against Ang II-induced cellular hypertrophy in vitro, as indicated by the significant reduction in ß/α-myosin heavy chain ratio. In vivo, INH improved heart dimensions, and reversed the increase in heart weight to tibia length ratio caused by Ang II. We found a significant increase in cardiac 19-HETE, as well as a significant reduction in AA and its metabolite, 20-HETE. In conclusion, 19-HETE, incubated with cardiomyocytes in vitro or induced in the heart by INH in vivo, provides cardioprotection against Ang II-induced hypertrophy. This further confirms the role of CYP, and their associated AA metabolites in the development of cardiac hypertrophy.