Cross-species comparison of the metabolism and excretion of selexipag

1. The metabolism of the prostacyclin receptor agonist selexipag (NS-304; ACT-293987) and its active metabolite MRE-269 (ACT-333679) has been investigated in liver microsomes and hepatocytes of rats, dogs, and monkeys. MRE-269 formation is the main pathway of selexipag metabolism, irrespective of species. Some interspecies differences were evident for both compounds in terms of both metabolic turnover and metabolic profiles. The metabolism of MRE-269 was slower than that of selexipag in all three species.

2. The metabolism of selexipag was also studied in bile-duct-cannulated rats and dogs after a single oral and intravenous dose of [¹⁴C]selexipag. MRE-269 acyl glucuronide was found in both rat and dog bile. Internal acyl migration reactions of MRE-269 glucuronide were identified in an experiment with the synthetic standard MRE-6001.

3. MRE-269 was the major component in the faeces of rats and dogs. In ex vivo study using rat and dog faeces, selexipag hydrolysis to MRE-269 by the intestinal microflora is considered to be a contributory factor in rats and dogs.

4. A taurine conjugate of MRE-269 was identified in rat bile sample. Overall, selexipag was eliminated via multiple routes in animals, including hydrolysis, oxidative metabolism, conjugation, intestinal deconjugation, and gut flora metabolism.     

mRNA levels of drug-metabolizing enzymes in 11 brain regions of cynomolgus macaques

The cynomolgus macaque is an important nonhuman primate species in drug metabolism studies, in part because of its evolutionary closeness to humans. Cytochromes P450 (P450s) have been investigated in the major drug-metabolizing organs, i.e., the liver and small intestine, but have not been fully investigated in the brain. However, recent investigations have indicated possible important roles for P450s in the brain. In this study, by using the quantitative polymerase chain reaction, we measured the mRNA levels of 38 cynomolgus drug-metabolizing enzymes, including 19 P450s, 10 UDP-glycosyltransferases, and 9 other enzymes, in 11 brain regions. Among these drug-metabolizing enzymes, expression of 32 enzyme mRNAs were detected in one or more brain regions, indicating their possible roles in the brain. Further investigation of metabolic activities would facilitate better understanding of the importance of these enzymes in the brain.     

Enhanced normograms and pregnancy outcome analysis in nonhuman primate developmental toxicity studies

The incidence of spontaneous pregnancy/infant losses is highly variable in long-tailed macaques (cynomolgus monkey), making it potentially difficult to ascertain test item-related effects in developmental toxicity studies. Therefore, pregnancy normograms had been developed by Jarvis et al. [1] to aid in the distinction of normal (e.g. test facility background) versus non-normal pregnancy outcomes. These normograms were mostly derived from embryo-fetal development studies and from PPND studies with a postnatal phase limited to seven days. However, the enhanced pre- and postnatal developmental (ePPND) study paradigm has essentially replaced these former study types. This work aims at providing enhanced normograms (e-normograms) in the context of regulatory ePPND studies. Survival functions for the prenatal phase (286 control pregnancies) and the postnatal phase (222 live infants) were estimated using the Kaplan-Meier estimator. Normograms were generated from survival curves and pseudo-study simulations. Data were available from two test facilities with comparable EU-compliant animal husbandry. Pregnancy duration/outcome as well as survival functions did not differ significantly between test facilities indicating that this husbandry system yields comparable developmental observations across different test facilities, at least in this NHP species. These novel e-normograms were developed for pregnant long-tailed macaques and provide an extended postnatal period up to three months, a new concept of separate normograms for the prenatal and the postnatal period, specific information on the perinatal phase events, a prediction of expected number of live infants for group size management, and the option to evaluate effects on pregnancy duration through distinction of live births and infant losses.     

