Evaluation of<i>Escherichia coli</i>Membrane Preparations of Canine CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 with Coexpressed Canine Cytochrome P450 Reductase

Locuson, Charles W.; Ethell, Brian T.; Voice, Michael W.; Lee, David; Feenstra, Kenneth L.

doi:10.1124/dmd.108.025312

Cited by 34 publications

(34 citation statements)

References 20 publications

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“…2A), whereas CYP2A25 exhibited much lower catalytic capability toward midazolam. Both enzymes would be minor contributors for the metabolism of midazolam in dog liver microsomes compared with other dog P450s, such as CYP3A12 (Locuson et al, 2009). Dog CYP2A13 and CYP2A25 exhibited low catalytic activities in hydroxylating testosterone at multiple sites (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Shou et al (2003) have expressed and characterized seven dog P450 enzymes (CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26) in baculovirus-infected Sf21 insect cells. More recently, the same seven dog P450s have also been expressed in Escherichia coli with coexpression of dog NADPHcytochrome P450 reductase (OR) (Locuson et al, 2009). Dog CYP2A enzymes have not been reported in any cDNA-directed expression system.…”

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confidence: 99%

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Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Zhou¹,

Linnenbach²,

Liu³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Dog CYP2A13 and CYP2A25 were coexpressed with dog NADPHcytochrome P450 reductase (OR) in baculovirus-infected Sf9 insect cells. CYP2A13 effectively catalyzed 7-ethoxycoumarin (7EC) deethylation and coumarin hydroxylation with apparent K m values of 4.8 and 2.1 M, respectively, similar to those observed using dog liver microsomes (7.5 and 0.75 M, respectively). CYP2A25 exhibited much lower affinity toward 7EC, with an apparent K m value of 150 M, which indicates that CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates. Similar to the dog CYP1A2 enzyme, CYP2A13 efficiently catalyzed phenacetin deethylation with a K m value of 3.9 M, which suggests that phenacetin is not a selective probe for dog CYP1A2 activity. Both dog CYP2A13 and CYP2A25 exhibited little or no catalytic activity toward other common cytochrome P450 probe substrates, including bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone. These results provided additional information about the selectivity of these commonly used probe substrates.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Zhou¹,

Linnenbach²,

Liu³

et al. 2010

Drug Metab Dispos

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“…P450 2A6 and P450 2B6 cDNAs were cotransformed and expressed with P450 reductase in Escherichia coli to produce active P450s (Locuson et al, 2009). EFV (50 M) was incubated with the membrane preparation for 60 min followed by termination of the reaction by using acetonitrile.…”

Section: Methodsmentioning

confidence: 99%

Dendritic Spine Injury Induced by the 8-Hydroxy Metabolite of Efavirenz

Tovar‐y‐Romo

Bumpus

Pomerantz

et al. 2012

J Pharmacol Exp Ther

121

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Despite combination antiretroviral therapies (cARTs), a significant proportion of HIV-infected patients develop HIV-associated neurocognitive disorders (HAND). Ongoing viral replication in the central nervous system (CNS) caused by poor brain penetration of cART may contribute to HAND. However, it has also been proposed that the toxic effects of long-term cART may contribute to HAND. A better understanding of the neurotoxic potential of cART is critically needed in light of the use of CNS-penetrating cARTs to contend with the virus reservoir in the brain. The efavirenz (EFV) metabolites 7-hydroxyefavirenz (7-OH-EFV) and 8-hydroxyefavirenz (8-OH-EFV) were synthesized and purified, and their chemical structures were confirmed by mass spectrometry and NMR. The effects of EFV, 7-OH-EFV, and 8-OH-EFV on calcium, dendritic spine morphology, and survival were determined in primary neurons. EFV, 7-OH-EFV, and 8-OH-EFV each induced neuronal damage in a dose-dependent manner. However, 8-OH-EFV was at least an order of magnitude more toxic than EFV or 7-OH-EFV, inducing considerable damage to dendritic spines at a 10 nM concentration. The 8-OH-EFV metabolite evoked calcium flux in neurons, which was mediated primarily by L-type voltage-operated calcium channels (VOCCs). Blockade of L-type VOCCs protected dendritic spines from 8-OH-EFV-induced damage. Concentrations of EFV and 8-OH-EFV in the cerebral spinal fluid of HIV-infected subjects taking EFV were within the range that damaged neurons in culture. These findings demonstrate that the 8-OH metabolite of EFV is a potent neurotoxin and highlight the importance of directly determining the effects of antiretroviral drugs and drug metabolites on neurons and other brain cells.

