Differences in Intestinal Hydrolytic Activities between Cynomolgus Monkeys and Humans: Evaluation of Substrate Specificities Using Recombinant Carboxylesterase 2 Isozymes

Cynomolgus monkeys, used as an animal model to predict human pharmacokinetics, occasionally show different oral absorption patterns to humans due to differences in their intestinal metabolism. In this study, we investigated the differences between intestinal hydrolytic activities in cynomolgus monkeys and humans, in particular the catalyzing activities of their carboxylesterase 2 (CES2) isozymes. For this purpose we used both human and monkey microsomes and recombinant enzymes derived from a cell culture system. Monkey intestinal microsomes showed lower hydrolytic activity than human microsomes for several substrates. Interestingly, in contrast to human intestinal hydrolysis, which is not enantioselective, monkey intestine showed preferential <i>R</i>-form hydrolysis of propranolol derivatives. Recombinant CES2 isozymes from both species, mfCES2v3 from monkeys and human hCE2, showed similar metabolic properties to their intestinal microsomes when expressed in HEK293 cells. Recombinant hCE2 and mfCES2v3 showed similar <i>K</i>_m values for both enantiomers of all propranolol derivatives tested. However, recombinant mfCES2v3 showed extreme <i>R</i>-enantioselective hydrolysis, and both hCE2 and mfCES2v3 showed lower activity for <i>O</i>-3-methyl-<i>n</i>-butyryl propranolol than for <i>O</i>-<i>n</i>-valeryl and <i>O</i>-2-methyl-<i>n</i>-butyryl propranolol. This lower hydrolytic activity was characterized by lower <i>V</i>_max values. Docking simulations of the protein–ligand complex demonstrated that the enantioselectivity of mfCES2v3 for propranolol derivatives was possibly caused by the orientation of its active site being deformed by an amino acid change of Leu107 to Gln107 and the insertion of Met309, compared with hCE2. In addition, molecular dynamics simulation indicated the possibility that the interatomic distance between the catalytic triad and the substrate was elongated by a 3-positioned methyl in the propranolol derivatives. Overall, these findings will help us to understand the differences in intestinal hydrolytic activities between cynomolgus monkeys and humans

Qualitative <i>De Novo</i> Analysis of Full Length cDNA and Quantitative Analysis of Gene Expression for Common Marmoset (<i>Callithrix jacchus</i>) Transcriptomes Using Parallel Long-Read Technology and Short-Read Sequencing

<div><p>The common marmoset (<i>Callithrix jacchus</i>) is a non-human primate that could prove useful as human pharmacokinetic and biomedical research models. The cytochromes P450 (P450s) are a superfamily of enzymes that have critical roles in drug metabolism and disposition via monooxygenation of a broad range of xenobiotics; however, information on some marmoset P450s is currently limited. Therefore, identification and quantitative analysis of tissue-specific mRNA transcripts, including those of P450s and flavin-containing monooxygenases (FMO, another monooxygenase family), need to be carried out in detail before the marmoset can be used as an animal model in drug development. <i>De novo</i> assembly and expression analysis of marmoset transcripts were conducted with pooled liver, intestine, kidney, and brain samples from three male and three female marmosets. After unique sequences were automatically aligned by assembling software, the mean contig length was 718 bp (with a standard deviation of 457 bp) among a total of 47,883 transcripts. Approximately 30% of the total transcripts were matched to known marmoset sequences. Gene expression in 18 marmoset <i>P450</i>- and 4 <i>FMO</i>-like genes displayed some tissue-specific patterns. Of these, the three most highly expressed in marmoset liver were <i>P450 2D-</i>, <i>2E</i>-, and <i>3A</i>-like genes. In extrahepatic tissues, including brain, gene expressions of these monooxygenases were lower than those in liver, although <i>P450 3A4</i> (previously <i>P450 3A21</i>) in intestine and <i>P450 4A11</i>- and <i>FMO1</i>-like genes in kidney were relatively highly expressed. By means of massive parallel long-read sequencing and short-read technology applied to marmoset liver, intestine, kidney, and brain, the combined next-generation sequencing analyses reported here were able to identify novel marmoset drug-metabolizing P450 transcripts that have until now been little reported. These results provide a foundation for mechanistic studies and pave the way for the use of marmosets as model animals for drug development in the future.</p></div

Gene Ontology (GO) classification of marmoset (<i>Callithrix jacchus</i>) sequences based on predicted gene ontology terms in liver, intestine, kidney, and brain from male (A) and female (B) marmosets.

<p>A total of 47,883 contigs with BLAST matches to known proteins were assigned to three main categories: cellular components, molecular function, and biological processes. RNA sequencing was done for mRNAs from liver, intestine, kidney, and brain of three male and three female marmosets. Only the top 100 genes are shown in this GO analysis.</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

Synthesis of double-strand cDNA for sequencing.

<p>First strand cDNA synthesis was accomplished by randomized primers, which enabled mRNA to be covered from the poly(A) side to the 5′-side near the start point of transcription.</p

Summary of sequence assembly.

<p>Summary of sequence assembly.</p

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

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

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