Female Cynomolgus Monkeys Research Articles

Preliminary investigation into long-term stress by isolated captivity-related changes of reproduction hormones in Cynomolgus monkey.

Stress profoundly affects physical and emotional well-being, extending its physiological influence to the female menstrual cycle, impeding the hypothalamus-pituitary-gonadal (HPG) axis, and affecting fertility by suppressing sex-stimulating hormones. In this study, we meticulously analyzed menstrual cycles and corresponding hormonal fluctuations in three female Cynomolgus monkeys. The preliminary findings indicated lower-than-normal levels of cortisol, follicle-stimulating hormone (FSH), and estradiol. Anovulatory bleeding occurred in one monkey, which could be linked to stress. In contrast to cortisol, alkaline phosphatase (ALP), which is correlated to cortisol levels, was consistently elevated in menstruating monkeys, suggesting its potential as a stress indicator. The non-menstruating group exhibited stress-related weight loss, emphasizing the observed ALP trends. Non-menstruating monkeys may experience more stress than menstruating monkeys. The implications of this study extend beyond the confines of primate studies and offer a valuable method for enhancing the welfare of female Cynomolgus monkeys.

Correlation analysis of positron emission tomography/computed tomography-magnetic resonance imaging of cannabinoid type 1 receptor in the lumbar spine and brain of aged osteoporosis female cynomolgus monkeys.

Although cannabinoid receptor 1 (CB1R) antagonists can inhibit bone loss in osteoporosis mouse models, different strains of mice show different bone mass phenotypes after knock out the CB1R gene. The relationship between CB1R and bone metabolism is complex, and its regulatory role in bone metabolism and as a therapeutic target for osteoporosis requires further investigation. Based on lumbar spine volumetric bone mineral density (vBMD) data of healthy female cynomolgus monkeys aged 1-25 years, naturally aged postmenopausal female osteoporotic monkeys and normal young monkeys were screened by detecting lumbar vertebrae vBMD and estradiol levels in this study. Positron emission tomography-computed tomography (PET/CT) and magnetic resonance imaging (MRI) scans were performed on the lumbar spine and brain of the two groups of monkeys using the probe [11C]OMAR, which specifically targets CB1R, and the difference in the CB1R expression of osteoporotic monkeys was evaluated. The vBMD values of two standard deviations (SDs) below the peak bone value (428.1±53.8 g/cm3) were set as the reference standard for osteoporosis vBMD. Of the 49 healthy female cynomolgus monkeys, 4 postmenopausal older osteoporotic monkeys (18-26 years) and 5 young control monkeys (6-7 years) were selected, and the mean vBMD of the lumbar spine of the two groups was 295.07±19.11 and 419.72±16.14 g/cm3, respectively (P<0.0001). Radioactive uptake in the lumbar spine was linearly and negatively correlated with vBMD (r=-0.7977; P=0.01). Dynamic PET/MR imaging of the brains showed that CB1R was upregulated in the osteoporosis group, and there was a negative linear correlation between the vBMD and area under the time-radioactivity curve (AUC) of the thalamus (r=-0.8506; P=0.0153) and prefrontal cortex (r=-0.8306; P=0.0207). In this study, PET/CT-MRI molecular imaging technology revealed that CB1R was upregulated in the lumbar spine and brain of the osteoporosis monkeys and that CB1R may be regulated by the brain-bone axis. CB1R antagonist may be a potential drug for the treatment of osteoporosis.

Anti-Hemojuvelin Monoclonal Antibody DISC-0974 Elicits a Durable and Consistent Response with Repeated Dosing in Cynomolgus Monkeys

Introduction DISC-0974 is a monoclonal antibody (mAB) targeting hemojuvelin and is currently in clinical trials evaluating effects on anemia in patients with myelofibrosis or chronic kidney disease. Inhibition of hemojuvelin (HJV) results in decreased hepcidin and reduction in ferroportin degradation, enabling more iron to be exported out of cells and into the bloodstream for transport to other cells and tissues. Methods A study was conducted in healthy, naïve cynomolgus monkeys to evaluate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 following once-monthly dosing for 6 months. Male and female cynomolgus monkeys (n=6/sex/dose group) were intravenously administered DISC-0974 at doses of 0, 0.3, 1, or 6 mg/kg on Days 1, 29, 57, 85, 113, and 141. An additional IV dose was administered on Day 165 for males and Day 166 for females. Dose levels were selected where the high dose would maximally mobilize iron for sustained periods over the treatment regimen, with lower doses selected to provide a dose-response. Male and female monkeys (n=4/sex/group) were euthanized on Day 168 and 169, respectively, and subgroups were monitored for an additional 6 months (n=2/sex/group) to evaluate reversibility of any effects. PK was evaluated after the 1 st, 3 rd, and 6 th dose in all monkeys, as well as during the 6-month period after the final dose in the remaining monkeys. PD effects (i.e., serum iron and transferrin saturation [TSAT]) were monitored at the same timepoints as those collected for PK. Results DISC-0974 was well tolerated, and no DISC-0974-related adverse effects were observed. Systemic exposure, based on area under the curve (AUC) and maximum plasma concentrations (C max), increased with dose. Systemic exposures were achieved that reached and exceeded those observed in clinical trials. The high dose of 6 mg/kg achieved systemic exposures, based on AUC, that were &amp;gt;15-fold those observed in humans. Half-lives were consistent with an mAb, and repeated exposure did not result in accumulation with monthly dosing. Increases in serum iron (up to approximately 3-fold baseline) (Figure 1) and TSAT (&amp;gt;90%), representative of PD activity, correlated with systemic exposures of DISC-0974, with similar activity maintained after each of the 7 monthly doses. Conclusion Overall, these data demonstrate that DISC-0974 is well tolerated after repeated monthly exposure in cynomolgus monkeys, and an appropriate and durable PK/PD profile was observed, supporting clinical development of chronic once-monthly dosing.

