ENZYME-SELECTIVE EFFECTS OF NITRIC OXIDE ON AFFINITY AND MAXIMUM VELOCITY OF VARIOUS RAT CYTOCHROMES P450

ABSTRACT: Nitric oxide (NO) has recently been shown to decrease cytochrome P450 (P450) enzyme activity rapidly (<30 min), concentration dependently, and enzyme-selectively in the rat liver. Interestingly, among all the studied P450 enzymes, only CYP2D1 was not affected by NO donors. However, these studies were conducted using only a single concentration of the substrates, thus lacking information about the possible simultaneous changes in both maximum velocity (V max ) and affinity (K m ) of the enzymes. In the present study, we systematically evaluated the effects of NO on the enzyme kinetic parameters of marker substrates for a range of P450 enzymes, including 2D1. Livers were perfused (1 h) in the absence In addition to cytokines, inflammatory states induce the expression of inducible nitric-oxide synthase and result in excessive production of nitric oxide (NO) in both hepatocytes and Kupffer cells One of the novel findings of our IPRL studies (Vuppugalla and Mehvar, 2004a,b) was the apparent lack of effect of NO donors on CYP2D1 enzyme, along with different degrees and time courses of inhibition or reversal of activities for other P450 enzymes. These studies were carried out only with a single substrate concentration. Therefore, one may argue that the observed enzyme-selective effects of NO may have been due to different positions of substrate concentrations along their respective Michaelis-Menten kinetic curves. This is especially true if NO alters both V max and K m of P450 enzymes. Article, publication date, and citation information can be found a

Current trends in drug metabolism and pharmacokinetics.

Pharmacokinetics (PK) is the study of the absorption, distribution, metabolism, and excretion (ADME) processes of a drug. Understanding PK properties is essential for drug development and precision medication. In this review we provided an overview of recent research on PK with focus on the following aspects: (1) an update on drug-metabolizing enzymes and transporters in the determination of PK, as well as advances in xenobiotic receptors and noncoding RNAs (ncRNAs) in the modulation of PK, providing new understanding of the transcriptional and posttranscriptional regulatory mechanisms that result in inter-individual variations in pharmacotherapy; (2) current status and trends in assessing drug-drug interactions, especially interactions between drugs and herbs, between drugs and therapeutic biologics, and microbiota-mediated interactions; (3) advances in understanding the effects of diseases on PK, particularly changes in metabolizing enzymes and transporters with disease progression; (4) trends in mathematical modeling including physiologically-based PK modeling and novel animal models such as CRISPR/Cas9-based animal models for DMPK studies; (5) emerging non-classical xenobiotic metabolic pathways and the involvement of novel metabolic enzymes, especially non-P450s. Existing challenges and perspectives on future directions are discussed, and may stimulate the development of new research models, technologies, and strategies towards the development of better drugs and improved clinical practice

Hepatic Disposition and Effects of Nitric Oxide Donors: Rapid and Concentration-Dependent Reduction in the Cytochrome P450-Mediated Drug Metabolism in Isolated Perfused Rat Livers

Various mechanisms, including high levels of cytokines and nitric oxide (NO), have been proposed as mediators for inflammation-induced cytochrome 450 down-regulation. However, the contribution of each of these mediators to the observed effects is controversial. We used an isolated perfused rat liver (IPRL) model to test the direct effects of NO donors on CYP450 down-regulation in the absence of cytokines or other confounding in vivo factors. Our hypothesis was that NO rapidly and concentration-dependently decreases CYP450 activities in IPRL. Livers were perfused (60 min) with 50 to 500 muM sodium nitroprusside (SNP) or 100 to 500 muM isosorbide dinitrate ( ISDN) as NO donors, and the perfusate and biliary disposition of SNP, ISDN, and generated nitrate/nitrite (NOx) were determined. Additionally, at the end of perfusion, catalytic activities and protein levels of various cytochrome isoenzymes were measured. Both SNP and ISDN exhibited linear hepatic disposition with extraction ratios of similar to0.30 and 0.50, respectively. Furthermore, although in small amounts, both NO donors and NOx were found in the bile. Except for CYP2D1, the catalytic activities of all the studied isoenzymes were substantially ( up to 85%) decreased by both NO donors. However, the apoprotein levels of isoenzymes remained largely unchanged. Additionally, the inhibitory effects of NO donors were concentration-dependent, with the concentrations of SNP producing one-half of maximum inhibition being in the order of 2C11 \u3e 2B1/2 \u3e 2E1 = 3A2 \u3e 1A1/2. These studies indicate that the effects of NO on the down-regulation of cytochrome 450 catalytic activity are rapid, concentration-dependent, and isoenzyme-selective

