Cytochrome P450-dependent Pathway Research Articles

Kidney Response to Chemotherapy-Induced Heart Failure: mRNA Analysis in Normotensive and Ren-2 Transgenic Hypertensive Rats.

The aim of the present study was to perform kidney messenger ribonucleic acid (mRNA) analysis in normotensive, Hannover Sprague–Dawley (HanSD) rats and hypertensive, Ren-2 renin transgenic rats (TGR) after doxorubicin-induced heart failure (HF) with specific focus on genes that are implicated in the pathophysiology of HF-associated cardiorenal syndrome. We found that in both strains renin and angiotensin-converting enzyme mRNA expressions were upregulated indicating that the vasoconstrictor axis of the renin–angiotensin system was activated. We found that pre-proendothelin-1, endothelin-converting enzyme type 1 and endothelin type A receptor mRNA expressions were upregulated in HanSD rats, but not in TGR, suggesting the activation of endothelin system in HanSD rats, but not in TGR. We found that mRNA expression of cytochrome P-450 subfamily 2C23 was downregulated in TGR and not in HanSD rats, suggesting the deficiency in the intrarenal cytochrome P450-dependent pathway of arachidonic acid metabolism in TGR. These results should be the basis for future studies evaluating the pathophysiology of cardiorenal syndrome secondary to chemotherapy-induced HF in order to potentially develop new therapeutic approaches.

Degradation of diclofenac by B. subtilis through a cytochrome P450-dependent pathway

Diclofenac exerts the highest level of acute toxicity among tested non-steroidal anti-inflammatory drugs. Although there are many reports on the microbial degradation of diclofenac, gaps remain in the knowledge related to the degradation process, especially that by Bacillus subtilis . In this study, we used B. subtilis 168trpc2 and its cytochrome P450 genetic defect strains B. subtilis Δ yrhJ and B. subtilis Δ yjiB to identify the key limiting protein and factor in diclofenac degradation. The results showed that in 72 h, B. subtilis 168trpc2 degraded 88.3% of diclofenac, whereas B. subtilis Δ yrhJ and B. subtilis Δ yjiB degraded 10% and 21%, respectively. These results illustrate that cytochrome P450 is essential for diclofenac degradation. The hydroxylation product of B. subtilis 168trpc2 is 5-OH-diclofenac, whereas the genetic defect strains generate the hydroxylation products 5-OH-diclofenac and 4ʹ-OH-diclofenac. Hydroxydiclofenac can subsequently be dechlorinated; through CO 2 loss and C-N bond cleavage wherein dechlorination is the central step. This study provides direct evidence that cytochrome P450 determines the position of diclofenac hydroxylation and that hydroxydiclofenac can be subsequently degraded.

Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway

The 1-deoxysphingolipids (1-deoxySLs) are atypical sphingolipids (SLs) that are formed when serine palmitoyltransferase condenses palmitoyl-CoA with alanine instead of serine during SL synthesis. The 1-deoxySLs are toxic to neurons and pancreatic β-cells. Pathologically elevated 1-deoxySLs cause the inherited neuropathy, hereditary sensory autonomic neuropathy type 1 (HSAN1), and are also found in T2D. Diabetic sensory polyneuropathy (DSN) and HSAN1 are clinically very similar, suggesting that 1-deoxySLs may be implicated in both pathologies. The 1-deoxySLs are considered to be dead-end metabolites, as they lack the C1-hydroxyl group, which is essential for the canonical degradation of SLs. Here, we report a previously unknown metabolic pathway, which is capable of degrading 1-deoxySLs. Using a variety of metabolic labeling approaches and high-resolution high-accuracy MS, we identified eight 1-deoxySL downstream metabolites, which appear to be formed by cytochrome P450 (CYP)4F enzymes. Comprehensive inhibition and induction of CYP4F enzymes blocked and stimulated, respectively, the formation of the downstream metabolites. Consequently, CYP4F enzymes might be novel therapeutic targets for the treatment of HSAN1 and DSN, as well as for the prevention of T2D.

Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803

Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.

