Development of a new device for artificial insemination in cynomolgus macaques.

In cynomolgus macaques, an important animal species for biomedical research, efficient reproduction has been hampered partly due to the difficulties of artificial insemination (AI) using straw tubes developed for humans or farm animals, because cynomolgus macaques have a complex cervical canal structure. In this study, taking into consideration the unique structure of the macaque cervical canal, we developed a novel device for AI, comprised of a syringe and an outer cylinder. At 24 and 48 h after using this device to inject semen into one female, viable sperm were observed in the oviduct where the sperm meets the oocytes. We then attempted AI using this new device on 10 females that were at pre-ovulation, and pregnancy was successful in three animals (30% pregnancy rate). These results show that the newly developed device can be used for AI in cynomolgus macaques.

Muscle wasting associated with pathologic change is a risk factor for the exacerbation of joint swelling in collagen-induced arthritis in cynomolgus monkeys.

BACKGROUND: Not only joint destruction but also muscle wasting due to rheumatoid cachexia has been problem in terms of quality of life of patients with rheumatoid arthritis (RA). In the present study, we performed histopathological examination and assessed relationships between characteristic parameters relating to muscle and joint swelling in a collagen-induced arthritis (CIA) model using cynomolgus monkeys (CMs). METHODS: Female CMs were used and CIA was induced by twice immunizations using bovine type II collagen with Freund's complete adjuvant. Arthritis level was evaluated from the degree of swelling at the peripheral joints of the fore and hind limbs. Food consumption, body weight, and serum biochemical parameters were measured sequentially. Five or 6 animals per time point were sacrificed at 2, 3, 5 and 9 weeks after the first immunization to obtain quadriceps femoris specimens for histopathology. Pimonidazole hydrochloride was intravenously administered to determine tissue hypoxia in skeletal muscle. RESULTS: Gradual joint swelling was observed and the maximum arthritis score was noted at Week 5. In histopathology, necrosis of muscle fiber in the quadriceps femoris was observed only at Week 2 and the most significant findings such as degeneration, atrophy, and regeneration of muscle fiber were mainly observed at Week 5. Food consumption was decreased up to Week 4 but recovered thereafter. Body weight decreased up to Week 5 and did not completely recover thereafter. A biphasic increase in serum cortisol was also observed at Weeks 2 and 5. Histopathology showed that muscle lesions were mainly composed of degeneration and atrophy of the muscle fibers, and ATPase staining revealed that the changes were more pronounced in type II muscle fiber than type I muscle fiber. In the pimonidazole experiment, mosaic pattern in skeletal muscle was demonstrated in the intact animal, but not the CIA animal. Increased arthritis score was accompanied by a decrease in serum creatinine, a marker that reflects muscle mass. CONCLUSIONS: Muscle wasting might exacerbate joint swelling in a collagen-induced arthritis model of cynomolgus monkeys.

Targeted delivery of immunogen to primate m cells with tetragalloyl lysine dendrimer.

Effective uptake of Ags by specialized M cells of gut-associated lymphoid tissues is an important step in inducing efficient immune responses after oral vaccination. Although stable nontoxic small molecule mimetics of lectins, such as synthetic multivalent polygalloyl derivatives, may have potential in murine M cell targeting, it remains unclear whether synthetic multivalent polygalloyl derivatives effectively target nonhuman and human M cells. In this study, we evaluated the ability of a tetragalloyl derivative, the tetragalloyl-D-lysine dendrimer (TGDK), to target M cells in both in vivo nonhuman primate and in vitro human M-like cell culture models. TGDK was efficiently transported from the lumen of the intestinal tract into rhesus Peyer's patches by M cells and then accumulated in germinal centers. Oral administration of rhesus CCR5-derived cyclopeptide conjugated with TGDK in rhesus macaque resulted in a statistically significant increase in stool IgA response against rhesus CCR5-derived cyclopeptide and induced a neutralizing activity against SIV infection. Furthermore, TGDK was specifically bound to human M-like cells and efficiently transcytosed from the apical side to the basolateral side in the M-like cell model. Thus, the TGDK-mediated vaccine delivery system represents a potential approach for enabling M cell-targeted mucosal vaccines in primates.