Characterizing bacterial and fungal communities along the longitudinal axis of the intestine in cynomolgus monkeys

ABSTRACT The gut microbiota develops specialized local communities along the gastrointestinal (GI) tract of the host. Among these microbes, intestinal bacteria and fungi interact closely with the host and play vital roles in their health. However, the gut biogeography of bacteria and fungi has rarely been studied simultaneously, and their correlations have not been reported. In this study, we collected the intestinal luminal contents from different gastrointestinal regions (the ileum, caecum, and colon) of wild-type female cynomolgus monkeys and performed 16S and 18S rRNA gene sequencing to determine the gut biogeography of bacterial and fungal communities. The richness and diversity of bacteria and fungi increased gradually from the small to the large intestine. The bacterial and fungal composition of ileum differed significantly from that of the colon. Specifically, Bacteroidia and Spirochaetia were abundant in the caecum and colon, while Actinobacteria and Cyanobacteria were enriched in the ileum. As for fungal taxa, the genera Aspergillus , Wallemia , and Cryptococcus_f_Tremellaceae were enriched in the colon, while the genus Fusarium was abundant in the caecum. Consequently, the bacterial and fungal functions of the ileum differed from those of the caecum and colon. Notably, we characterized the correlations between intestinal bacteria and fungi at different gastrointestinal regions of cynomolgus monkeys using Spearman correlation analysis. In summary, our results contribute to a more comprehensive understanding of the composition and function of intestinal bacteria and fungi in non-human primates (NHPs) and reveal their correlations along the longitudinal axis of the intestine. IMPORTANCE Gut microbiota varies along the gastrointestinal (GI) tract and exerts profound influences on the host’s physiology, immunity, and nutrition. Given that gut microbes interact with the host closely and the gastrointestinal function differed from the small to the large intestine, it is essential to characterize the gut biogeography of the microbial community. Here, we focused on intestinal bacteria and fungi in cynomolgus monkeys and determined their spatial distribution along the GI tract by performing 16S and 18S rRNA gene sequencing. The composition and function of bacterial and fungal communities differed significantly at different biogeographic sites of the intestine, and the site-specific correlations between intestinal bacteria and fungi were revealed. Thus, our studies characterized the gut biogeography of bacteria and fungi in NHPs and revealed their site-specific correlations along the GI tract.

PET imaging of dopamine transporters and D2/D3 receptors in female monkeys: effects of chronic cocaine self-administration.

Brain imaging studies using positron emission tomography (PET) have shown that long-term cocaine use is associated with lower levels of dopamine (DA) D2/D3 receptors (D2/D3R); less consistent are the effects on DA transporter (DAT) availability. However, most studies have been conducted in male subjects (humans, monkeys, rodents). In this study, we used PET imaging in nine drug-naïve female cynomolgus monkeys to determine if baseline measures of DAT, with [18F]FECNT, and D2/D3R availability, with [11C]raclopride, in the caudate nucleus, putamen and ventral striatum were associated with rates of cocaine self-administration and if these measures changed during long-term (~13 months) cocaine self-administration and following time-off (3-9 months) from cocaine. Cocaine (0.2 mg/kg/injection) and 1.0 g food pellets were available under a multiplefixed-interval (FI) 3-min schedule of reinforcement. In contrast to what has been observed in male monkeys, baseline D2/D3R availability was positively correlated with rates of cocaine self-administration only during the first week of exposure; DAT availability did not correlate with cocaine self-administration. D2/D3R availability decreased ~20% following cumulative intakes of 100 and 1000 mg/kg cocaine; DAT availability did not significantly change. These reductions in D2/D3R availability did not recover over 9 months of time-off from cocaine. To determine if these reductions were reversible, three monkeys were implanted with osmotic pumps that delivered raclopride for 30 days.We found that chronic treatment with the D2/D3R antagonist raclopride increased D2/D3R availability in the ventral striatum but not in the other regions when compared to baseline levels. Over 13 months of self-administration, tolerance did not develop to the rate-decreasing effects of self-administered cocaine on food-reinforced responding, but number of injections and cocaine intake significantly increased over the 13 months. These data extend previous findings to female monkeys and suggest sex differences in the relationship between D2/D3R availability related to vulnerability and long-term cocaine use.