Adverse drug interactions as a high‐risk factor for lethal post‐transplant complications in Chinese population

Metabolism of triazole antifungal agents is highly competitive to conventional post-transplant immunosuppressants like cyclosporine A (CsA) via the cytochrome P450-dependent pathway. We present the first report on lethal complications that may arise due to this type of drug interaction. A retrospective survey identified 10 of 104 cases (9.62%) that suffered life-threatening complications associated with the interaction between CsA and itraconazole or voriconazole following allogeneic hematopoietic stem cell transplantation (allo-HSCT) at our center. According to the close drug monitoring, all 10 patients experienced supratherapeutic levels of CsA even with a preemptive CsA dosage reduction and prompt dose adjustment. Six patients developed grade I to III acute graft-versus-host disease (aGVHD) and eventually died from either idiopathic pneumonia syndrome or diffuse alveolar hemorrhage; another four patients died from CSA-associated neurological complications. Impaired hepatic and renal function was noted in only one of these 10 cases. The high frequency as well as the unpredictability of severe complications lead us to suggest that triazole should always be replaced by another antifungal medication (e.g., amphotericin B or Echincandins) while patients receive CsA after HSCT, especially in the Chinese population.

Inhibition of the antithrombotic effects of clopidogrel by proton pump inhibitors: Facts or fancies?

Clopidogrel plus aspirin is considered the antiplatelet treatment of choice in patients with acute coronary syndrome, whether or not they are undergoing a percutaneous coronary intervention (PCI). The same treatment is mandatory in all patients undergoing a PCI with stent implantation. Clopidogrel is a pro-drug that needs metabolic activation through a cytochrome P450-dependent pathway, with an extensive involvement of the CYP 2C19 isoenzyme. Proton pump inhibitors (PPIs) reduce the risk of gastrointestinal bleeding in patients receiving dual antiplatelet therapy. In the past two years some scientific evidences have suggested a possible negative interference of PPIs on antiplatelet effect of clopidogrel because of the competitive inhibition of the CYP 2C19 isoenzyme. Few studies testing platelet reactivity in patients receiving both clopidogrel and a PPI have demonstrated a reduced inhibitory effect of the association on platelet aggregation. Moreover, results from retrospective observational studies have shown a higher incidence of major cardiovascular events in patients receiving both clopidogrel and PPIs. These data have not been confirmed neither by the only prospective randomized study comparing clopidogrel plus omeprazole with clopidogrel alone, nor by the retrospective analysis of the TRITON TIMI 38 trial, where PPIs did not affect the clinical outcome of patients given clopidogrel or prasugrel. Nevertheless both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) have discouraged the concomitant use of clopidogrel and PPIs. Important questions concerning a true interference between the two classes of drugs still remain unanswered and need to be addressed by adequately powered studies.

Excretion and Metabolism of Lersivirine (5-{[3,5-Diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile), a Next-Generation Non-Nucleoside Reverse Transcriptase Inhibitor, after Administration of [14C]Lersivirine to Healthy Volunteers

Lersivirine [UK-453,061, 5-((3,5-diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl)oxy)benzene-1,3-dicarbonitrile] is a next-generation non-nucleoside reverse transcriptase inhibitor, with a unique binding interaction within the reverse transcriptase binding pocket. Lersivirine has shown antiviral activity and is well tolerated in HIV-infected and healthy subjects. This open-label, Phase I study investigated the absorption, metabolism, and excretion of a single oral 500-mg dose of [14C]lersivirine (parent drug) and characterized the plasma, fecal, and urinary radioactivity of lersivirine and its metabolites in four healthy male volunteers. Plasma C(max) for total radioactivity and unchanged lersivirine typically occurred between 0.5 and 3 h postdose. The majority of radioactivity was excreted in urine (approximately 80%) with the remainder excreted in the feces (approximately 20%). The blood/plasma ratio of total drug-derived radioactivity [area under the plasma concentration-time profile from time zero extrapolated to infinite time (AUC(inf))] was 0.48, indicating that radioactive material was distributed predominantly into plasma. Lersivirine was extensively metabolized, primarily by UDP glucuronosyltransferase- and cytochrome P450-dependent pathways, with 22 metabolites being identified in this study. Analysis of precipitated plasma revealed that the lersivirine-glucuronide conjugate was the major circulating component (45% of total radioactivity), whereas unchanged lersivirine represented 13% of total plasma radioactivity. In vitro studies showed that UGT2B7 and CYP3A4 are responsible for the majority of lersivirine metabolism in humans.

Funktionsdiagnostik vor Leberresektion - teuer und ohne klinische Relevanz?