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“…The reported studies in dogs mainly focus on the expression and catalytic activity of metabolic enzymes in the liver (Kyokawa et al, 2001;Mills et al, 2010). Less is known about the intestinal expression distribution of cytochromes P450 (P450s), UDP-glucuronosyltransferases (UGTs) (Bock et al, 2002;Mealey et al, 2008), and transporters (Conrad et al, 2001) or about P450 substrate specificity (Fraser et al, 1997;Turpeinen et al, 2007;Locuson et al, 2009) and intersubject variability in expression.…”

Section: Introductionmentioning

confidence: 99%

Expression Profiles of Metabolic Enzymes and Drug Transporters in the Liver and along the Intestine of Beagle Dogs

Haller¹,

Schuler²,

Lazic³

et al. 2012

Drug Metab Dispos

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ABSTRACT:Beagle dogs are widely used in preclinical pharmacokinetic, safety, and formulation studies. However, little is known about intestinal and hepatic distribution of major enzymes and transporters involved in oral absorption and presystemic drug metabolism. We characterized mRNA levels of CYP3A12, CYP3A26, CYP2D15, UGT1A6, ABCB1 (MDR1), ABCC1 (MRP1), ABCG2 (BCRP), SLC15A1 (PEPT1), and SLC22A1 (OCT1) in dog liver and along the intestine by real-time quantitative reverse transcription-polymerase chain reaction. Tissue protein levels of CYP2D15, MDR1, and PEPT1 were obtained by Western blot. Gene distribution and expression variability was statistically described by a generalized additive mixed model smoothing function and correspondence analysis. Results were compared with the expression pattern known for the human orthologs. Hepatic mRNA levels for metabolic enzymes were generally higher than those for membrane transporters, whereas in the intestine the opposite was observed. Hepatic mRNA levels followed the order CYP2D15 > UGT1A6 Ϸ CYP3A26 > ABCB1 Ϸ SLC15A1 Ϸ SLC22A1 > ABCG2 > ABCC1 Ϸ CYP3A12. Along the gut, the genes were differentially distributed with greatest expression in duodenum/upper jejunum (ABCG2), middle jejunum (ABCB1 and SLC15A1), or in cecum/colon (ABCC1 and CYP2D15). CYP3A12, CYP3A26, SLC22A1, and UGT1A6 had a rather uniform expression. Intestinal mRNA profiles of CYP2D15, ABCB1, and SLC15A1 correlated with the respective protein levels. Canine CYP3A12/26, CYP2D15, and ABCB1 colonic distributions differed from those of human orthologs, whereas UGT1A6, ABCC1, ABCG2, SLC15A1, and SLC22A1 were comparable to those of humans in both small and large intestine. We aim to apply these data to better interpret pharmacokinetic studies in dogs with respect to their human relevance.

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Evaluation ofEscherichia coliMembrane Preparations of Canine CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 with Coexpressed Canine Cytochrome P450 Reductase

Cited by 34 publications

References 20 publications

Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Dendritic Spine Injury Induced by the 8-Hydroxy Metabolite of Efavirenz

Expression Profiles of Metabolic Enzymes and Drug Transporters in the Liver and along the Intestine of Beagle Dogs

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