Short-term inhibitory effects of nitric oxide on cytochrome P450-mediated drug metabolism: time dependency and reversibility profiles in isolated perfused rat livers. Drug Metab Dispos 32

ABSTRACT: Nitric oxide (NO) is implicated as a mediator in the decreased catalytic activities of cytochrome P450 (P450) enzymes during inflammation or infection. Here, we examined the time course and the reversibility of the NO effect on P450s using isolated perfused rat livers. Livers were perfused at a constant rate with the NO donor sodium nitroprusside (SNP) for 0.5 or 1 h, followed by washout periods of 0 to 2.5 h. At the end of perfusion, microsomes were prepared and analyzed for P450 activities and other metabolic markers. Whereas 0.5 h of NO exposure caused an irreversible decline (ϳ30%) in total P450 content, a greater decline after 1 h of NO (ϳ55%) was mostly (ϳ30%) reversible, a pattern identical to that observed for the microsomal heme content. NO exposure also caused an enzyme-selective and time-dependent decline in P450 activities. Whereas the pattern of decline and reversibility of activities were qualitatively similar for CYP3A2, 2C11, 2E1, and 1A1/2, they differed for 2B1/2 and 2D1 in that the decline in the activity was delayed (1 h) for 2B1/2 and not observed for 2D1. This may be attributed to the accessibility of heme or cysteine thiolate and/or the presence/reactivity of critical cysteinyl amino acid residues in various P450 enzymes. Additionally, for most enzymes, the activity showed a biphasic decline, one within 1 h of SNP perfusion and another after 2 h of washout. This was associated with an identical biphasic decline in the microsomal free thiols, presumably due to the rapid and slow reaction of NO and peroxynitrite, respectively, with critical P450 thiols. The short-term effects of NO on P450 are time-dependent and enzyme-selective, with both reversible and irreversible mechanisms

Enzyme-Selective Effects of Nitric Oxide on Affinity and Maximum Velocity of Various Rat Cytochromes P450

Nitric oxide ( NO) has recently been shown to decrease cytochrome P450 ( P450) enzyme activity rapidly (≤ 30 min), concentration dependently, and enzyme-selectively in the rat liver. Interestingly, among all the studied P450 enzymes, only CYP2D1 was not affected by NO donors. However, these studies were conducted using only a single concentration of the substrates, thus lacking information about the possible simultaneous changes in both maximum velocity (V-max) and affinity (K-m) of the enzymes. In the present study, we systematically evaluated the effects of NO on the enzyme kinetic parameters of marker substrates for a range of P450 enzymes, including 2D1. Livers were perfused ( 1 h) in the absence ( control) or presence of two NO donors with different mechanisms of NO release. At the end of the perfusion, microsomes were prepared and used for kinetic analysis. Except for 2D1, NO reduced the V-max of all the model reactions studied, although to a varying degree. However, the effects of NO donors on K-m were more diverse. Whereas the K-m values for testosterone 6β-hydroxylation (3A2) and 16α-hydroxylation (2C11) significantly decreased, the values for chlorzoxazone 6-hydroxylation (2E1), dextromethorphan N-demethylation ( 3A2), and high affinity ethoxyresorufin O-dealkylation (1A1/2) significantly increased in the presence of NO donors. Furthermore, the K-m values for the high-affinity component of dextromethorphan O-demethylation and benzyloxyresorufin O-dealkylation remained unchanged. These results indicate that NO can potentially change both the V-max and K-m of various substrates selectively and confirm our previous findings that the activity of CYP2D1 is not affected by NO donors