Genetic variants of CYP3A4 and CYP3A5 in cynomolgus and rhesus macaques.

Cynomolgus and rhesus macaques are frequently used in preclinical trials due to their close evolutionary relationships to humans. We conducted an initial screening for genetic variants in cynomolgus and rhesus macaque genes orthologous to human CYP3A4 and CYP3A5. Genetic screening of 78 Indochinese and Indonesian cynomolgus macaques and 34 Chinese rhesus macaques revealed a combined total of 42 CYP3A4 genetic variants, including 12 nonsynonymous variants, and 34 CYP3A5 genetic variants, including nine nonsynonymous variants. Four of these nonsynonymous variants were located at substrate recognition sites or the heme-binding region, domains essential for protein function, including c.886G>A (V296M) and c.1310G>A (S437N) in CYP3A4 and c.1437C>G (N479K) and c.1310G>C (T437S) in CYP3A5. The mutant proteins of these genetic variants were expressed in Escherichia coli and purified. Metabolic activity of these proteins measured using midazolam and nifedipine as substrates showed that none of these protein variants substantially influences the drug-metabolizing capacity of CYP3A4 or CYP3A5 protein. In Indonesian cynomolgus macaques, we also found IVS3+1delG in CYP3A4 and c.625A>T in CYP3A5, with which an intact protein cannot be produced due to a frameshift generated. Screening additional genomes revealed that two of 239 animals and three of 258 animals were heterozygous for IVS3+1delG of CYP3A4 and c.625A>T of CYP3A5, respectively. Some genetic variants were unevenly distributed between Indochinese and Indonesian cynomolgus macaques and between cynomolgus and rhesus macaques. Information on genetic diversity of macaque CYP3A4 and CYP3A5 presented here could be useful for successful drug metabolism studies conducted in macaques.

Effect of oral ketoconazole on oral and intravenous pharmacokinetics of simvastatin and its acid in cynomolgus monkeys.

Drugs with potential drug-drug interactions (DDIs) may have a limited scope of use and, at worst, may have to be withdrawn from the market. Therefore, during the drug discovery process it is important to select drug candidates with reduced potential for DDIs. In the present study, we evaluated the pharmacokinetics of simvastatin (SV), a typical substrate for cytochrome P450 (P450) 3A, and examined the DDI between SV and ketoconazole (KTZ), a P450 3A inhibitor, in monkeys. SV metabolism in monkey liver and intestinal microsomes was almost completely inhibited by addition of anti-P450 3A4 antiserum. A similar effect was seen in human microsomes, and the IC(50) values of KTZ for inhibition of SV metabolism were similar in monkey and human samples. In vivo, there were no significant differences in the pharmacokinetic parameters of SV and SVA after i.v. administration of SV in the presence of KTZ compared with those in controls, probably because of the limited systemic exposure to KTZ. In contrast, the pharmacokinetics of SV and SVA after p.o. administration of SV were significantly influenced by the presence of KTZ, and C(max) and area under the plasma concentration-time curve were approximately 5 to 10 times higher than those after p.o. dosing with SV alone. The increases in systemic SV exposure caused by a concomitant p.o. dose of KTZ in monkeys were similar to those observed in clinical studies, which suggests that monkeys might be a suitable animal model in which to predict DDIs involving P450 3A inhibition.

Extracellular high mobility group box chromosomal protein 1 is a coupling factor for hypoxia and inflammation in arthritis.

