Activity Of CYP2D1 Research Articles

Effects of vindoline on rat cytochrome P450 isoform activities in vitro.

A specific ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) method has been described for the simultaneous determination of the metabolites of tacrine, bupropion, diclofenac, dextromethorphan and midazolam, which are the five probe drugs of the five cytochrome P450 (CYP450) isoforms CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A4. The inhibition degree was determined by calculating the IC50 . The chromatographic separation was performed on a C18 column with a mobile phase consisting of 0.1% formic acid and acetonitrile. The mass spectrometric analysis was conducted in positive electrospray ionization mode. The IC50 values of CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A were 113.4, 83.78, 22.50, 9.081 and 52.76 μmol L-1 , respectively. The in vitro results demonstrated that vindoline could inhibit CYP2D1 activity in rats, and weak inhibitory effect on CYP2C11 and CYP3A, but had no obvious effects on CYP1A2 and CYP2B.

Evaluation of Cinnamon (Cinnamomum Verum) Effects on Liver CYP450 2D1 Activity and Hepatic Clearance in Diabetic Rats

Objectives: The present study assessed the effects of cinnamon on the activity of the liver CYP2D1 enzyme and hepatic clearance in the rat model of type 1 and 2 diabetes mellitus. Methods: Male Wistar rats were randomly categorized into 8 groups. Fourteen days after induction of diabetes type 1 and 2, type 1 groups received cinnamon and insulin plus cinnamon and type 2 groups received cinnamon and metformin plus cinnamon daily for 14 days. On day 28, rats were subjected to liver perfusion by buffer containing dextromethorphan as the CYP2D1 enzyme activity probe. Perfused samples were analyzed by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection to evaluate the CYP2D1 activity and hepatic clearance. Results: In the control group, enzyme activity and hepatic clearance changed from 0.0081 ± 0.00009 and 6.09 ± 0.2 mL/min to 0.0059 ± 0.0001 and 3.71 ± 0.07 mL/min in the untreated type 1 diabetic rats and to 0.0006 ± 0.0001 and 5.19 ± 0.02 mL/min in untreated type 2 ones. These pharmacokinetic (PK) parameters changed to 0.0069 ± 0.0005 and 6.27 ± 0.06 mL/min in treated type 1 and 0.0115 ± 0.0003 and 5.79 ± 0.11 mL/min in the treated type 2 rats with only cinnamon administration. Treatment with cinnamon plus insulin or metformin modulated these PK parameters to 0.0039 ± 0.00006 and 4.88 ± 0.13 mL/min in type 1 and 0.0092 ± 0.0005 and 6.13 ± 0.01 mL/min in type 2 diabetic rats. Conclusions: Cinnamon can act as an effective complementary medicine in order to normalize the metabolism and clearance processes in diabetes mellitus.

Evaluation of hepatic CYP2D1 activity and hepatic clearance in type I and type II diabetic rat models, before and after treatment with insulin and metformin.

Conversion in the metabolism of drugs occurs in diabetes mellitus. Considering the importance of metabolic enzymes' activities on the efficacy and safety of medicines, the changes in liver enzymatic activity of CYP2D1 and its related hepatic clearance, by using Dextromethorphan as probe in the animal model of type I and type II diabetes, before and after treatment, was assessed in this study. Male Wistar rats were randomly divided into 6 groups. Seven days after induction of diabetes type I and type II, treatment groups were received insulin and metformin daily for 14days, respectively. In day 21, rats were subjected to liver perfusion by Krebs-Henseleit buffer containing Dextromethorphan as CYP2D1 probe. Perfusate samples were analyzed by HPLC fluorescence method in order to evaluate any changes in CYP2D1 activity. The average metabolic ratio of dextromethorphan and hepatic clearance were changed from 0.012 ± 0.004 and 6.3 ± 0.1 in the control group to 0.006 ± 0.0008 and 5.2 ± 0.2 in the untreated type I diabetic group, and 0.008 ± 0.003 and 5.0 ± 0.6 in the untreated type II diabetic rats. Finally, the mean metabolic ratio and hepatic clearance were changed to 0.008 ± 0.001 and 5.4 ± 0.1, and 0.013 ± 0.003 and 6.1 ± 0.4 in the treated groups with insulin and metformin, respectively. In type I diabetic rats, corresponding treatment could slightly improve enzyme activity, whereas the hepatic clearance and enzyme activity reached to the normal level in type II group. Graphical abstract .