Sexual Dimorphic Rightward Lateralization of Cerebral Sulcal Infolding in Cynomolgus Monkeys (Macaca fascicularis)

Cerebral sulcal infolding exhibits unique species-related lateralization patterns. The present investigation aimed to characterize the asymmetric patterns of sulcal infolding in cynomolgus monkeys and their sexual dimorphism. Three-dimensional magnetic resonance (MR) images were acquired at 7-Tesla from the fixed brains of adult male (n = 5) and female (n = 5) monkeys. The gyrification index (GI) was estimated on MR images throughout the cerebral cortex (global-GI) or in the representative primary sulci (sulcal-GI). The global-GI did not differ between the sexes when the ipsilateral sides were compared. Although there was no sex difference in the sulcal-GI of the ipsilateral sides of any primary sulci, a significant right bias of the sulcal-GI was noted in the inferior rams of the arcuate sulcus and circular sulcus in males but not in females. Secondary sulcal emergence was examined to assess sulcal infolding asymmetry at the individual and population levels. Nonbiased asymmetric emergence was noted in the posterior supraprincipal dimple in both sexes and the rostral sulcus in females. Notably, the emergence of the superior postcentral dimple was significantly right-lateralized in females. The findings revealed right-biased sulcal infolding in male and female cynomolgus monkeys, although the lateralized cortical regions differed between the sexes.

Safety and Osteointegration of Titanium Screws Coated with a Fibroblast Growth Factor-2-Calcium Phosphate Composite Layer in Non-Human Primates: A Pilot Study.

Spinal instrumentation surgery for older patients with osteoporosis is increasing. Implant loosening may occur due to inappropriate fixation in osteoporotic bone. Developing implants that achieve stable surgical results, even in osteoporotic bone, can reduce re-operation, lower medical costs, and maintain the physical status of older patients. Fibroblast growth factor-2 (FGF-2) promotes bone formation; thus, coating pedicle screws with an FGF-2-calcium phosphate (FGF-CP) composite layer is hypothesized to enhance osteointegration in spinal implants. We designed a long-term implantation pilot study that estimated the safety and bone-forming efficacy of pedicle screws coated with an FGF-CP composite layer in cynomolgus monkeys. Titanium alloy screws, either uncoated (controls) or aseptically coated with an FGF-CP composite layer, were implanted in the vertebral bodies of six female adult cynomolgus monkeys (three monkeys per group) for 85 days. Physiological, histological, and radiographic investigations were performed. There were no serious adverse events, and no radiolucent areas were observed around the screws in either group. The bone apposition rate in the intraosseous region was significantly higher in the FGF-CP group than in the controls. Moreover, as analyzed by Weibull plots, the bone formation rate of the FGF-CP group exhibited a significantly higher regression line slope than the control group. These results demonstrated that there was significantly less risk of impaired osteointegration in the FGF-CP group. Our pilot study suggests that FGF-CP-coated implants could promote osteointegration, be safe, and reduce the probability of screw loosening.

Effects of selective dopamine D3 receptor partial agonist/antagonists on oxycodone self-administration and antinociception in monkeys.

Recent studies suggest that dopamine D3 receptors (D3R) may be a therapeutic target for opioid use disorders (OUD). This study examined the effects of the D3Rpartial agonist (±)VK4-40 and the D3R-selective antagonist (±)VK4-116, compared to the mu-opioid receptor antagonist naltrexone (NTX), in nonhuman primate models of OUD and antinociception. Adult male and female (N = 4/sex) cynomolgus monkeys were trained to self-administer oxycodone (0.003-0.1 mg/kg/injection) first under a fixed-ratio (FR) and then a progressive-ratio (PR) schedule of reinforcement during daily 1- and 4-hr sessions, respectively. Under the FR schedule, intravenous NTX (0.01-0.1 mg/kg), (±)VK4-116 (1.0-10 mg/kg), and (±)VK4-40 (1.0-10 mg/kg) were studied in combination with the peak oxycodone dose and a dose on the descending limb of the dose-effect curve; NTX and (±)VK4-40 were also studied at the peak of the PR dose-response curve (N = 4). Following saline extinction, each compound was examined on oxycodone-induced reinstatement. Finally, these compounds were assessed in adult male rhesus monkeys (N = 3) in a warm-water (38 °C, 50 °C, 54 °C) tail withdrawal assay. NTX decreased responding on the peak of the FR oxycodone dose-response curve, but increased responding on the descending limb. (±)VK4-40, but not (±)VK4-116, significantly decreased peak oxycodone self-administration; (±)VK4-40 did not increase responding on the descending limb. NTX and (±)VK4-40, but not (±)VK4-116, attenuated oxycodone-induced reinstatement. Under PR responding, NTX and (±)VK4-40 decreased breakpoints. Oxycodone-induced antinociception was attenuated by NTX, but not by (±)VK4-40 or (±)VK4-116. Together, these results suggest that further research evaluating the effects of (±)VK4-40 as a novel pharmacotherapy for OUD is warranted.

