Cloning, sequence analysis, and tissue expression of marmoset paraoxonase 1.

Paraoxonase (PON) plays roles in the metabolism of organophosphate xenobiotics and drugs. Despite the importance of marmosets for research into drug metabolism and pharmacokinetics, marmoset paraoxonase has not yet been fully characterized. Consequently, we identified the PON1 gene in the marmoset genome by sequence homology analysis. Marmoset PON1 cDNA containing an open reading frame (1065 bp) was successfully cloned from marmoset liver by reverse transcription-polymerase chain reaction. The deduced amino acid sequence (355 amino acids) has approximately 93% identity with the human ortholog and contains important amino acid residues for substrate binding and calcium ion coordination. According to a phylogenetic tree of PON1 amino acid sequences constructed using data from seven animal species, marmoset PON1 is closer to human PON1 than it is to the PON1 orthologs of experimental animals such as pigs, rabbits, rats, and mice. Marmoset PON1 mRNA was predominantly expressed in liver among the five tissues examined. Marmoset PON1 protein secreted into plasma was detected by immunoblotting. The paraoxon-hydrolyzing activity in plasma was higher in marmosets than in humans. Based on these data, we concluded that marmoset and human PON1 have similar characteristics with regard to genomic structure, amino acid sequences, and tissue distribution.

Human Aldehyde Oxidase 1-Mediated Carbazeran Oxidation in Chimeric TK-NOG Mice Transplanted with Human Hepatocytes.

Carbazeran is a potent phosphodiesterase inhibitor with species-dependent metabolic profiles in rats, dogs, and humans. In this study, we investigated the aldehyde oxidase (AOX)-mediated oxidation of carbazeran to 4-oxo derivatives in chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene with humanized livers (humanized-liver mice). Liver cytosolic fractions from humanized-liver mouse effectively catalyzed carbazeran 4-oxidation with high affinity for the substrate, similar to those of the human liver cytosolic fractions and recombinant human AOX1 protein. Furthermore, hepatocytes prepared from humanized-liver mice and humans also exhibited substantial metabolism via carbazeran 4-oxidation. After a single oral administration of carbazeran (10 mg/kg), plasma levels of 4-oxo-carbazeran, N-desethyl-4-oxo-carbazeran, and 6,7-dimethoxy-1-[4-(hydroxy)-piperidino]-4-phthalazinone (three human metabolites formed via 4-oxidation) were higher in humanized-liver mice than in the control mice. In contrast, plasma levels of O-desmethylcarbazeran (a major metabolite in dogs) in control mice were higher than those in the humanized-liver mice. Relative excreted amounts of the three 4-oxidation-derived human-specific metabolites in the urine and feces were greater for humanized-liver mice than control mice, whereas the relative excreted amounts of O-desmethylcarbazeran were predominant in the urine and feces of control mice. Thus, the production of carbazeran 4-oxo derivatives was elevated in humanized-liver mice compared with control mice, in agreement with our in vitro enzyme-mediated oxidation data. These results suggest that hepatic human AOX1 functions in humanized-liver mice at the in vivo level and that humanized-liver mice may be useful for predicting drug metabolism in humans, at least with regard to human AOX1-dependent metabolism. SIGNIFICANCE STATEMENT: We found that the production of carbazeran 4-oxo derivatives was higher in humanized-liver mice than in control mice. These results were supported by the fact that carbazeran was rapidly metabolized to 4-oxo-carbazeran in humanized-liver mouse hepatocytes expressing human aldehyde oxidase 1. These results suggest that human aldehyde oxidase 1 is functional in humanized-liver mice in vivo and that chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene transplanted with human hepatocytes may be a suitable model animal for predicting aldehyde oxidase-dependent biotransformation of drugs in humans.

Importance of cynomolgus monkeys in development of monoclonal antibody drugs.

Animal species used in the preclinical studies for development of monoclonal antibody (mAb) drugs are surveyed in this review. Relevant animal species for preclinical studies of mAb candidates are those express desired epitope of mAb candidates. Cynomolgus monkeys cross-react with mAb drugs much higher than other animal species commonly used in preclinical studies such as absorption, distribution, metabolism and excretion (ADME), efficacy, and toxicity studies, for development of new drugs. Moreover, plasma exposure of the mAb drugs in humans is predicted well from the exposure in the monkeys, and the placental transfer of immunoglobulin G (IgG, all the mAb drugs contain IgG) from mother to fetus is similar between humans and the monkeys from a viewpoint of time course and plasma level of IgG transferred. These observed findings indicate that the monkeys are the most suitable animal species used in the ADME and toxicity studies for development of new mAb drugs.

Genetic polymorphisms of drug-metabolizing cytochrome P450 enzymes in cynomolgus and rhesus monkeys and common marmosets in preclinical studies for humans.

Cynomolgus monkeys (Macaca fascicularis, Old World Monkeys) and common marmosets (Callithrix jacchus, New World Monkeys) have been widely, and expectedly, used as non-human primate models in drug development studies. Major drug-metabolizing cytochrome P450 (P450) enzymes information is now available that supports these primate species as animal models, and it is established that multiple forms of cynomolgus monkey and common marmoset P450 enzymes have generally similar substrate recognition functionality to human P450 enzymes. This research update provides information on genetic polymorphisms of P450 enzymes in cynomolgus monkey and common marmoset like human P450 enzymes. Information on rhesus monkeys (Macaca mulatta), another macaque species used in drug metabolism studies, is also included for comparison. Among a variety of cynomolgus monkey P450 variants investigated, typical examples include individual pharmacokinetic data for efavirenz and R-warfarin associated with cynomolgus monkey P450 2C9 (formerly 2C43) and 2C19 (2C75) variants, respectively, and for R-omeprazole and S-warfarin associated with marmoset P450 2C19 variants. These findings provide a foundation for understanding the individual pharmacokinetic and toxicological results in non-human primates as preclinical models and will help to further support understanding of molecular mechanisms of human P450 function. In addition to these polymorphic P450 enzymes, effects of aging on some drug clearances mediated by cynomolgus monkey and common marmoset P450 enzymes were found in elder animals or animals pretreated with rifampicin. This review describes genetic and acquired individual differences in cynomolgus monkey and common marmoset P450 enzymes involved in drug oxidation associated with pharmacological and/or toxicological effects.

Cortical Processing of Level Cues for Spatial Hearing is Impaired in Children with Prelingual Deafness Despite Early Bilateral Access to Sound.

Bilateral cochlear implantation aims to restore binaural hearing, important for spatial hearing, to children who are deaf. Improvements over unilateral implant use are attributed largely to the detection of interaural level differences (ILDs) but emerging evidence of impaired sound localization and binaural fusion suggest that these binaural cues are abnormally coded by the auditory system. We used multichannel electroencephalography (EEG) to assess cortical responses to ILDs in two groups: 13 children who received early bilateral cochlear implants (CIs) simultaneously, known to protect the developing auditory cortices from unilaterally driven reorganization, and 15 age matched peers with normal hearing. EEG source analyses indicated a dominance of right auditory cortex in both groups. Expected reductions in activity to ipsilaterally weighted ILDs were evident in the right hemisphere of children with normal hearing. By contrast, cortical activity in children with CIs showed: (1) limited ILD sensitivity in either cortical hemisphere, (2) limited correlation with reliable behavioral right-left lateralization of ILDs (in 10/12 CI users), and (3) deficits in parieto-occipital areas and the cerebellum. Thus, expected cortical ILD coding develops with normal hearing but is affected by developmental deafness despite early and simultaneous bilateral implantation. Findings suggest that impoverished fidelity of ILDs in independently functioning CIs may be impeding development of cortical ILD sensitivity in children who are deaf but do not altogether limit benefits of listening with bilateral CIs. Future efforts to provide consistent/accurate ILDs through auditory prostheses including CIs could improve binaural hearing for children with hearing loss.

Simultaneous bilateral cochlear implants: Developmental advances do not yet achieve normal cortical processing.

BACKGROUND: Simultaneous bilateral cochlear implantation promotes symmetric development of bilateral auditory pathways but binaural hearing remains abnormal. To evaluate whether bilateral cortical processing remains impaired in such children, cortical activity to unilateral and bilateral stimuli was assessed in a unique cohort of 16 children who received bilateral cochlear implants (CIs) simultaneously at 1.97 ± 0.86 years of age and had ~4 years of CI experience, providing the first opportunity to assess electrically driven cortical development in the absence of reorganized asymmetries from sequential implantation. METHODS: Cortical activity to unilateral and bilateral stimuli was measured using multichannel electro-encephalography. Cortical processing in children with bilateral CIs was compared with click-elicited activity in 13 normal hearing children matched for time-in-sound. Source activity was localized using the Time Restricted, Artefact and Coherence source Suppression (TRACS) beamformer method. RESULTS: Consistent with dominant crossed auditory pathways, normal P1 activity (~100 ms) was weaker to ipsilateral stimuli relative to contralateral and bilateral stimuli and both auditory cortices preferentially responded to the contralateral ear. Right hemisphere dominance was evident overall. Children with bilateral CIs maintained the expected right dominance but differences from normal included: (i) minimal changes between ipsilateral, contralateral and bilateral stimuli, (ii) weaker than normal contralateral stimulus preference, (iii) symmetric activity to bilateral stimuli, and (iv) increased occipital lobe recruitment during bilateral relative to unilateral stimulation. Between-group contrasts demonstrated lower than normal activity in the inferior parieto-occipital lobe (suggesting deficits in sensory integration) and greater than normal left frontal lobe activity (suggesting increased attention), even during passive listening. CONCLUSIONS: Together, findings suggest that early simultaneous bilateral cochlear implantation promotes normal-like auditory symmetry but that abnormalities in cortical processing consequent to deafness and/or electrical stimulation through two independent speech processors persist.

Marmoset Flavin-Containing Monooxygenase 3 in the Liver Is a Major Benzydamine and Sulindac Sulfide Oxygenase.

Common marmosets (Callithrix jacchus) are potentially primate models for preclinical drug metabolism studies because there are similarities in the molecular characteristics of cytochrome P450 enzymes between this species and humans. However, characterization of non-cytochrome P450 enzymes has not been clarified in marmosets. Here, we report characterization of flavin-containing monooxygenases FMO1-FMO5 identified in marmoset tissues. Marmoset FMO forms shared high amino acid sequence identities (93%-95%) and phylogenetic closeness with human homologous FMO forms. FMO1 and FMO3 mRNA were abundantly expressed in the liver and kidneys among five marmoset tissues examined, where FMO3 protein was detected by immunoblotting. FMO inhibition assays using preheated tissue microsomes indicated that benzydamine N-oxygenation and sulindac sulfide S-oxygenation in the marmoset liver was mainly catalyzed by FMO3, the major hepatic FMO. Marmoset FMO3 protein heterologously expressed in Escherichia coli effectively catalyzed benzydamine N-oxygenation and sulindac sulfide S-oxygenation comparable to marmoset liver microsomes. These results indicate that the FMO3 enzyme expressed in marmoset livers mainly metabolizes benzydamine and sulindac sulfide (typical human FMO substrates), suggesting its importance for FMO-dependent drug metabolism in marmosets.

Cortical Representation of Interaural Time Difference Is Impaired by Deafness in Development: Evidence from Children with Early Long-term Access to Sound through Bilateral Cochlear Implants Provided Simultaneously.

Accurate use of interaural time differences (ITDs) for spatial hearing may require access to bilateral auditory input during sensitive periods in human development. Providing bilateral cochlear implants (CIs) simultaneously promotes symmetrical development of bilateral auditory pathways but does not support normal ITD sensitivity. Thus, although binaural interactions are established by bilateral CIs in the auditory brainstem, potential deficits in cortical processing of ITDs remain. Cortical ITD processing in children with simultaneous bilateral CIs and normal hearing with similar time-in-sound was explored in the present study. Cortical activity evoked by bilateral stimuli with varying ITDs (0, ±0.4, ±1 ms) was recorded using multichannel electroencephalography. Source analyses indicated dominant activity in the right auditory cortex in both groups but limited ITD processing in children with bilateral CIs. In normal-hearing children, adult-like processing patterns were found underlying the immature P1 (∼100 ms) response peak with reduced activity in the auditory cortex ipsilateral to the leading ITD. Further, the left cortex showed a stronger preference than the right cortex for stimuli leading from the contralateral hemifield. By contrast, children with CIs demonstrated reduced ITD-related changes in both auditory cortices. Decreased parieto-occipital activity, possibly involved in spatial processing, was also revealed in children with CIs. Thus, simultaneous bilateral implantation in young children maintains right cortical dominance during binaural processing but does not fully overcome effects of deafness using present CI devices. Protection of bilateral pathways through simultaneous implantation might be capitalized for ITD processing with signal processing advances, which more consistently represent binaural timing cues.SIGNIFICANCE STATEMENT Multichannel electroencephalography demonstrated impairment of binaural processing in children who are deaf despite early access to bilateral auditory input by first finding that foundations for binaural hearing are normally established during early stages of cortical development. Although 4- to 7-year-old children with normal hearing had immature cortical responses, adult patterns in cortical coding of binaural timing cues were measured. Second, children receiving two cochlear implants in the same surgery maintained normal-like input from both ears, but this did not support significant effects of binaural timing cues in either auditory cortex. Deficits in parieto-occiptal areas further suggested impairment in spatial processing. Results indicate that cochlear implants working independently in each ear do not fully overcome deafness-related binaural processing deficits, even after long-term experience.

Strong Induction of Cytochrome P450 1A/3A, But not P450 2B, in Cultured Hepatocytes from Common Marmosets and Cynomolgus Monkeys by Typical Human P450 Inducing Agents.

BACKGROUND: Common marmosets (Callithrix jacchus) and cynomolgus monkeys (Macaca fascicularis) are used as non-human primate models in preclinical studies for drug development. OBJECTIVE: The assessment of P450 induction in hepatocytes from marmosets and cynomolgus monkeys was performed using typical P450 inducers. METHODS: Induction of cytochrome P450 1-4 family enzymes was analyzed in two lots of cultured hepatocytes from common marmosets and cynomolgus monkeys after 24-h treatment with typical human P450 inducing agents by real-time reverse transcription-polymerase chain reaction. RESULTS: Marmoset P450 3A4 mRNA and P450 2C8/2C19 mRNA in hepatocytes were strongly (>10- fold) and weakly (>2) induced by rifampicin, respectively. Marmoset 1A1 and 1A2 mRNA were induced strongly (>200-fold) by ß-naphthoflavone and omeprazole. Marmoset P450 2B6 mRNA was induced (~5-fold) by a constitutive androstane receptor agonist, but not by phenobarbital. Cynomolgus monkey P450 3A4 mRNA and P450 1A1 mRNA in cultured hepatocytes were also induced by rifampicin and omeprazole, respectively, but P450 2B6 mRNA was not induced by phenobarbital. CONCLUSION: These results indicate that P450 1A/3A induction by typical human P450 inducers in hepatocytes from marmosets and/or cynomolgus monkeys are similar to those of humans (except for P450 2B induction by phenobarbital in humans), suggesting that marmosets and cynomolgus monkeys might be suitable models for evaluating the drug interactions in preclinical studies.

Cytochrome P450 2A6 Phenotyping Using Dietary Caffeine Salivary Metabolite Ratios and Genotyping Using Blood on Storage Cards in Non-smoking Japanese Volunteers.

BACKGROUND: A simple method of genotyping and phenotyping cytochrome P450 2A6 (CYP2A6) was previously reported using individual blood samples and urinary caffeine metabolite ratios of 1,7-dimethyluric acid (17U) to 1-methylxanthine (1X). OBJECTIVE: Blood spotted onto storage cards and salivary caffeine metabolites were analyzed in 27 healthy non-smoking Japanese volunteers with no prior abstention from dietary caffeine intake. METHODS: 1,7-Dimethylxanthine (17X), 17U, 1X, and caffeine levels in spot saliva samples were determined in Japanese non-smokers by high-performance liquid chromatography under normal dietary caffeine consumption. RESULTS: 17U/17X ratios in saliva were almost constant over time, but those of 17U/1X were variable in two subjects tested before and 1-2.5 h after caffeine treatment (a cup of black tea). In seven subjects, 17U/17X ratios in saliva were highly correlated with those in plasma (r = 0.98, p < 0.01) and well correlated with those in urine samples (r = 0.78, p < 0.05). The average 17U/17X ratios, but not 17U/1X ratios, in saliva under dietary caffeine consumption obtained from subjects with CYP2A6*1/*4 (n=11) and CYP2A6*4/*4 (whole-gene deletion, n=2) genotypes were significantly lower than those from subjects with wild-type CYP2A6*1/*1 (n=14). Genotyping was done by a multiplex real-time polymerase chain reaction method using blood spotted onto storage cards. CONCLUSION: The present results suggest that the decreased CYP2A6 function associated with the whole-gene deletion genotype (determined using blood samples) could be detected using 17U/17X ratios, but not 17U/1X ratios, in spot saliva samples under normal dietary caffeine consumption in Japanese non-smokers, just as it could be detected using urinary 17U/1X ratios.