Drug-metabolizing activity of CYP2C93 protein determined using human CYP2C substrates.

<p>CPR, cytochrome P450 reductase. N.D., not determined.</p><p>In each reaction, 5 pmol of the recombinant protein was used with substrate (50 µM diclofenac, 100 µM flurbiprofen, 100 µM paclitaxel, 200 µM <i>S</i>-mephenytoin, or 1 mM tolbutamide) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016923#s2" target="_blank"><i>Materials and Methods</i></a>.</p><p>The recombinant cynomolgus and rhesus monkey CYP2C93 proteins were analyzed along with cynomolgus monkey CYP2C8, CYP2C43, CYP2C75, and CYP2C76. CYP2C93v1 and CYP2C93v2 correspond to SV1 and SV2 transcripts of CYP2C93, respectively.</p

Kinetic analysis for oxidations of typical human CYP2C9 substrates catalyzed by monkey CYP2C93.

<p>Each substrate (0–5000 µM tolbutamide, 0–200 µM diclofenac, and 0–200 µM flurbiprofen) was incubated with recombinant CYP2C93v1 (of rhesus monkey) at 37°C for 15 min in the presence of an NADPH-generating system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016923#s2" target="_blank"><i>Materials and Methods</i></a>. Kinetic parameters were calculated from a fitted curve by non-linear regression (mean ± SE).</p

Phylogenetic tree of CYP2C amino acid sequences.

<p>The phylogenetic tree was created by the neighbour-joining method using CYP2C amino acid sequences of human (h), cynomolgus monkey (mf), rhesus monkey (mm), dog (d), and rat (r), found in GenBank. For cynomolgus monkey CYP2C93, the amino acid sequence was predicted from the SV1 cDNA (c.102 was filled in with thymine). Human CYP2A6 amino acid sequence was used as outgroup.</p

CYP2C93 amino acid sequences and hepatic expression of CYP2C93 transcripts.

<p>(<b>A</b>) Multiple alignment of cynomolgus monkey (mf) and rhesus monkey (mm) CYP2C amino acid sequences. For cynomolgus monkey CYP2C93, the amino acid sequences were predicted from transcript variant SV1 cDNA (c.102T was filled in with thymine). The broken and solid lines above the sequences indicate the putative heme-binding region and the six putative substrate recognition sites (SRSs), respectively. Asterisks under the sequences indicate identical amino acids. (<b>B</b>) Hepatic expression of two CYP2C93 transcript variants in cynomolgus monkeys and rhesus monkeys. Expression of normal transcript SV1 and aberrant transcript SV2 was analyzed in livers of 7 cynomolgus monkeys and 10 rhesus monkeys by RT-PCR and gel electrophoresis of the PCR products. SV1 was expressed in 5 rhesus monkeys, including animal number 1 which expressed SV1 faintly, whereas only SV2 was expressed in the cynomolgus monkeys. For these animals, genotyping of c.102T>del and IVS2-1G>T was determined. None of these animals possessed c.102T>del. All the cynomolgus monkeys were homozygous for IVS2-1G>T, but only one rhesus monkey possessed this allele (as a heterozygote).</p

Measurement of CYP2C93 mRNA tissue expression.

<p>Real-time RT-PCR was performed using the probe and primer set specific for CYP2C8, CYP2C43, CYP2C75, CYP2C76, and CYP2C93 mRNA. The expression level of each CYP2C mRNA was normalized to the 18S rRNA level and represents the average ± SD from three independent amplifications. (<b>A</b>) CYP2C93 mRNA expression was measured in cynomolgus monkey tissues, brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus. Among these tissues, CYP2C93 mRNA was predominantly expressed in liver. (<b>B</b>) Hepatic expression of CYP2C8, CYP2C43, CYP2C75, CYP2C76, and CYP2C93 mRNAs was measured in two rhesus monkeys expressing normal transcript CYP2C93 SV1. CYP2C93 mRNA was expressed at a lower level than other CYP2C mRNAs, but the difference in expression levels of CYP2C93 mRNA and other CYP2Cs varied in the two animals. CYP2C18 mRNA was excluded from the analysis due to its hepatic expression level substantially lower than other CYP2C mRNAs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016923#pone.0016923-Uno5" target="_blank">[16]</a>.</p

Genomic structure of the macaque <i>CYP2Cs</i>.

<p>The location and direction of <i>CYP2C8</i>, <i>CYP2C76</i>, and <i>CYP2C93</i> in the macaque <i>CYP2C</i> cluster were determined by PCR-amplification patterns using the macaque <i>CYP2C</i> BAC clones, and by the BLAT analysis of the rhesus monkey genome data. <i>CYP2C93</i>, along with <i>CYP2C76</i>, was located at the end of the gene cluster, the location of which corresponds to the intergenic region in the human genome.</p

Sequence identity of CYP2C93 cDNA and amino acids as compared to human and other macaque CYP2Cs.

<p>Sequence identity of CYP2C93 cDNA and amino acids as compared to human and other macaque CYP2Cs.</p

Exon-intron boundary sequences of <i>CYP2C93</i>.

<p>Exon and intron sequences are indicated in capital and lower case letters, respectively.</p><p>The dinucleotide sequence at the highly conserved GU-AG motif is shown as underlined bold lettering.</p

Thalidomide Increases Human Hepatic Cytochrome P450 3A Enzymes by Direct Activation of the Pregnane X Receptor

Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. ((2013) Chem. Res. Toxicol. 26, 486−489) using the model substrate midazolam in various <i>in vitro</i> and <i>in vivo</i> models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pretreatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting the activation of upstream transcription factors involved in detoxication, e.g., the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of coregulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 coregulator-derived nuclear receptor-interaction motifs and coregulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significant modulation of coregulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation