Sex- and age-dependent gene expression in human liver: An implication for drug-metabolizing enzymes.

Sex and age differences in hepatic expression of drug-metabolizing enzyme genes could cause variations in drug metabolism, but has not been fully elucidated, especially in Asian population. In this study, the global expression of human hepatic genes was analyzed by microarrays in 40 Japanese subjects (27 males and 13 females). Thirty-five sex-biased genes were identified (P < 0.005). Whereas, 60 age-biased genes in two age groups, <60 years and ≥70 years (P < 0.001), were identified in males. By Gene Ontology analysis, the sex-biased genes were related to protein catabolism and modification, while the age-biased genes were related to transcription regulation and cell death. Quantitative polymerase chain reaction confirmed the female-biased expression of drug-metabolizing enzyme genes BChE, CYP4X1, and SULT1E1 (≥1.5-fold, P < 0.05). Further analysis of drug-metabolizing enzyme genes indicated that expression of CYP2A6 and CYP3A4 in females in the ≥70 age group was less than in the <60 age group (≥1.5-fold, P < 0.05), and this trend was also observed for PXR expression in males (≥1.5-fold, P < 0.05). The results presented provide important insights into hepatic physiology and function, especially drug metabolism, with respect to sex and age.

Expression profile of early estradiol-responsive genes in cynomolgus macaque liver: implications for drug-metabolizing enzymes.

Estrogen plays important roles in estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, the major drug metabolizing organ, estrogen is known to regulate expression of some drug-metabolizing enzymes. Due to the lack of information on the role of estrogen in hepatic gene expression in primate species, we previously investigated the late response of hepatic gene expression to estradiol in cynomolgus macaques. To understand the early response of hepatic gene expression to estradiol, in this study, microarray analysis was conducted using cynomolgus macaque liver samples collected at 1 h and 5 h after estradiol injection. Comparison of expression profiles in estradiol and solvent (control)-treated ovariectomized cynomolgus macaques revealed 27 differentially expressed genes (>2.0-fold), including 18 at 1 h and 9 at 5 h after estradiol injection. As indicated by Gene Ontology analysis, these genes were related to oxidoreductase activity and transferase activity, partly representing important aspects of drug-metabolizing enzymes. Further analysis by quantitative polymerase chain reaction revealed that estradiol down-regulated CYP2A24, CYP2C76, and CYP2E1 (>2.0-fold) at 1 h and up-regulated GSTM5 (>2.0-fold) at 5 h after estradiol injection. These results suggest that the short-term estradiol treatment influenced expression of hepatic genes, including drug-metabolizing enzyme genes, in cynomolgus macaque liver.

Effect of estradiol on gene expression profile in cynomolgus macaque liver: implications for drug-metabolizing enzymes.

Estrogen regulation of gene expression is essential for physiological function of estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, estrogen is responsible for sex-dependent gene expression of drug-metabolizing enzymes in rodents. However, the influence of estrogen on hepatic gene expression has not been fully investigated in primates, including human. Macaque, including cynomolgus macaque, is an important species for comparative studies aimed at understanding human physiology due to its evolutionary closeness to human. To identify estrogen-responsive genes in primate liver, therefore, hepatic gene expression was compared, by microarray analysis, in ovariectomized cynomolgus macaques treated with estradiol or solvent (control). The analysis identified 98 estradiol-responsive genes; 47 and 51 were up- and down-regulated by estradiol, respectively (≥2.0-fold, P < 0.05). Expression of drug-metabolizing enzyme genes was also influenced by estradiol treatment; estradiol enhanced expression of GSTM5 (3.8-fold, P < 0.05) and CYP3A8(4) (2.7-fold, P < 0.01), but lowered expression of CYP4F12 (2.2-fold, P < 0.01), as verified by quantitative polymerase chain reaction. In particular, CYP3A8(4), orthologous to human CYP3A4, is an essential drug-metabolizing enzyme in cynomolgus macaque liver. These results suggest that expression of hepatic genes, including drug-metabolizing enzyme genes, is at least partly regulated by estradiol in cynomolgus macaque.

Effects of metoprolol on epinephrine-induced takotsubo-like left ventricular dysfunction in non-human primates.

Takotsubo cardiomyopathy, alternatively known as stress cardiomyopathy, is an increasingly recognized clinical syndrome characterized by acute reversible apical ventricular dysfunction. To elucidate the mechanism, we tried to make a new model of takotsubo-like cardiomyopathy in non-human primates. Echocardiography revealed that repeated intravenous infusion of epinephrine overdose in cynomolgus monkeys induced takotsubo-like cardiomyopathy, which is characterized by progressive left ventricle and depressed systolic function with severe hypokinesis in apical regions and hyperkinesis in the basal region. Although this cardiac dysfunction almost normalized after a month even without any treatment, metoprolol, a beta-blocker, improved the decreased ejection fraction earlier than in the control. Luxol fast blue staining, which is useful for estimating myocytolysis, showed that increased myocytolysis was observed in the apical ventricle of the epinephrine-infused heart. Metoprolol diminished epinephrine-induced cardiomyocytolysis. To explain the mechanism of takotsubo myopathy and the effect of metoprolol, gene expressions in apical or basal ventricle were compared. Heart failure-related genes, such as brain natriuretic peptide, connective tissue growth factor and osteopontin; calcium signaling-related genes, such as ryanodine receptor 2, sarcoendoplasmic reticulum Ca(2+)-ATPase 2A2 and adenylate cyclase 7; renin-angiotensin system-related genes, such as angiotensinogen, angiotensin II receptor, type 1 and type 2; and mitochondria-related genes, such as peroxisome proliferator-activated receptor-gamma co-activator-1alpha, cytochrome c and transcription factor A mitochondrial, were significantly changed at the apical ventricle rather than at the basal ventricle. The changes of some genes improved with metoprolol treatment. These results indicate that this model is valuable in understanding the pathogenesis of takotsubo cardiomyopathy and the effectivity of beta-blockers.

A null allele impairs function of CYP2C76 gene in cynomolgus monkeys: a possible genetic tool for generation of a better animal model in drug metabolism.

The monkey CYP2C76 gene does not correspond to any of the human CYP2C genes, and its enzyme is at least partly responsible for the species difference occasionally seen in drug metabolism between monkeys and humans. To establish a line and/or lines of monkeys that are expected to show metabolic patterns highly similar to humans, we set out to find monkeys that lacked CYP2C76 activity. By genetic screening of 73 monkeys and a database search of expressed sequence tags, we found a total of 10 nonsynonymous genetic variants in the coding region of CYP2C76, including a null genotype (c.449TG>A). Some of the variants were differently distributed between two animal groups originating from different geographical regions (Indochina and Indonesia). After screening 170 additional genomic samples, we identified a total of eight animals (six males and two females) that were heterozygous for c.449TG>A, which could be used for establishing a homozygous line. If the homozygotes show drug-metabolizing properties more similar to humans than wild-type monkeys, the homozygotes may serve as a better animal model for drug metabolism. The data presented in this article provide the essential genetic information to perform a successful study by using cynomolgus monkeys and present a possible tool to generate a better animal model for drug metabolism.

Sex-related differences in the expression of mfGSTA2, a novel GST identified in cynomolgus monkey (Macaca fascicularis).

Glutathione S-transferase (GST) is one of the most important phase II drug-metabolizing enzymes, catalyzing the conjugation of electrophilic substrates to glutathione. Unlike in humans, a surprisingly limited number of GST genes have been identified in monkeys. The identification of additional GST genes in this model system would prove to be advantageous, because monkeys remain an important predictor of drug effects and toxicities in humans during preclinical studies. In this study, we report the identification and characterization of the following six cDNAs in cynomolgus monkeys: mfGSTA1, mfGSTA2, mfGSTM5, mfMGST1, mfGSTO1, and mfGSTZ1. These cDNAs encode GSTs highly homologous (approximately 95%) to human GST cDNAs. Among these, the mfGSTA1, mfGSTM5, mfMGST1, mfGSTO1, and mfGSTZ1 cDNAs correspond to a single human GST counterpart, whereas the mfGSTA2 cDNA is highly similar to human GSTA1 and GSTA2 cDNAs. An analysis of tissue samples indicates that these GST genes are predominantly expressed in the liver along with some extrahepatic expression as determined by real-time reverse transcriptase-polymerase chain reaction. It is interesting to note that mfGSTA2 is significantly differentially expressed between males and females in the jejunum, where a striking 8-fold higher expression level is observed in males. These results suggest that a potential sex difference in the metabolism of drugs may be mediated by mfGSTA2. This also provides a basis for the investigation of sex-dependent drug metabolism in monkeys.

Ginsenoside Rb1 reduces neurodegeneration in the peri-infarct area of a thromboembolic stroke model in non-human primates.

Ginsenoside Rb1 (GRb1), a major component of the traditional herb ginseng, has been reported to show a neuroprotective effect in a rodent ischemic model. The purpose of this study was to investigate effects of GRb1 on early and delayed brain injuries in a non-human primate thromboembolic stroke model. Thromboembolic stroke was induced by occlusion of the middle cerebral artery by injection of an autologous blood clot into the left internal carotid artery. GRb1 (300 microg/kg per day, i.v.) and vehicle were administered from 7 days before embolization to the day following embolization (total: 8 times). Neurological deficits were observed at 1, 6, and 24 h and at 2, 4, and 7 days after embolization. At 7 days after embolization, neuron damage in the peri-infarct area and core region were assessed by NeuN, TUNEL, and GFAP staining. GRb1 improved the skeletal muscle coordination score of the neurologic deficits (median: GRb1 vs vehicle = 10 vs 12, P<0.05). In the GRb1 group, positive neurons expressed by NeuN staining were noted in the ischemic peri-infarct area, and TUNEL- and GFAP-positive cells significantly decreased, when compared with vehicle. These results demonstrated that GRb1 ameliorated both early and delayed injuries in the thromboembolic stroke model in non-human primates.

Characterization of cynomolgus monkey cytochrome P450 (CYP) cDNAs: is CYP2C76 the only monkey-specific CYP gene responsible for species differences in drug metabolism?

Cynomolgus monkey CYP2C76 does not have a corresponding ortholog in humans, and it is at least partly responsible for differences in drug metabolism between monkeys and humans. To determine if CYP2C76 is the only monkey-specific CYP gene, we identified cynomolgus monkey cDNAs for CYP2A23, CYP2A24, CYP2E1, CYP2J2, CYP3A5, CYP3A8, CYP4A11, CYP4F3, CYP4F11, CYP4F12, and CYP4F45. These CYP cDNAs showed a high sequence identity (93-96%) to the homologous human CYP cDNAs. The monkey CYPs were preferentially expressed in liver among the analyzed tissues. Moreover, all five analyzed monkey CYPs (CYP2A23, CYP2A24, CYP2E1, CYP3A5, and CYP3A8) metabolized typical substrates for human CYPs in the corresponding subfamilies. These results suggest that these 11 monkey CYP cDNAs are closely related to the human CYP cDNAs and thus, unlike CYP2C76, are not apparent monkey-specific cDNAs.

Left ventricular volume and function in cynomolgus monkeys using real-time three-dimensional echocardiography.

BACKGROUND: The purpose of this study was to investigate the feasibility of evaluating cardiac function by real time three-dimensional (RT3D) echocardiography in isoflurane-anesthetized male cynomolgus monkeys. Additionally differences between inhibitory effects of beta-blockers and a Ca channel blocker on left ventricular (LV) function were examined. METHODS AND RESULTS: End-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF) in the control (without any drug effect) were not significantly changed by repetitive measurement at a 30-day interval. Propranolol and metoprolol (0.1 and 0.3 mg/kg/10 minutes, i.v.) caused a dose-dependent increase in ESV, but little effect on EDV, resulting in a decrease in EF. Verapamil (0.1 and 0.3 mg/kg/10 minutes, i.v.) increased both EDV and ESV, but decreased EF was noted at 0.3 mg/kg. CONCLUSIONS: These results demonstrate the feasibility of RT3D echocardiography in providing reproducible estimations of LV volume and EF in monkeys when evaluating drugs that may affect cardiac function.

Evaluation of QT interval using a linear model in individual cynomolgus monkeys.

INTRODUCTION: The cynomolgus monkey, one of a number of primate species phylogenetically close to humans, is commonly used in cardiovascular research, but a method for determination of the RR interval-corrected QT interval in this species needs greater consideration. The objectives of this study were to determine a method for evaluating QT interval in cynomolgus monkeys individually, disregarding RR interval change artifacts, and to investigate prerequisite information for this method. METHODS: The physiological QT-RR relationship for practical evaluation of QT interval was recorded and analyzed by 24-hour telemetric ECG monitoring. A linear model for log-transformed QT and RR intervals was used to correct the QT interval from RR interval change artifacts for each animal. Sample size was also estimated based on the simulation results. RESULTS: Histograms showed that both QT and RR intervals had a right-heavy tail distribution. QT interval corrected individually by the linear model formula showed smaller within-animal variability than QTb and QTf, which were corrected by Bazett's formula and Fridericia's formula. The simulation results showed that the individual correction factor, beta(i), could be reliably estimated when at least 24 pairs of QT-RR baseline data were available. DISCUSSION: As with humans, QT interval in cynomolgus monkeys varies widely between individuals. Therefore, a method for correcting QT interval individually should be considered, whenever extensive untreated data are available.

Effects of phosphodiesterase (PDE) inhibitors on human ether-a-go-go related gene (hERG) channel activity.

It is presumed that phosphodiesterase (PDE) inhibitors have two mechanisms for inhibition of hERG currents in the acute applications to cells: direct channel block, and downregulation of human ether-a-go-go related gene (hERG) activities by PKA-dependent pathway mediated phosphorylation through their inhibitory effects against PDE enzymes. However, it is unknown whether PDE inhibition contributes to the inhibitory effects of PDE inhibitors on hERG currents. This study examined the effects of various PDE inhibitors on hERG currents using both the whole-cell and perforated patch-clamp techniques in hERG transfected CHO-K1 cells. The study also investigated the contribution of the PKA-dependent pathway to the inhibitory effects of PDE inhibitors on hERG currents. Of the PDE inhibitors tested, vinpocetine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), vesnarinone, rolipram and dipyridamole decreased hERG currents in a concentration-dependent manner. Vinpocetine and vesnarinone markedly decreased the hERG current with an IC (50)of 0.13 and 20.6 microm, respectively, at comparatively low concentrations. Furthermore, vinpocetine caused a cumulative block of hERG currents. Milrinone, amrinone and zaprinast had no effect on the hERG current up to 100 microm. Of the PDE3 inhibitors (vesnarinone, amrinone and milrinone), only vesnarinone showed an hERG inhibitory effect. The inhibitory effects of vinpocetine and vesnarinone were not significantly affected by the co-application of protein kinase inhibitors. Furthermore, the protein kinase activators had no effect on hERG currents. It is concluded that vinpocetine and vesnarinone block the hERG channel directly, and that the inhibitory effect on intracellular PDE in the PKA-dependent pathway may not be involved in the inhibition of hERG currents in hERG transfected CHO-K1 cells.

CYP2C76, a novel cytochrome P450 in cynomolgus monkey, is a major CYP2C in liver, metabolizing tolbutamide and testosterone.

Monkeys are widely used as a primate model to study drug metabolism because they generally show a metabolic pattern similar to humans. However, the paucity of information on cytochrome P450 (P450) genes has hampered a deep understanding of drug metabolism in the monkey. In this study, we report identification of the CYP2C76 cDNA newly identified in cynomolgus monkey and characterization of this CYP2C along with cynomolgus CYP2C20, CYP2C43, and CYP2C75. The CYP2C76 cDNA contains the open reading frame encoding a protein of 489 amino acids that are only approximately 80% identical to any human or monkey P450 cDNAs. Gene and protein expression of CYP2C76 was confirmed in the liver of cynomolgus and rhesus monkeys but not in humans or the great apes. Moreover, CYP2C76 is located at the end of the CYP2C gene cluster in the monkey genome, the region of which corresponds to the intergenic region adjacent to the CYP2C cluster in the human genome, strongly indicating that this gene does not have the ortholog in humans. Among the four CYP2C genes expressing predominantly in the liver, the expression level of CYP2C76 was the greatest, suggesting that CYP2C76 is a major CYP2C in the monkey liver. Assays for the capacity of CYP2C76 to metabolize drugs using several substrates typical for human CYP2Cs revealed that CYP2C76 showed unique metabolic activity. These results suggest that CYP2C76 contributes to overall drug-metabolizing activity in the monkey liver and might account for species difference occasionally seen in drug metabolism between monkeys and humans.

Effects of intra-arterial urokinase on a non-human primate thromboembolic stroke model.

One of the most important prognostic factors in the thrombolytic treatment of acute ischemic stroke is to re-canalize. The purpose of this study was to evaluate the effectiveness and safety of urokinase in a primate thromboembolic stroke model. Thromboembolic stroke was accomplished via occlusion of the middle cerebral artery (MCA) obtained by injecting an autologous blood clot into the left internal carotid artery in 21 male cynomolgus monkeys. Animals were randomly assigned to the following treatment groups: Group 1: vehicle (saline), Group 2: urokinase (40,000 IU), Group 3: urokinase (120,000 IU,) over 2 or 6 h via intra-internal carotid catheter starting 1 h after embolization, respectively. In the urokinase-treated groups, neurologic deficits were improved in consciousness and skeletal muscle coordination, but not sensory and motor systems. The infarction size in Group 2 (11.9 +/- 3.9% of the hemisphere) and 3 (7.6 +/- 2.5%) were significantly smaller than that (24.7 +/- 3.5%) in Group 1. However, 2 of 5 animals in Group 3 died. In conclusion, urokinase improved neurologic deficits and reduced cerebral infarction on thromboembolic stroke in the cynomolgus monkey.

Evaluation of cardiac function in primates using real-time three-dimensional echocardiography as applications to safety assessment.

INTRODUCTION: A timed non-invasive determination of cardiac function is potentially important for safety pharmacology and toxicity studies. The objectives of this study were to evaluate the accuracy of real-time three-dimensional (RT3D) echocardiography measurements of the left ventricular (LV) volume and LV function and to investigate the effects of some drugs on LV function in cynomolgus monkeys. METHODS: RT3D echocardiography was performed (SONOS 7500, Philips Med Sys) under isoflurane inhalation. RT3D echocardiography measurements and reconstructions were obtained using Tom-Tec (4DLV analysis). We determined end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), stroke volume (SV), cardiac output (CO) and heart rate as assessments of LV function. EDV, calculated from two-dimensional (2D) echocardiography and RT3D echocardiography, and the actual LV volume were evaluated and compared. Furthermore, each parameter was determined before and after intravenous infusion (5 or 10 min) of propranolol, verapamil and dobutamine. RESULTS: A strong correlation was found between the actual LV volume and that calculated from RT3D echocardiography (r=0.96, p<0.001). Propranolol (0.1 mg/kg/10 min, n=5) caused an increase in ESV, but not EDV, resulting in a decrease in EF and SV, while verapamil produced increases in both EDV and ESV. Dobutamine (0.01 mg/kg/5 min, n=5) produced decreases in both EDV and ESV and thereby the increased CO resulted from the increased SV. DISCUSSION: These results demonstrate that RT3D echocardiography provides a feasible and accurate estimation of LV volume and EF for safety pharmacology and toxicity studies.

Post-ischemic cyclooxygenase-2 expression is regulated by the extent of cerebral blood flow reduction in non-human primates.

We determined whether up to 24 h of ischemia could induce the expression of cyclooxygenase-2 (COX-2) in the brain of nonhuman primates. Randomized animals were subjected to either a 2 h ischemia (group II; n=3) or a 24 h ischemia (group III; n=3). Three animals in group I served as controls. In group III, regional cerebral blood flow (CBF) and the cerebral glucose metabolic rate (CMRglc) were evaluated using positron emission tomography. Upregulation of COX-2 mRNA expression was observed after 2 h of ischemia, but disappeared by 24 h in the ischemic temporal cortex, in which both CMRglc and CBF were markedly reduced. In the ischemic parietal cortex, where CMRglc was preserved, COX-2 expression persisted even 24 h after ischemia. This study is the first to demonstrate neuronal COX-2 induction within potentially viable hypoperfused brain areas in nonhuman primates.

Development of telemetry system in the common marmoset--cardiovascular effects of astemizole and nicardipine.

The purpose of this study was to evaluate a telemetry system for examining the cardiovascular system in the conscious common marmoset. Parameters obtained were blood pressure, heart rate, respiratory rate, ECG, body temperature and locomotor activity, and these were continuously recorded on a data recorder via the telemetry system and then processed by a computerized system. Diurnal rhythms of blood pressure, heart rate, body temperature and locomotor activity were observed in this system. We studied the effects of astemizole (antihistamine) and nicardipine (Ca2+ channel blocker) on cardiovascular parameters. Astemizole at 30 mg/kg (p.o.) and at 1 to 3 mg/kg (i.v.), prolonged QT interval and induced ventricular extrasystole. Torsades de pointes occurred in one of three cases at 3 mg/kg (i.v.) and 30 mg/kg (p.o.), while it did not affect the blood pressure, respiratory rate and body temperature. Nicardipine at 30 mg/kg (p.o.) caused sustained hypotension and tachycardia. These results demonstrate the usefulness of the telemetry system using the common marmoset for evaluating the cardiovascular effects of drugs under physiological conditions.