Hepatic failure after liver resection is a complication that is dreaded by surgeons and has a poor outcome. Inadequate functional reserve of the remaining liver parenchyma leads to the inability to regenerate and finally to the progression of liver failure. In order to predict the functional reserve of the remaining liver parenchyma, many different liver function tests have been established. Basis for most liver function assessments are metabolic liver functions such as cytochrome p450 dependent pathways or the extraction and biliary excretion of dye. Nuclear imaging of the liver parenchyma does not only allow visualisation of the liver but also accumulation of information on hepatocyte volume that might be a better predictor for the hepatic reserve and the regenerative capacity compared to the liver volume alone. However, to date no single method has been proven to be able to predict safe limits of resectability. If an underlying liver disease is excluded the resectability is mostly limited by volumetric analysis and technical feasibility of liver resection. In patients with underlying liver disease cirrhosis should be excluded. In case of liver cirrhosis, only Child-Pugh-Turcott A patients with normal bilirubin levels and without portal hypertension should be considered for liver resection.

New Approaches to the Modulation of the Cyclooxygenase-2 and 5-Lipoxygenase Pathways

The eicosanoid family comprises a number of biologically active lipid mediators involved in the regulation of inflammation and cancer cell growth. Eicosanoid biosynthesis is usually initiated by the release of arachidonic acid (AA) from membrane phospholipids in response to the interaction of a phospholipase-A(2) (PLA(2)) stimulus with a receptor on the cell membrane. The free released AA is subsequently metabolized by three major enzymatic pathways: the cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450-dependent pathways. The COX pathway transforms AA into prostaglandins (PGs) and is of particular clinical relevance because it is the main target for non-steroidal anti-inflammatory drugs (NSAIDs). Of interest, COX-2, one of the two COX isoforms, is primarily involved in inflammation and cancer and for this reason selective COX-2 inhibitors have been developed. The efficacy of these compounds is similar to that of traditional NSAIDs but with a lower risk of gastrointestinal toxicity and bleeding. On the other hand, emerging information has recognized the role of other AA metabolites derived from the 5-LO pathway, the leukotrienes (LTs), in mediating and maintaining inflammation. Consequently, drugs able to inhibit 5-LO are now included among the effective pharmacological therapies, especially in asthma and allergic inflammation. Moreover, COX-2 and 5-LO pathways appear to act in parallel in the regulation of cell proliferation and neo-angiogenesis and both COX-2 and 5-LO inhibitors are being investigated as potential anticancer drugs. This review article will update the progress achieved in the knowledge of COX-2 and 5-LO and discuss the emerging approaches for the pharmacological modulation of these pathways.

Biotransformation of the flame retardant tetrabromo-bisphenol A by human and rat sub-cellular liver fractions

The comparative in vitro metabolism of the flame retardant tetrabromo-bisphenol A was studied in rat and human using a [ 14C]-radio-labelled molecule. Tetrabromo-bisphenol A is metabolised into the corresponding glucuronide (liver S9 fractions) and several other metabolites produced by cytochrome P450 dependent pathways (liver microsomes and liver S9 fractions). No major qualitative differences were observed between rat and human, regardless of the selected concentration, within the 20–200 μM range. Tetrabromo-bisphenol A undergoes an oxidative cleavage near the central carbon of the molecule, that leads to the production of hydroxylated dibromo-phenol, hydroxylated dibromo-isopropyl-phenol and glutathione conjugated dibromo-isopropyl-phenol. The main metabolites of tetrabromo-bisphenol A are two molecules of lower polarity than the parent compound, characterised as a hexa-brominated compound with three aromatic rings and a hepta-brominated dimer-like compound, respectively. Both structures, as well as the lower molecular weight metabolites resulting from the breakdown of the molecule, suggest the occurrence of chemically reactive intermediates formed following a first step oxidation of tetrabromo-bisphenol A.

Clopidogrel and Ticlopidine: P2Y12Adenosine Diphosphate-Receptor Antagonists for the Prevention of Atherothrombosis