Abstract 2793: Preclinical pharmacokinetics in cynomolgus monkeys and first in human dose prediction of DB-1303, a HER2-targeting antibody-drug conjugate

Abstract Background: DB-1303 is a novel antibody-drug conjugate comprised of trastuzumab biosimilar, enzymatically cleavable peptide-linker, and a proprietary topoisomerase I inhibitor P1003. It is designed to have high plasma stability, low free payload in circulation and wide therapeutic index. Herein, the preclinical pharmacokinetics (PK) characteristics of DB-1303 in cynomolgus monkeys is reported, and human PK exposure in a first in human study was predicted using population PK model with allometric scaling. Material and Methods: The PK parameters of DB-1303 were obtained from single PK and repeated TK studies in female and male cynomolgus monkeys. Concentrations of DB-1303 ADC, total antibody and released payload P1003 were measured using ELISA or LC-MS/MS. In a PK study, single dose of DB-1303 at 1, 3 and 10 mg/kg were determined in cynomolgus monkeys via IV infusion. In TK studies, cynomolgus monkeys were intravenously administered with DB-1303 at 50, 80 mg/kg once every 3 weeks for 2 repeated doses, or 10, 40, 80 mg/kg once every 3 weeks (Q3W) for 3 repeated doses followed by a 4-week recovery. A population PK model was developed based on the collective PK/TK data in monkeys. Results: The non-compartment analysis PK parameters of three analytes (DB-1303 ADC, total antibody and released payload P1003) are summarized following single and multiple doses of DB-1303 in cynomolgus monkeys. To predict the human PK for DB-1303, a target mediated drug disposition (TMDD) model was constructed, incorporating binding to serum HER2 and subsequent elimination of the complex into a standard 2-compartmental PK model. The 2-compartment linear parameters were scaled from cynomolgus monkey population PK parameters using allometric scaling exponents of 1 for volumes and 1 for clearance. The binding of DB-1303 to HER2 was incorporated into the model and assumed to be the same for HER2 ECD and transmembrane domain. The PK profiles for DB-1303 in human at the proposed doses in the first in human study are predicted. The safety margins from the monkey HNSTD for each proposed human dose are calculated. Conclusions: DB-1303 exhibited favorable PK characteristics of DB-1303 in cynomolgus monkeys, and is predicted to have large safety margins at the selected doses in the first in human study. At the time of presentation, the first-in-human phase 1/2 study in patients with advanced solid tumors is in progress (NCT05150691). Citation Format: Rong Shi, Shengchao Lin, Yu Zhang, Bing Li, Haiqing Hua, Yang Qiu. Preclinical pharmacokinetics in cynomolgus monkeys and first in human dose prediction of DB-1303, a HER2-targeting antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2793.

Age-related changes in lumbar bone mineral density measured using quantitative computed tomography in healthy female cynomolgus monkeys.

Cynomolgus monkeys are widely used in studies related to osteoporosis, and there is no evidence of age-related changes in volumetric bone mineral density (vBMD) measured using quantitative computed tomography (QCT) in nonhuman primates. This study aimed to describe changes in the characteristics of lumbar vBMD with age, to analyze the relationship between lumbar vBMD and body composition, and to investigate the precision of QCT measurements in healthy female cynomolgus monkeys. Age-related changes in lumbar vBMD were described using cubic regression models, and the accumulated bone loss rates (ABLR) of the lumbar spine were calculated. Spearman rank correlation and ridge regression analysis were used to investigate the relationship of the average lumbar vBMD and body components. Thirty animals were selected to analyze the short-term in vivo precision of the QCT measurements. The precision was expressed as the root-mean-square coefficient of variation (RMS-CV%) or root-mean-square standard deviation (RMS-SD). A total of 72 healthy female cynomolgus monkeys, aged 1-25 years, were included in this study. The average lumbar vBMD of female cynomolgus monkeys increased with age until the age of 10 years, around which it reached peak bone mass, with a relatively marked decline after the age of 13 years. The ABLRs of female cynomolgus monkeys in the premenopausal (13-19 years) and postmenopausal age groups (20-25 years) were -4.9% and -21.2%, respectively. Ridge regression analysis showed that age and subcutaneous adipose tissue (SAT) contributed positively to the average lumbar vBMD in animals aged ≤10 years, whereas in animals aged >10 years, age contributed negatively to lumbar vBMD. The RMS-CV% (RMS-SD) of the lumbar vBMD measured using QCT ranged from 0.47% to 1.60% (1.91-6.31 mg/cm3). Age-related changes in lumbar vBMD measured using QCT in healthy female monkeys showed similar trends to those in humans. Age and SAT may affect the lumbar vBMD in female cynomolgus monkeys. QCT revealed good precision in measuring the lumbar vBMD in female cynomolgus monkeys.