Assessment of the inhibition risk of paris saponins, bioactive compounds from Paris polyphylla, on CYP and UGT enzymes via cocktail inhibition assays

Paris saponins, also known as polyphyllins, are natural compounds extracted from Paris polyphylla, which have many pharmacological activities, such as anti-inflammation and anti-cancer. In particular, paris saponin I, II, VII and polyphyllin VI are the components of the quality standard for Paris polyphylla. However, the inhibition risk of polyphyllins on cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGT) remains unclear. Therefore, this report investigated the potential inhibitory effects of paris saponin I, II, VII and polyphyllin VI on the activities of CYP (CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1 and CYP3A2) and UGT (UGT1A1, UGT1A3, UGT1A6, PROG and AZTG) through cocktail inhibition assays in vitro. In the study of CYP, polyphyllin VI exhibited weak inhibition on CYP2D1 activity in rat liver microsomes with IC50 value at 45.2 μM, while paris saponin VII weakly inhibited CYP2C11 and CYP2E1 activities with IC50 value at 42.0 and 67.7 μM, respectively. In the study of UGT, none of the four steroidal saponins showed significant inhibition risk. In conclusion, paris saponin I, II, VII and polyphyllin VI have very low potential to cause the possible toxicity and drug interactions involving CYP and UGT enzymes, indicating that they are safe enough to take with drugs.

Assessment of CYP2D6 re-activation after inhibitory effect of MDMA using tramadol as a probe.

Abstract Background In recent years, the use of tramadol as a probe drug for human cytochrome p450 2D6 (CYP2D6) has been investigated. The objective of this study was to assess the recovery of rat CYP2D1 enzymatic activity after mechanism-based inhibition induced by a single dose of ecstasy (MDMA, 3,4-methylenedioxymethamphetamine) and evaluation of the tramadol ability as a probe drug. CYP2D1 is orthologous in rats to human CYP2D6 and was employed in the current study. Methods A total of 16 male rats were selected and divided into control and treatment groups. The control group did not receive MDMA, while rats in the treatment group received a single dose of MDMA (1 mg/kg) and were subsequently divided into groups that were tested at 1 h, 10 days or 30 days post-administration. The rats were subjected to liver perfusion with Krebs-Heinslet buffer containing tramadol for 60 min and the tramadol and M1 levels were determined by HPLC-fluorescence. Results The enzymatic activity of CYP2D1 for the 1-h group decreased significantly when compared with the control group (p<0.05). Moreover, enzymatic activity increased non-significantly in the 10- and 30-day groups in comparison with the control group. The concentration and AUC0−60 of tramadol increased in the 1-h and 10-day groups when compared with the control group but decreased in the 30-day group; however, none of these changes was statistically significant (p>0.05). The M1 metabolic ratio in the 1-h group decreased significantly when compared with the control group (p<0.05). The M1 metabolic ratio of the 10-day group increased and of the 30-day group decreased, but neither of these changes were significant. Conclusions Regardless of the genotype, the enzymatic activity of rat CYP2D1 recovered by 10 days post-administration of MDMA. It appears that tramadol, irrespective of its stereoselectivity, is not able to appraise rat hepatic CYP2D1 activity. It can be extrapolated that tramadol is a not suitable probe drug for human hepatic CYP2D1 because CYP2D1 in rats is orthologous to human CYP2D6. Further animal and human studies are required to confirm this hypothesis.

Inhibition of Re Du Ning Injection on Enzyme Activities of Rat Liver Microsomes Using Cocktail Method

ObjectiveRe Du Ning Injection (RDN), a Chinese materia medica injection, is made from the extracts of Lonicerae Japonicae Flos, Gardeniae Fructus, and Artemisiae Annuae Herba. Since last decade, RDN has been widely used in China for the treatment of viral infection, fever, and inflammation. To assess the potential interacting of RDN with co-administered drugs, the inhibitory effects of RDN on the enzyme activities (CYP1A1, CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2) of rat liver microsomes were investigated by a cocktail method. MethodsA sensitive and specific LC-MS method capable of simultaneous quantification of five metabolites in rat liver microsomes was developed and validated. Then RDN (0.625%–1.0%) was incubated with rat liver microsomes and specific substrates. The enzyme activities were expressed as the formation rate of the specific metabolites of the substrates (pmol · mg · protein−1 · min−1). ResultsRDN competitively inhibited the activities of CYP1A2 and CYP2C11, with inhibition constant (Ki) values determined to be 0.18% and 0.63%, respectively. RDN exhibited the mixed inhibition on the activity of CYP2D1, with a Ki value of 0.15%. The activities of CYP1A1 and CYP3A1/2 were not markedly inhibited even by 1.0% RDN. ConclusionRDN could inhibit the rat enzyme activities of CYP1A2, 2C11, and 2D1 in vitro with different inhibition modes, which is worthy of promoting safety and efficacy of RDN.