Ticlopidine and clopidogrel belong to the same chemical family of thienopyridine adenosine diphosphate (ADP)-receptor antagonists. They have shown their efficacy as platelet antiaggregant and antithrombotic agents in many animal models, both ex vivo and in vivo. Although ticlopidine was discovered more than 30 years ago, it was only recently that the mechanism of action of ADP-receptor antagonists was characterized in detail. Ticlopidine and clopidogrel both behave in vivo as specific antagonists of P2Y (12), one of the ADP receptors on platelets. Metabolic steps that involve cytochrome P450-dependent pathways are required to generate the active metabolite responsible for this in vivo activity. The active moiety is a reactive thiol derivative that targets P2Y (12) on platelets. The interaction is irreversible, accounting for the observation that platelets are definitely antiaggregated, even if no active metabolite is detectable in plasma. The interaction is specific for P2Y (12); other purinoceptors such as P2Y (1) and P2Y (13) are spared. This results in inhibition of the binding of the P2Y (12) agonist 2-methylthio-ADP and the ADP-induced downregulation of adenylyl cyclase. Platelet aggregation is affected not only when triggered by ADP but also by aggregation inducers when used at concentrations requiring released ADP as an amplifier. The efficacy and safety of clopidogrel has been established in several large, randomized, controlled trials. The clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE) trial demonstrated the superiority of clopidogrel over acetylsalicylic acid (ASA) in patients at risk of ischemic events, including ischemic stroke, myocardial infarction (MI), and peripheral arterial disease. The clopidogrel in unstable angina to prevent recurrent ischemic events (CURE) trial showed a sustained, incremental benefit when clopidogrel was added to standard therapy (including ASA) in patients with unstable angina and non-Q-wave MI. The clopidogrel for the reduction of events during observation (CREDO) trial demonstrated the benefit of continuing clopidogrel (plus ASA) for 12 months, as opposed to 1 month, after percutaneous coronary intervention. The proven efficacy of clopidogrel, coupled with its favorable safety and tolerability profile, has prompted its evaluation in an extensive, ongoing clinical trial program that will help to further characterize the benefit of clopidogrel in patients with a range of atherothrombotic profiles.

Clopidogrel: a review of its mechanism of action

The search for active antiplatelet drugs within the original chemical class of the thienopyridines, led to the discovery of clopidogrel, a novel ADP-selective agent whose antiaggregating properties are several times higher than those of ticlopidine. The antiaggregating properties of this compound are well known and, very recently, new results have clarified its mechanism of action. Clopidogrel is active only after intravenous or oral administration, and no circulating activity has been found in the plasma of treated animals or human volunteers. Experiments in rats have demonstrated that the antiaggregating activity was caused by a shortlasting metabolite generated in the liver by a cytochrome P450-dependent pathway. The antiaggregating property of clopidogrel is caused by an inhibition of the binding of ADP to its platelet receptors, and more specifically to the low affinity receptors, the high affinity binding sites being unaffected by clopidogrel. Several events in the ADP activation process, including adenylyl cyclase down-regulation, protein tyrosine phosphorylation, activation of the GPIIb-IIIa complex, fibrinogen binding, aggregation and release, were inhibited by clopidogrel and indicate their close relationship with the activation of a low affinity receptor by ADP. In contrast, binding of ADP to its high affinity binding sites (clopidogrel-resistant receptors) induced shape change, cytosolic calcium increase and phosphorylations of several other proteins, some events which were clopidogrel-sensitive. Thus, clopidogrel not only constitutes a potent antithrombotic drug in humans but also a good tool to study the effect of ADP on platelets.

Eicosanoids and heart disease

The discovery of prostaglandins over 5 decades ago heralded a new era in the study of mediators or factors involved in physiological and pathophysiological processes. Prostaglandins, however, do not represent the sole products derived from arachidonic acid or other fatty acid precursors; instead these fatty acids can undergo metabolism to numerous bioactive compounds derived from cyclooxygenase, lipoxygenase, as well as the recently discovered epoxygenase cytochrome P-450 dependent pathways. Collectively, these products are commonly referred to as eicosanoids and have been implicated in numerous biological phenomena. It has long been hypothesized that the prostaglandins play an important role in cardiovascular regulation. For instance, the concept of a balance between the vasoconstrictor, prothrombotic properties of some arachidonic acid derived products and the opposing actions of others led to the development of drug therapies against thromboembolic disorders. Aspirin, for example, a well-known cyclooxygenase inhibitor, has been shown to be efficacious against myocardial infarction and stroke of embolic origin. Moreover, cyclooxygenase- and lipoxygenase-derived products have been implicated in various types of cardiac dysfunction including coronary constriction, arrhythmogenesis, anaphylactic reactions, or ischemic and reperfusion injury.In view of the evolving complexity of arachidonic acid metabolism and increasing evidence that eicosanoids, either alone, or through interaction with other substances, represent important mediators in either the development of heart disease or the myocardial response to injury, we organized this symposium entitled Eicosanoids and Heart Disease concurrent with the 31st Annual Meeting of the Canadian Federation of Biological Societies. A one-half day symposium precludes the possibility of detailed coverage of the many areas in cardiovascular disease in which eicosanoid participation could be implicated and which ideally one would hope to cover. The organizers' aim was to address, in the form of research presentations or reviews, current areas of investigation dealing with selected topics in heart disease.The success of this symposium was made possible by the participation of several distinguished scientists. We would also like to thank the Pharmacological Society of Canada, the Canadian Heart Foundation, Upjohn Canada, Sterling Drug, and Ciba-Geigy U.S. A. for financial support. The organizers thank Dr. J. Burka for serving as Guest Editor and Mrs. L. Hendrickson of this Journal for coordinating the submission of the manuscripts.