Effects of Age and Multiple Vaginal Births on Lower Urinary Tract Structure and Function in Nonhuman Primates.

The relative roles of urinary sphincter damage, aging, and childbirth in stress urinary incontinence (SUI), have not been established. This study was performed to elucidate the roles of these factors. The study included: (1) 8 female cynomolgus monkeys (17-19 years of age and 7-8 vaginal births each); (2) six 5-yearold nulliparous monkeys with surgically created chronic urinary sphincter dysfunction; and (3) six 5-year-old, nulliparous, nosurgery controls. Sedated abdominal leak point pressure (ALPP) and maximum urethral sphincter pressures (MUP) were measured. Sphincters, bladders, and pelvic support muscles were quantified for collagen content. Additionally, bladders were analyzed for collagen fiber thickness, length, and angle using CT-FIRE analysis of Picrosirius red-stained tissues. Resting MUP values were similar in the controls and older multiparous monkeys (P>0.05). However, aging and multiple births reduced pudendal nerve-stimulated increases in MUP (P<0.05 vs. controls). ALPP values were lower in the older multiparous versus younger groups of monkeys (P<0.05). Sphincter collagen content was greater, and muscle content less, in the injury model (P<0.05 vs. controls). However, these measures were not affected by age and childbirth (P>0.05 vs. young groups). Bladder collagen content was greater, and muscle content less, in the old multiparous monkeys (P<0.05 vs. younger groups). Additionally, collagen fibers were thicker and more angular in the bladders of the older multiparous monkeys than in the other nonhuman primate groups (P<0.05). Pelvic support muscles had higher collagen and lower muscle content in the older multiparous monkeys than in the younger groups of monkeys (P<0.05). SUI, associated with aging and multiple childbirths, appeared to be more strongly associated with bladder dysfunction, reduced pelvic muscle support, and the compensatory response to neural stimulation than with selective urinary sphincter dysfunction.

Delay discounting as a behavioral phenotype associated with social rank in female and male cynomolgus monkeys: Correlation with kappa opioid receptor availability

Cocaine use disorder (CUD) is a significant problem worldwide, with no FDA-approved treatments. Epidemiological data indicate that only about 17 % of people that use cocaine will meet DSM criteria for CUD. Thus, the identification of biomarkers predictive of eventual cocaine use may be of great value. Two potentially useful predictors of CUD are social hierarchies in nonhuman primates and delay discounting. Both social rank and preference for a smaller, immediate reinforcer relative to a larger, delayed reinforcer have been predictive of CUD. Therefore, we wanted to determine if there was also a relationship between these two predictors of CUD. In the present study, monkeys cocaine-naive responded under a concurrent schedule of 1- vs. 3-food pellets and delivery of the 3-pellet option was delayed. The primary dependent variable was the indifference point (IP), which is the delay that results in 50 % choice for both options. In the initial determination of IP, there were no differences based on sex or social rank of the monkeys. When the delays were redetermined after ~25 baseline sessions (range 5–128 sessions), dominant females and subordinate males showed the largest increases in IP scores from the first determination to the second. Because 13 of these monkeys had prior PET scans of the kappa opioid receptor (KOR), we examined the relationship between KOR availability and IP values and found that the change in IP scores from the first to the second determination significantly negatively predicted average KOR availability in most brain regions. Future studies will examine acquisition to cocaine self-administration in these same monkeys, to determine if IP values are predictive of vulnerability to cocaine reinforcement.

Spatially resolved gene regulatory and disease-related vulnerability map of the adult Macaque cortex

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell transcriptomic and chromatin accessibility (single-cell ATAC) data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult female cynomolgus monkey brain, and integrated this dataset with Stereo-seq (spatial enhanced resolution omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

The Histology of Background Pathological Processes in Cynomolgus Monkeys (Macaca fascicularis) from Control Groups in Toxicology Studies

Histological samples of organs obtained from 72 (35 male and 37 female) cynomolgus monkeys (M. fascicularis) from negative control groups in 12 toxicological studies were analyzed retrospectively. The most common processes where focal accumulations of exogenous pigment were found in 76.4% of animals in the lungs, non-atrophic chronic gastritis, which was observed in 37.4% of animals, focal chronic quiescent colitis was noted in 36.1% of cases, infiltrate in the liver were observed in 33.3% of animals, focal chronic quiescent gastritis was identified in 30.5% of animals, lymphoplasmacytic infiltrates in the kidneys were found in 26.4% of animals. Identification and accumulation of information on background pathology findings in primates from control groups are essential since these data allow differentiating spontaneous pathology from abnormalities associated with administration of a drug test, to correctly interpret the results of toxicology studies.