Effects of early maternal care on adolescent attention bias to threat in nonhuman primates

Attention bias towards threat using dot-probe tasks has mainly been reported in adults with stress-related disorders such as PTSD and other anxiety disorders, in some cases associated with early life stress or traumatic experiences. Studies during adolescence are scarce and inconsistent, which highlights the need to increase our understanding of the developmental processes that predict attentional biases, given that this is a time of emergence of psychopathology. Here, we use a translational nonhuman primate model of early life stress in the form of infant maltreatment to examine its long-term impact on attentional biases during adolescence using the dot-probe task and identify interactions with early life risk factors, such as prenatal exposure to stress hormones and emotional/stress reactivity during infancy. Maltreated animals showed higher reaction times to social threat than animals that experienced competent maternal care, suggesting interference of negative valence stimuli on attentional control and cognitive processes. Higher emotional reactivity during infancy in Maltreated animals predicted attention bias towards threat, whereas higher levels of prenatal cortisol exposure was associated with bias away (avoidance of) threat in maltreated and control groups. Our findings suggest that different postnatal experiences and early biobehavioral mechanisms regulate the development of emotional attention biases during adolescence.     

Interleukin-1β and tumor necrosis factor-α affect cytochrome P450 expression in cynomolgus macaque hepatocytes

The cynomolgus macaque, partly due to its evolutionary closeness to humans, is an important nonhuman primate species used in drug metabolism studies. In humans, expressions of cytochromes P450 (P450s), including the important drug-metabolizing enzyme P450 3A4, are affected by various cytokines. However, this phenomenon has not been fully investigated in cynomolgus macaques. In this study, the effects of cytokines on P450 expression were investigated using the quantitative polymerase chain reaction to evaluate mRNA expression. Hepatocytes from cynomolgus macaques were treated with lipopolysaccharide and various cytokines, including interleukin (IL)-1β, IL-2, IL-6, interferon-γ, and tumor necrosis factor-α, and the expression levels of 11 P450s were compared with those of solvent-treated controls. Tumor necrosis factor-α significantly decreased cynomolgus P450 2C8 and 2C76 mRNA expression in multiple lots of cynomolgus hepatocytes investigated. IL-1β significantly decreased cynomolgus P450 1A1, 2C8, 2C19, and 2C76 mRNA expression, but increased P450 3A5 mRNA expression in multiple lots of hepatocytes. Moreover, P450 1A1-and 2C19-mediated drug oxidations were significantly and dose-dependently suppressed by IL-1β, under the present limited conditions. These results suggest that cytokines can influence hepatic P450 mRNA expression levels in cynomolgus macaques, just as cytokines are reported to affect P450 expression in humans.     

Modulation of local field potentials and neuronal activity in primate hippocampus during saccades

Primates use saccades to gather information about objects and their relative spatial arrangement, a process essential for visual perception and memory. It has been proposed that signals linked to saccades reset the phase of local field potential (LFP) oscillations in the hippocampus, providing a temporal window for visual signals to activate neurons in this region and influence memory formation. We investigated this issue by measuring hippocampal LFPs and spikes in two macaques performing different tasks with unconstrained eye movements. We found that LFP phase clustering (PC) in the alpha/beta (8–16 Hz) frequencies followed foveation onsets, while PC in frequencies lower than 8 Hz followed spontaneous saccades, even on a homogeneous background. Saccades to a solid grey background were not followed by increases in local neuronal firing, whereas saccades toward appearing visual stimuli were. Finally, saccade parameters correlated with LFPs phase and amplitude: saccade direction correlated with delta (≤4 Hz) phase, and saccade amplitude with theta (4–8 Hz) power. Our results suggest that signals linked to saccades reach the hippocampus, producing synchronization of delta/theta LFPs without a general activation of local neurons. Moreover, some visual inputs co‐occurring with saccades produce LFP synchronization in the alpha/beta bands and elevated neuronal firing. Our findings support the hypothesis that saccade‐related signals enact sensory input‐dependent plasticity and therefore memory formation in the primate hippocampus.     

Effects of social subordination and oestradiol on resting‐state amygdala functional connectivity in adult female rhesus monkeys