Selective Effects of Nitric Oxide on the Disposition of Chlorzoxazone and Dextromethorphan in Isolated Perfused Rat Livers

The rapid and direct effects of nitric oxide ( NO) donors sodium nitroprusside ( SNP) and isosorbide dinitrate ( ISDN) on the hepatic and biliary disposition of chlorzoxazone (CZX), a marker of CYP2E1, and dextromethorphan (DEM), a marker of CYP2D1, were studied in a single-pass isolated perfused rat liver model. Livers (n = 30) were perfused with constant concentrations of NO donors (0-120 min) in addition to infusion of CZX or DEM (60-120 min), and periodical outlet and bile samples were collected. Both ISDN and SNP significantly reduced ( 30 and 60%, respectively) the hepatic extraction ratio of CZX and decreased ( 50 and 70%, respectively) the recovery of the CYP2E1-mediated metabolite, 6-hydroxychlorzoxazone, in the outlet perfusate and bile. As for DEM, both NO donors increased ( up to 3.5-fold) the recovery of the CYP2D1-mediated metabolite dextrorphan (DOR) in the outlet perfusate. However, this was associated with a simultaneous decrease (50 +/- 75%) in the excretion of the metabolite into the bile, thus resulting in no change in the overall recovery of DOR as a result of NO donor treatment. The decrease in the biliary excretion of DOR was caused by NO-induced simultaneous reductions in both the conjugation of DOR and biliary clearance of DOR conjugate. Additionally, both SNP and ISDN significantly reduced the metabolism of DEM to 3-hydroxymorphinan, which is mostly regulated by CYP3A2. These studies in an intact liver model confirm the selectivity of the inhibitory effects of NO donors on cytochrome P450 enzymes, which was recently reported in microsomal studies, and expand these inhibitory effects to conjugation pathways

Short-Term Inhibitory Effects of Nitric Oxide on Cytochrome P450-mediated Drug Metabolism: Time Dependency and Reversibility Profiles in Isolated Perfused Rat Livers

Nitric oxide (NO) is implicated as a mediator in the decreased catalytic activities of cytochrome P450 (P450) enzymes during inflammation or infection. Here, we examined the time course and the reversibility of the NO effect on P450s using isolated perfused rat livers. Livers were perfused at a constant rate with the NO donor sodium nitroprusside (SNP) for 0.5 or 1 h, followed by washout periods of 0 to 2.5 h. At the end of perfusion, microsomes were prepared and analyzed for P450 activities and other metabolic markers. Whereas 0.5 h of NO exposure caused an irreversible decline (∼30%) in total P450 content, a greater decline after 1 h of NO (∼55%) was mostly (∼30%) reversible, a pattern identical to that observed for the microsomal heme content. NO exposure also caused an enzyme-selective and time-dependent decline in P450 activities. Whereas the pattern of decline and reversibility of activities were qualitatively similar for CYP3A2, 2C11, 2E1, and 1A1/2, they differed for 2B1/2 and 2D1 in that the decline in the activity was delayed (1 h) for 2B1/2 and not observed for 2D1. This may be attributed to the accessibility of heme or cysteine thiolate and/or the presence/reactivity of critical cysteinyl amino acid residues in various P450 enzymes. Additionally, for most enzymes, the activity showed a biphasic decline, one within 1 h of SNP perfusion and another after 2 h of washout. This was associated with an identical biphasic decline in the microsomal free thiols, presumably due to the rapid and slow reaction of NO and peroxynitrite, respectively, with critical P450 thiols. The short-term effects of NO on P450 are time-dependent and enzyme-selective, with both reversible and irreversible mechanisms