Analysis of serum bone turnover markers in female cynomolgus monkeys of different ages.

ObjectiveThe purpose of this study was to examine bone turnover markers, estradiol, parathyroid hormone, and 25 hydroxyvitamin D, in cynomolgus monkeys at different ages to improve our understanding of the changes in bone turnover markers throughout the life cycle of cynomolgus monkeys and to provide a basis for the establishment of a non-human primate model of osteoporosis.MethodsTotal Body Bone Mineral Density and Total Body Bone Mineral Content were measured using Dual-Energy X-Ray Absorptiometry in cynomolgus monkeys at different ages. Serum bone turnover marker' levels were measured using enzyme immunoassays at each age group, and the relationship between bone turnover markers and age was assessed by Spearman rank correlation analysis to investigate the relationship between bone turnover markers and age in female cynomolgus monkeys.ResultsTotal Body Bone Mineral Density in female cynomolgus monkeys peaked at 10 years of age and then formed a plateau that was maintained until old age. Procollagen I Aminoterminal Propeptide, Bone Alkaline Phosphatase, Osteocalcin, and C-Terminal Telopeptide Of Type I Collagen peaked at 1 to 3 years of age and gradually decreased with age, leveling off by 10 years of age. Estradiol, parathyroid hormone, and 25 hydroxyvitamin D, follicle-stimulating hormone, luteinizing hormone, were not significantly different among age groups.ConclusionThis paper provides data on trends in bone turnover markers throughout the life cycle of female cynomolgus monkeys, which are similar to human changes.

A safety and pharmacodynamic study of the highly selective aldosterone synthase inhibitor PB6440 in the cynomolgus monkey

Abstract Background Aldosterone is an important mediator of hypertension, particularly resistant hypertension, heart failure, and chronic kidney disease. PB6440 is a potent inhibitor of aldosterone synthase (CYP11B2) with high selectivity over the closely related enzyme CYP11B1. In previous studies in cynomolgus monkeys, PB6440 exhibited excellent oral bioavailability and a marked suppression of aldosterone synthesis at doses as low as 1 mg/kg/day. Importantly, no effect was observed on cortisol production nor significant changes noted in plasma concentrations of the steroid precursors 11-deoxycortisol or deoxycorticosterone (DOC), which are dependent on CYP11B1 activity. The purpose of the current study was to assess the safety and pharmacodynamics of higher doses of PB6440 in the cynomolgus monkey in order to determine a therapeutic index prior to initiation of studies in humans. Methods Male and female cynomolgus monkeys (2/sex/dose group) were administered 0, 10, 30 or 100 mg/kg PB6440 once-daily for 14 days by oral gavage. Assessment of safety was based on mortality, clinical observations, body weights, and clinical and anatomic pathology. Blood samples were also collected for pharmacodynamic analysis, including aldosterone, cortisol, 11-deoxycortisol, DOC, and ACTH. Results PB6440 was well tolerated at all doses. There were no deaths and no reports of clinical observations at any dose level. Mild decreases in body weight were observed in PB6440-treated animals, which were likely due to the diuretic effects of the compound. PB6440 caused an increase in adrenal weights which was associated with hypertrophy of the adrenal zona fasciculata, a finding which has previously been observed with other aldosterone synthase inhibitors. PB6440 led to marked declines in basal aldosterone levels of −97%, −97% and −98% from baseline at 10, 30 and 100 mg/kg/day, respectively, at 24-hours following the final dose. Despite these clear declines in circulating aldosterone levels, no meaningful changes in cortisol, 11-deoxycortisol, DOC, or ACTH levels were observed, supporting lack of CYP11B1 inhibition. PB6440 led to expected decreases in plasma sodium (up to 7 nmol/L at the high dose). No effect on plasma potassium levels were observed at any dose level. Conclusions PB6440 was well tolerated in the cynomolgus monkey at doses that are much higher than needed for potent aldosterone suppression based on previous studies. At the highest dose level tested (100 mg/kg), which led to a 98% reduction in basal aldosterone levels, no evidence of significant CYP11B1 inhibition was observed. The absence of an increase in plasma potassium in the presence of a plasma sodium decline merits further study. Thus, PB6440 is a highly selective novel aldosterone synthase inhibitor for the potential treatment of hypertension, heart failure, and chronic kidney disease in humans. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): PhaseBio Pharmaceuticals Inc.

Lymphocyte subsets and immunoglobulin levels in peripheral blood from cynomolgus monkeys (Macaca fascicularis) of different age groups.