OBJECTIVE: Tissue hypoxia is closely associated with arthritis pathogenesis, and extracellular high mobility group box chromosomal protein 1 (HMGB-1) released from injured cells also has a role in arthritis development. This study was thus undertaken to investigate the hypothesis that extracellular HMGB-1 may be a coupling factor between hypoxia and inflammation in arthritis. METHODS: Concentrations of tumor necrosis factor alpha, interleukin-6, vascular endothelial growth factor, lactic acid, lactate dehydrogenase, and HMGB-1 were measured in synovial fluid (SF) samples from patients with inflammatory arthropathy (rheumatoid arthritis and pseudogout) and patients with noninflammatory arthropathy (osteoarthritis). The localization of tissue hypoxia and HMGB-1 was also examined in animal models of collagen-induced arthritis (CIA). In cell-based experiments, the effects of hypoxia on HMGB-1 release and its associated cellular events (i.e., protein distribution and cell viability) were studied. RESULTS: In SF samples from patients with HMGB-1-associated inflammatory arthropathy (i.e., samples with HMGB-1 levels >2 SD above the mean level in samples from patients with noninflammatory arthropathy), concentrations of HMGB-1 were significantly correlated with those of lactic acid, a marker of tissue hypoxia. In CIA models in which the pathologic phenotype could be attenuated by HMGB-1 neutralization, colocalization of HMGB-1 with tissue hypoxia in arthritis lesions was also observed. In cell-based experiments, hypoxia induced significantly increased levels of extracellular HMGB-1 by the cellular processes of secretion and/or apoptosis-associated release, which was much more prominent than the protein release in necrotic cell injury potentiated by oxidative stress. CONCLUSION: These findings indicate that tissue hypoxia and its resultant extracellular HMGB-1 might play an important role in the development of arthritis.

Nonhuman primate intestinal villous M-like cells: an effective poliovirus entry site.

Humans and some Old World monkeys, chimpanzees, and cynomolgus macaques, are susceptible to oral poliovirus (PV) infection. Interestingly, rhesus macaques, although sensitive to injected PV, are not susceptible to gut infection. Not much is known about the initial event of gut infection by PV in rhesus macaques so far. Here, we show that PV can efficiently enter the lamina propria (LP) by penetrating across intestinal villous M-like cells in rhesus macaques. We found by immunofluorescence analysis that PV effectively invades LP rather than germinal centers (GCs) in rhesus macaques despite expressing PV receptor CD155 on cells within GCs and LP. Furthermore, energy dispersive X-ray spectroscopy demonstrated that gold-labeled PV is spatiotemporally internalized into villous M-like cells and engulfed by macrophage-like cells in LP. These results suggest that rhesus macaques may be resistant to productive gut PV infection owing to a defective translocation of PV to GCs.

Clinical pathology data from cynomolgus monkeys from China in which diarrhea was observed during quarantine.

Shin Nippon Biomedical Laboratories, Ltd. (SNBL) imported and quarantined 3,148 cynomolgus monkeys (aged 2.5 to 6.5 years) from China in 2002. The hematology and blood biochemistry data obtained from these monkeys on Day 32 of quarantine were analyzed separately by sex [2,890 animals in which no abnormalities were observed during the 35-day quarantine period (normal group), and 258 animals which exhibited diarrhea 1 to 12 times (diarrhea group)]. The values obtained for all parameters were within the normal range (mean +/- SD), and no significant abnormalities were noted in either sex. The clinical pathology data from 11 animals (6 males and 5 females) exhibiting diarrhea repeatedly (10 to 12 times) were statistically analyzed, and significant differences were noted in PLT and ALP in both sexes. The PLT values of these animals were within the normal group mean +/- 2 SD, and were considered within the normal range. A significant difference was noted in some individual ALP values (males: Nos. 2 and 3, females: Nos. 1, 3, and 4). The clinical pathology data obtained from the normal group in this study basically correspond to the widely reported results already obtained from healthy cynomolgus monkeys, from which it can be concluded that the cynomolgus monkeys from China were generally healthy and presenting no particular abnormality. The clinical pathology data from the normal group will serve as valuable baseline data for experimenters using cynomolgus monkeys.

CYP2C76 deficiency is embryonic lethal in cynomolgus macaques: The potential role of CYP2C76 in early embryogenesis.