Studies on the in vitro biological N-oxidation of trimethoprim.

In vivo studies on the metabolism of trimethoprim have shown that N-oxidation to isomeric N-oxides occurs, with considerable inter-species variation in the proportion of each N-oxide excreted. No in vitro studies on the metabolism of trimethoprim have been reported to date. We now report a sensitive and specific HPLC method for the simultaneous determination of the isomeric N-oxides of trimethoprim. In vitro metabolic studies with hepatic microsomes from several animal species have been conducted. Optimum incubation conditions have been established and the use of potential metabolic inducers, activators and inhibitors has demonstrated that the N-oxidation of trimethoprim to its isomeric N-oxides is mediated via a cytochrome P450 dependent pathway.

Glucagon activation of the thiol:Protein disulfide oxidoreductase in isolated, rat, hepatic microsomes

The hepatic, microsomal, thiol:protein disulfide oxidoreductase catalyzes the glutathione (GSH) reduction of protein disulfides to sulfhydryl groups. In the presence of physiological concentrations of glucagon this activity increased from 2.3 to 6.4 fold in isolated microsomes. The stimulation had a P 50 for glucagon of 7.8 × 10 −10 M which was only observed at microsomal protein concentrations of less than 100 ug/ml and in the presence of a GSH reducing system. This latter observation suggests that the stimulation may be inhibited by the presence of oxidized glutathione. These data support the hypothesis that glucagon may act in part by stimulating the reduction of protein disulfides by the thiol:protein disulfide oxidoreductase. There is an active interest in elucidating the mechanism of the transduction of hormonal action. Among the mechanisms which have been most actively studied have been those schemes which involve changes in the phosphorylation status of critical enzymes in various metabolic pathways (1). Recently Ziegler (2,3) has suggested an alternative mechanism in which the regulation of the activity of these pathways is mediated by changes in the oxidation-reduction state of various enzymes in the particular metabolic pathway. Even though to date there have been no published reports presenting data directly supporting this mechanism (3), a recent study from our laboratory would suggest that changes in the ratio of protein disulfides to sulfhydryl groups may be important in hormone action (4). In this studies we observed that in isolated, hepatic microsomes, the ATP-dependent calsium pump is inactivated through direct oxidation by a NADPH-dependent system (5). This inhibition appears to be catalyzed by a cytochrome P-450 dependent pathway. Further, the pump activity can be partially restored by reduced glutathione, presumably through a microsomal, thiol:protein disulfide oxidoreductase (5, MS submited). Finally we have found that physiological concentrations of norepinephrine (10 −8 M), when added to the isolated microsomal preparation, can significantly increase this NADPH-dependent inhibition of the calcium pump (4). Together these observations are consistent with the hypothesis that the hormonal action of norepinephrine in the hepatocyte may be mediated by inactivation of the calcium pump and that this inactivation is due to the reversible oxidation of critical sulfhydryls in one or more components of the calcium pump system. Since our previous studies suggested that the modulation of oxidative processes could mediate the transduction of the action of norepinephrine, it is possible that the activities of reductive enzymes are also modulated by hormones (3). In the current study, we have therefore examined in incubations of hepatic microsomes, the effect of various concentrations of glucagon on the activity of the GSH dependent microsomal enzyme, thiol:protein disulfide oxidoreductase (EC 1.8.4.2). This enzymes catalyzes the GSH reduction of protein disulfides to sulfhydryls. We find that in this preparation the addition of physiological concentrations the hormone (10 −9) stimulated from 2.3–6.4 fold the GSH reduction of endogenous and exogenous protein disulfides.

Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [ 14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methylviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxy-ethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.