Preclinical studies demonstrate that chronic stress modulates the effects of oestradiol (E2) on behaviour through the modification of the amygdala and the medial prefrontal cortex (mPFC) neuronal structure. Clinical studies suggest that alterations in amygdala functional connectivity (FC) with the mPFC may be associated with stress‐related phenotypes, including mood and anxiety disorders. Thus, identifying the effects of stress and E2 on amygdala‐mPFC circuits is critical for understanding the neurobiology underpinning the vulnerability to stress‐related disorders in women. In the present study, we used a well‐validated rhesus monkey model of chronic psychosocial stress (subordinate social rank) to examine effects of E2 on subordinate (SUB) (i.e. high stress) and dominant (DOM) (i.e. low stress) female resting‐state amygdala FC with the mPFC and with the whole‐brain. In the non‐E2 treatment control condition, SUB was associated with stronger left amygdala FC to subgenual cingulate (Brodmann area [BA] 25: BA25), a region implicated in several psychopathologies in people. In SUB females, E2 treatment strengthened right amygdala‐BA25 FC, induced a net positive amygdala‐visual cortex FC that was positively associated with frequency of submissive behaviours, and weakened positive amygdala‐para/hippocampus FC. Our findings show that subordinate social rank alters amygdala FC and the impact of E2 on amygdala FC with BA25 and with regions involved in visual processing and memory encoding.     

Evidence in primates supporting the use of chemogenetics for the treatment of human refractory neuropsychiatric disorders

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Influence of conventional forceps extraction and extraction with an extrusion instrument on cementoblast loss and external root resorption of replanted monkey incisors

Cementoblast loss and root resorption on the root surface was registered histologically after removal and immediate replantation of maxillary central incisors in monkeys ( Cecropithecus aethiops ). Incisors were removed with either a conventional extraction technique using forceps or with a special designed extrusion instrument and in both cases teeth were immediately replanted. Altogether 18 monkeys were used for the experiments in which 9 monkeys were sacrificed after 3 d and 9 animals after 8 wk. Histological analysis was made in horizontal sections perpendicular to the long axis of the tooth using either 24 measuring points or 12 sections. Histometric analysis showed extensive cementoblast loss in the periodontal ligament (PDL) and with a maximum of damage occurring on the corner surfaces of the root. Extracted teeth showed significantly more extensive cementoblast loss than extruded teeth. Root resorption was also found in the corner locations and was significantly more frequent in extracted than in extruded teeth. There was a similarity in the distribution of cementoblast loss and root resorption indicating that root resorption develops in the same areas where cementoblast loss takes place, i.e. locations that are more compressed during removal of the tooth and must have caused by mechanical damage to the PDL exerted on the root surface during tooth removal.     

Sub-gingival microflora in Macaca mulatta species of rhesus monkey

The Macaca mulatta species of rhesus monkey is one of several non-human primate (nhp) models for periodontal disease. This report presents the bacteriology of the gingival sulci in M. mulatta monkeys. Three sub-gingival sites (maxillary right central incisor, the disto-buccal of the mandibular left second molar and mesio-buccal of the mandibular right second molar) of 9 monkeys were evaluated clinically before scaling and 7 days after scaling. Plaque samples were obtained from sub-gingival sites before clinical examination and studied bacteriologically by dark field microscopy, selective and non-selective culture, and by primary phenotypic characterizations of culture isolates. Several gingival sites presented with mild gingival inflammation. Anaerobic and facultatively anaerobic bacteria were the predominant flora colonizing the gingival sulci. The major microbial groups were Haemophilus species (100% of sites; percentage of total anaerobic count (TAC); 21-51), Peptostreptococcus micros (89%, 7.5–29.5), Actinomyces sp. (85%, 7–27), Fusobacterium nucleatum (90%, 5–8), Actinobacillus actinomycetemcomitans (73%, 1.3–12), black-pigmented anaerobic rods ( BPAR ) (80%, 0.6–6.5) and oral streptococci (80%, 0.2–1.0). Microbial groups detected less often were Wolinella sp. (66%, 0–2.6), Capnocytophaga sp. (30%), Eikenella corrodens (4.7%, 0), Campylobacter sp. (28%, 0–0.1) and spirochetes (4.7%, 0–0.07). Seven days after gingival sites were scaled, the plaque score and indices for gingival inflammation declined significantly. The gingival flora after scaling were characterized by lower proportions of the Actinomyees sp., P. micros and BPAR; and increased proportions of the oral streptococci, relative to pre-scaling levels. The major microbial groups at scaled gingival sites were the Haemophilus sp., oral streptococci, F. nucleatum and A. actinomycetemcomitans. The mutual proportions of microbial groups varied non-significantly within gingival sites and between monkeys. In conclusion, the M. mulatta gingival sulci are colonized by microbial species that resemble putative pathogens of periodontal disease, and the composition and character of the gingival flora are similar to the gingival flora of other Old World monkey models.