Immunotoxicity assessment is an important part of non-clinical safety evaluation of biotechnology-derived pharmaceuticals. The reference ranges of evaluated parameters, which depend on the sex, age and geographical origin of animals, play a significant role in interpreting the study results. The aim of this study was to determine the reference ranges of parameters commonly used for non-clinical immunotoxicity studies in cynomolgus monkeys (Macaca fascicularis) of different ages. The percentages of CD3+, CD4+, CD8+, CD20+, CD16/56+ lymphocytes, and the serum levels of immunoglobulins A, M, G and E in clinically healthy cynomolgus monkeys of both sexes and different ages (37-104 months) were analysed. The lymphocyte subsets were assessed by flow cytometry and serum immunoglobulin levels were determined by enzyme-linked immunosorbent assay. Statistical analysis showed a weak rank correlation of CD3+, CD20+ lymphocyte levels with the ages of female and male cynomolgus monkeys, and no significant correlation with age for the combined data of both sexes. The serum levels of immunoglobulins A, M, G and E did not have a significant rank correlation with age for the combined data of both sexes, whereas IgA levels in females and IgG levels in males were weakly correlated with age. Overall, these results justify the use of animals aged three to eight years for non-clinical immunotoxicity evaluation of biotechnology-derived pharmaceuticals. It is important to avoid the formation of animal groups with extreme ages (three years or eight years) in one group during randomization.

Abstract 5589: BYON4228 is a pan-allelic SIRPα antibody that potentiates killing of antibody-opsonized tumor cells and lacks binding to T cells

Abstract Preclinical data have established CD47-SIRPα interactions as a myeloid immune checkpoint in cancer, which is corroborated by preliminary evidence from the first clinical studies with CD47 blockers. However, the ubiquitously expressed CD47 mediates functional interactions with other ligands as well, and therefore targeting of the primarily myeloid cell-restricted inhibitory immunoreceptor SIRPα may represent a better strategy. Here, we present preclinical results on a novel clinical candidate, BYON4228. BYON4228 is an antibody directed against SIRPα and recognizes both of the common allelic variants of human SIRPα which maximizes its potential clinical application in the broad human population. Notably, in contrast to several other anti-SIRPα antibodies in development, BYON4228 does not recognize the closely related T cell-expressed SIRPγ that has been reported to mediate interactions with CD47 as well, which are known to be instrumental in T cell extravasation and activation. BYON4228 binds to the N-terminal part of SIRPα and its epitope overlaps with the CD47-binding site. BYON4228 therefore prevents binding of CD47 to SIRPα and thus blocks inhibitory signaling through the CD47-SIRPα axis. Functional studies show that BYON4228 potentiates both macrophage- and neutrophil-mediated elimination of hematologic and solid cancer cells in vitro in the presence of several different tumor targeting antibodies like trastuzumab, rituximab, daratumumab and cetuximab, illustrating the broad potential clinical benefit and application of BYON4228. Single intravenous infusion of up to 100 mg/kg BYON4228 to male and female cynomolgus monkeys was well tolerated and did not elicit any adverse effects. Collectively, this defines BYON4228 as a pan-allelic anti-SIRPα antibody without T cell SIRPγ recognition that promotes the destruction of antibody-opsonized cancer cells. Clinical studies are planned to start in 2022. Citation Format: Mary van Helden, Seline Zwarthoff, Hugo Olsman, Marc Paradé, Ellen Mattaar, Karin de Laat, Imke Lodewijks, Paul Boersema, Lilian Driessen-Engels, Jorien Sanderink, Wendy Kappers, Diels van den Dobbelsteen, Ruud Ubink, Roel Arends, Gerard Rouwendal, Gijs Verheijden, Miranda van der Lee, Wim Dokter, Timo Van den Berg. BYON4228 is a pan-allelic SIRPα antibody that potentiates killing of antibody-opsonized tumor cells and lacks binding to T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5589.

Abstract 3431: Dose range finding study in non-human primates confirms the unique mechanism of action of CTX-8371, a novel bispecific antibody blocking PD-1 and PD-L1