Effect of Dimethoate on the Activity of Hepatic CYP450 Based on Pharmacokinetics of Probe Drugs

Background: Dimethoate (DM), one of the most widely used systemic organophosphate insecticide, has been reported to exert toxic effects after long-time subchronic exposure. This study aims at investigating the toxic effect of DM on liver after repeated administration of low doses of DM in rats. Methods: Twenty Sprague-Dawley rats were randomly divided into the control group (n = 10) and the DM group (n = 10). After 2 weeks' exposure to DM at low dosage (5 mg/kg), biochemical parameters of hepatic functions were measured, histology and CYP450 expressed in liver was detected. The activities of CYP1A2, CYP2C11, CYP2D1, and CYP3A2 were evaluated by the Cocktail method. Results: The level of AChE (acetylcholinesterase) was significantly decreased, hepatic functions were damaged and the mRNA level of CYP2D1 was significantly increased in the DM group (p < 0.05). The pharmacokinetics of probe drug revealed AUC_(0-t), AUC_(0-_∞₎, t_1/2 and C_max of metoprolol was shorten in the DM group (p < 0.05). However, there were no statistical differences in MRT, t_1/2, CL and T_max for phenacetin, tolbutamide and midazolam. Conclusions: A low dosage of DM could induce the activity of CYP2D1 in liver and increase the metabolism of metoprolol when exposed for 2 weeks.

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 (< or =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 (Vmax) and affinity (Km) 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 Vmax of all the model reactions studied, although to a varying degree. However, the effects of NO donors on Km were more diverse. Whereas the Km values for testosterone 6beta-hydroxylation (3A2) and 16alpha-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 Km 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 Vmax and Km of various substrates selectively and confirm our previous findings that the activity of CYP2D1 is not affected by NO donors.

Expression of Four Rat CYP2D Isoforms inSaccharomyces cerevisiaeand Their Catalytic Specificity

We cloned four cDNAs belonging to the CYP2D subfamily to express these enzymes in yeast cells and to compare their catalytic activities simultaneously. Three are believed to be alleles of CYP2D1, 2D2, and 2D3, respectively, based on high nucleotide sequence similarity, while CYP2D4 had both sequences of CYP2D4 and CYP2D18. Expression plasmids carrying CYP2D cDNAs were transformed intoSaccharomyces cerevisiae.Typical P450 CO-difference spectra with absorbance maximum at 448 nm were recorded with microsomal preparations from the yeast cells expressing the four CYP2D forms. A catalytic study of these CYP2D forms was done with debrisoquine, bufuralol, and lidocaine. CYP2D2 had the highest debrisoquine 4-hydroxylation (2.2 nmol/min/nmol P450) activity, similar to that (2.2 nmol/min/nmol) of human CYP2D6 expressed in yeast cells. CYP2D3 had high lidocaine N-deethylation (43 nmol/min/nmol P450) activity, and both CYP2D3 and 2D2 exhibited high lidocaine 3-hydroxylation (2.4 and 1.6 nmol/min/nmol P450, respectively) activity. Bufuralol 1′-hydroxylation catalytic capabilities were comparable among the four isoforms. The activity of CYP2D1 was relatively low toward the three substrates (debrisoquine, 0.091; bufuralol, 1.5; lidocaine 3-hydroxylation, 0.019; lidocaine N-deethylation, 2.8 nmol/min/nmol P450). These findings indicate that debrisoquine, a typical substrate for CYP2D forms, was mainly metabolized by CYP2D2 but not CYP2D1 in rat liver and that the CYP2D forms have different substrate specificity.

The influence of anesthetic agents on rat hepatic cytochromes P450 in vivo.

The objective of this study was to identify which anesthetics when used acutely will affect cytochrome P450 (CYP) activity in male Sprague-Dawley rats in vivo. The anesthetics tested were fentanyl citrate, alpha-chloralose, ketamine, urethane (ethyl carbamate), halothane, and ether. CO2 anesthesia was used as the control comparator. Theophylline was used as a probe for CYP1A activity, phenobarbital for CYP2B/2C, flecainide for CYP2D1, and ethosuximide for CYP3A activity. All probes were administered via tail vein injection after anesthetic-induced loss of the righting reflex. Single sample probe clearances were estimated, and used as an index of CYP activity. Fentanyl citrate, alpha-chloralose, halothane, and ether did not have statistically significant effects on any of the CYP activities. Ketamine did not significantly affect CYP1 or CYP2B/2C activity. However, it decreased the clearance of flecainide (i.e. CYP2D1 activity) by 13.4% (p < 0.001) and the clearance of ethosuximide (i.e. CYP3A activity) by 17.6% (p < 0.0001). Urethane increased the clearance of theophylline by 91.5% (p < 0.0001), and decreased the clearance of ethosuximide by 40.5% (p < 0.0001) though it did not affect CYP2B/2C or CYP2D1 activities significantly. From this data, we conclude that a single dose of ketamine mildly inhibits the activity of CYP2D1 and CYP3A, and a single dose of urethane strongly inhibits CYP3A but increases CYP1A activity.

Gliclazide hydroxylation by rat liver microsomes.