Cynomolgus macaques are an important primate species for drug metabolism studies; however cynomolgus CYP2C76, an important drug-metabolizing enzyme, accounts for drug metabolism differences to humans, so that CYP2C76-null animals might show drug-metabolizing properties more similar to humans. In this study, attempts were made to produce CYP2C76-null animals by assisted reproduction technology. Oocytes and sperm collected from the heterozygotes for the null allele (c.449TG > A) were subjected to intracytoplasmic sperm injection, and the embryos produced were cultured in vitro through the blastocyst stage. Preimplantation genetic diagnosis using a biopsied portion of the blastocyst revealed that none of the 32 blastocysts analyzed were homozygotes. In contrast, 2 of the 20 embryos analyzed were homozygotes at the 8-cell stage, indicating that CYP2C76-null embryos most likely stop developing between the 8-cell and blastocyst stage. By polymerase chain reaction, expression of CYP2C76 mRNA was detected in oocytes and blastocysts, but not in 2-, 4-, 8-, or 16/32-cell stage embryos. Metabolic assays showed that CYP2C76 metabolized progesterone. These results indicated that CYP2C76 null was likely embryonic lethal, suggesting its potential role during early embryogenesis in cynomolgus macaques.

Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: implications for cytochrome P450 genes.

Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (<1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.

Newly identified CYP2C93 is a functional enzyme in rhesus monkey, but not in cynomolgus monkey.

Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP) 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C93 cDNA contained an open reading frame of 490 amino acids approximately 84-86% identical to human CYP2Cs. CYP2C93 was located in the genomic region, which corresponded to the intergenic region in the human genome, indicating that CYP2C93 does not correspond to any human genes. CYP2C93 mRNA was expressed predominantly in the liver among 10 tissues analyzed. The CYP2C93 proteins heterologously expressed in Escherichia coli metabolized human CYP2C substrates, diclofenac, flurbiprofen, paclitaxel, S-mephenytoin, and tolbutamide. In addition to a normal transcript (SV1), an aberrantly spliced transcript (SV2) lacking exon 2 was identified, which did not give rise to a functional protein due to frameshift and a premature termination codon. Mini gene assay revealed that the genetic variant IVS2-1G>T at the splice site of intron 1, at least partly, accounted for the exon-2 skipping; therefore, this genotype would influence CYP2C93-mediated drug metabolism. SV1 was expressed in 6 of 11 rhesus monkeys and 1 of 8 cynomolgus monkeys, but the SV1 in the cynomolgus monkey was nonfunctional due to a rare null genotype (c.102T>del). These results suggest that CYP2C93 can play roles as a drug-metabolizing enzyme in rhesus monkeys (not in cynomolgus monkeys), although its relative contribution to drug metabolism has yet to be validated.

Characteristics of troponins as myocardial damage biomarkers in cynomolgus monkeys.

Recently, troponin T (TnT) and troponin I (TnI) have been reported as suitable biomarkers of myocardial injury for pre-clinical toxicity studies. The purpose of the present study was to investigate the characteristics of troponins as myocardial damage biomarkers in cynomolgus monkeys. Initially, tissue distribution of biomarkers was investigated in nine organs (including the heart, liver, and kidneys) collected from naive cynomolgus monkeys. The results showed that TnT and TnI were distributed specifically in the heart, and were not detected in other tissues. Secondly, changes in blood biomarker levels and histopathological changes in cardiac tissue were investigated following myocardial injury induced by concomitant administration of isoproterenol (ISO) and vasopressin (VASO). Compared with pre-dosing, TnT and TnI were markedly increased in the ISO + VASO groups, in which severe histopathological changes including necrosis and vacuolation of muscle fibers were observed. In order to investigate the relationship of biomarker levels with the severity of myocardial injury, Spearman's correlation coefficient was calculated between C(max) and AUC and necrosis and vacuolation scores in the heart. A high correlation between necrosis and vacuolation in the heart and TnT and TnI levels was noted. These results suggest that TnT and TnI possess high sensitivity and specificity for myocardial injury in cynomolgus monkeys, and are useful biomarkers for detection of drug-induced myocardial injury in cynomolgus 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.

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.