Abstract Multiple antibodies blocking PD-1 or PD-L1 have been approved for clinical use. However, not all patients successfully respond to monotherapy and the need for combinations has become more apparent. CTX-8371 combines PD-1/PD-L1 targeting in one bispecific, tetravalent molecule. CTX-8371 potently blocks PD-1/PD-L1 in vitro and in vivo. This robust activity may be explained by the unique mechanism of action (MOA) of CTX-8371 which involves cell surface PD-1 cleavage. Here we present further in vivo evidence of PD-1 cleavage by CTX-8371 detected during a dose range finding (DRF) study in cynomolgus macaques. Results of this study as well as in vitro receptor occupancy (RO) and the PK/PD profile of CTX-8371 in tumor-bearing mice are described. CTX-8371 was administered to female cynomolgus monkeys (n=3/group) in a single intravenous infusion at 2 mg/kg or weekly infusions at 10 or 50 mg/kg, for a total of 2 doses. PBMCs were isolated before, 24 hours after the first dose, or 24 hours after the second dose (for 10 and 50 mg/kg groups). Immunophenotyping, including PD-1 and PD-L1, was performed on PBMCs by flow cytometry. Mean serum concentration-time data was analyzed to derive PK parameters using noncompartmental and two-compartment models. PK was also determined in tumor-bearing mice using an MSD ELISA-based assay and non-compartmental analysis. In monkeys, CTX-8371 administration resulted in a marked decrease of PD-1+CD4+ and PD-1+CD8+ T cells in PBMCs on days 2 and 9 at all dose levels, indicating PD-1 cleavage from the cell surface. These results confirm in vitro, and in vivo MOA data obtained from mouse studies. Clearance and half-life of CTX-8371 were calculated to be ~6.87 mL/day/kg and 5.45 days, respectively, which fall within the expected ranges for a human IgG1 antibody in non-human primates (NHP) with linear PK. CTX-8371 infusion was well tolerated in cynomolgus monkeys. In tumor-bearing mice, the dose which results in 50% of maximum tumor growth inhibition was found to be 1-2 mg/kg. The minimum effective dose, at which at least 50% of animals exhibit greater than 50% tumor growth inhibition over two consecutive days, was 1mg/kg. It was observed that 11% (1/9), 78% (7/9) and 100% (9/9) of mice in 0.1 mg/kg, 1 mg/kg and 10 mg/kg dose groups, respectively, meet the above criteria. CTX-8371 also showed dose-dependent in vitro RO on T cells from peripheral blood of human, cynomolgus monkey, and transgenic hPD-1hPD-L1 mice. Data presented here confirm the unique MOA of CTX-8371, cleavage of PD-1 from the cell surface. The dual blockade of PD-1 and PD-L1, combined with PD-1 cleavage, may offer superior benefit compared to current checkpoint inhibitor therapies. RO data as well as murine and NHP PK/PD analyses will guide a projected human dose for future development. Citation Format: Diana I. Albu, Xianzhe Wang, Yan Qin, Vivian Li, Purushothama Nanjappa, Amy Daniel Ulumben, Ruturaj Jadhav, Jason Kong, Austin Ablicki, Neal Schilling, Thomas Schuetz, Susan Kalled, Bing Gong, Nelly Kuklin. Dose range finding study in non-human primates confirms the unique mechanism of action of CTX-8371, a novel bispecific antibody blocking PD-1 and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3431.

TransCon IL-2 β/γ, a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog, demonstrates improved pharmacokinetics and profound expansion of cytotoxic immune cells in non-human primates.

2563 Background: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe side effects including cytokine release syndrome (CRS) and vascular leak syndrome (VLS). This is believed to be due to activation of IL-2Rα+ endothelial cells and inflammatory eosinophils as well as high Cmax due to the short half-life requiring frequent high dose administrations. Also, potent activation of immunosuppressive IL-2Rα+ regulatory T cells (Tregs) may limit IL-2’s efficacy. TransCon IL-2 β/γ is a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog designed to optimally address these shortcomings. Methods: Naïve male and female cynomolgus monkeys received 2-3 doses of TransCon IL-2 β/γ intravenously Q2W. Blood samples were taken to assess plasma concentrations of IL-2 β/γ (active drug), serum cytokines, hematology and immunophenotyping. Histopathology was also performed. Results: Administration of TransCon IL-2 β/γ induced robust increases in absolute lymphocyte counts (ALC), with no/minor increases in eosinophil counts. The increase in ALC was primarily driven by potent activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells and Tregs. At doses with maximum pharmacodynamic (PD) responses, TransCon IL-2 β/γ induced on average &gt; 20-fold ALC increases, &gt; 19-fold CD8+ T cell increases, &gt; 50-fold Effector Memory CD8+ T cell increases, &gt; 18-fold NK cell increases and &gt; 400-fold γδ T cell increases compared to pre-dose levels. These changes corresponded with a low Cmax and a long effective half-life ( &gt; 30 hours) for IL-2 β/γ. A clear induction of soluble CD25 and chemokine MCP-1 was seen after dosing, while levels of IL-5, IL-6, IFN-γ and TNF-α remained low. Further, histopathology showed no signs of vascular damage, tissue necrosis, pulmonary edema or eosinophilia. Conclusions: In cynomolgus monkeys, TransCon IL-2 β/γ demonstrated remarkable PD responses in CD8+ T cells, NK cells and γδ T cells over Tregs and eosinophils. No signs of VLS or CRS were observed. These results are likely due to the selective IL-2Rβ/γ bias along with the slow and sustained release of IL-2 β/γ resulting in low Cmax and long circulatory half-life enabled by the TransCon prodrug technology. The massive expansion of γδ T cells, which are known to possess considerable anti-tumor activity, is to our knowledge a unique finding for TransCon IL-2 β/γ among IL-2 variants in clinical development. Altogether, the responses seen in monkeys are suggestive of a potentially substantial improvement in the therapeutic index beyond what has been achieved by aldesleukin or new IL-2 variants to date. TransCon IL-2 β/γ is currently being evaluated in a clinical Phase 1/2 trial as monotherapy and in combination with pembrolizumab (NCT05081609).