1. The metabolism of gliclazide to hydroxygliclazide has been investigated in Sprague-Dawley rat liver microsomes. 2. The kinetics of hydroxygliclazide formation are consistent with Michaelis-Menten kinetics (mean (+/- SD, n = 3) apparent K(m) and Vmax = 256 +/- 27 microM and 1.85 +/- 0.10 nmol/ min/mg respectively). 3. Tolbutamide competitively inhibited hydroxygliclazide formation (Ki = 840 microM) and gliclazide competitively inhibited hydroxytolbutamide formation (Ki = 240 microM) with Ki similar to K(m). Therefore gliclazide and tolbutamide may be metabolized by the same enzyme in the rat. In nine livers the formation of hydroxygliclazide correlated with the formation of hydroxytolbutamide (rs = 0.82, p < 0.01). 4. Diclofenac (Ki = 64 microM), phenytoin (Ki = 38 microM), mephenytoin (Ki = 66 microM), glibenclamide (Ki = 14 microM) and glipizide (Ki = 189 microM) were fully competitive inhibitors of gliclazide hydroxylation. The rank order of Ki constants differed for gliclazide and tolbutamide suggesting that gliclazide and tolbutamide hydroxylases are not identical enzymes. 5. Quinine (Ki = 0.3 microM) and quinidine (Ki = 4.3 microM) were partially competitive inhibitors of hydroxygliclazide formation. Hydroxylation of gliclazide was related to the activity of CYP2D1 as assessed by dextrorphan production from dextromethorphan (rs = 0.83, p = 0.01). 6. In the rat gliclazide is metabolized to hydroxygliclazide by at least two cytochrome P450 isoforms, including tolbutamide hydroxylase and 2D1, which have similar affinities for gliclazide.

An evaluation of cytochrome P450 isoform activities in the female dark agouti (DA) rat: Relevance to its use as a model of the CYP2d6 poor metaboliser phenotype

The female dark agouti (DA) rat lacks CYP2D1, the equivalent enzyme in the rat to human CYP2D6 (debrisoquine hydroxylase), and shows impaired metabolism of a number of CYP2D6 substrates. However, from the data available in the literature it is not entirely clear whether the enzyme deficiency in the DA rat is restricted to CYP2D1, and whether factors such as age and substrate concentration are important determinants of interstrain differences in the activity of this enzyme. Given that the female DA rat is used as a model of the human CYP2D6 poor metaboliser phenotype, there is a need for a systematic evaluation of the P450 activities in the DA rat, and of its suitability as a model of the PM phenotype. In the present study metoprolol was used as a probe substrate to investigate CYP2D1 activity since both the α-hydroxylation and O-demethylation of this drug are catalysed by CYP2D6 in man. Formation of α-hydroxymetoprolol (AHM) and O-demethylmetoprolol (ODM) was 10- and 2.5-fold lower in liver microsomes from female DA rats compared with microsomes from agematched female Wistar rats, the latter representing the extensive metaboliser strain. Kinetic analysis suggested that in both strains of rat both the α-hydroxylation and O-demethylation of metoprolol were catalysed by more than one enzyme. By using quinine as a specific inhibitor of the enzyme, CYP2D1 was identified as an intermediate affinity site in the Wistar strain and was shown to have impaired activity in the DA strain. The activities of lower and higher affinity sites were similar in the two strains. Thus, the only difference between the two strains with respect to both routes of metoprolol metabolism appeared to be in the activity of CYP2D1. Interstrain differences were found to be highly dependent on the choice of substrate concentration, being more marked at lower concentrations. We have also investigated the metabolism of a number of probe compounds for some of the other P450 isoforms commonly involved in drug metabolism to determine the selectivity of the deficiency in the DA strain. p-Nitrophenol hydroxylation and erythromycin N-demethylation were catalysed at higher rates by DA than by Wistar liver microsomes, indicating higher levels of activity of CYP2E1 and CYP3A in the former strain. Felodipine oxidation, tolbutamide hydroxylation and both the hydroxylation and N-demethylation of S-mephenytoin were catalysed at similar rates by microsomes from the two strains, indicating similar activities of enzymes in the CYP2C and CYP3A families. However, both the hydroxylation and N-demethylation of R-mephenytoin were impaired in the DA strain. This indicates that at least one other isoform of P450, thought also to be a member of the CYP2C or CYP3A families, in addition to CYP2D1 is deficient in the DA strain. Our findings indicate that whilst the female DA rat could be used as a preliminary screen to identify CYP2D6 substrates, because of interspecies differences in metabolism it could not be used to provide quantitative information regarding the contribution of CYP2D6 to an oxidation in man. In addition, a small number of false positives would be identified owing to other enzyme deficiencies; no false negatives would be expected. Comparisons between strains should be performed using female, age-matched animals and low substrate concentrations.