Non-Clinical Safety Evaluation of Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells in Cynomolgus Monkeys.

Purpose: In recent years, exosomes have been proved to be used to treat many diseases. However, due to the lack of uniform quality control standards for exosomes, the safety of exosomes is still a problem to be solved, especially now more and more exosomes are used in clinical trials, and its non-clinical safety evaluation is particularly important. However, there is no safety evaluation standard for exosomes at present. Therefore, this study will refer to the evaluation criteria of therapeutic biological products, adopt non-human primates to evaluate the non-clinical safety of human umbilical cord mesenchymal stem cell exosomes from the general pharmacology and immunotoxicity, aiming at establishing a safety evaluation system of exosomes and providing reference for the clinical application of exosomes in the future. Methods: 3.85 × 1012 exosomes derived from human umbilical cord mesenchymal stem cells were injected into cynomolgus monkeys intravenously. The changes of general clinical conditions, hematology, immunoglobulin, Th1/Th2 cytokines, T lymphocytes and B lymphocytes, and immune organs were observed before and within 14 days after injection. Results: The results showed that exosomes did not have obvious pathological effects on the general clinical conditions, blood, coagulation function, organ coefficient, immunoglobulin, Th1/Th2 cytokines, lymphocytes, major organs, and major immune organs (spleen, thymus, bone marrow) of cynomolgus monkeys. However, the number of granulocyte-macrophage colonies in exosomes group was significantly higher than that in control group. Conclusion: To sum up, the general pharmacological results and immunotoxicity results showed that the injection of 3.85 × 1012 exosomes may have no obvious adverse reactions to cynomolgus monkeys. This dose of exosomes is relatively safe for treatment, which provides basis research for non-clinical safety evaluation of exosomes and provides reliable research basis for future clinical application of exosomes.

Relationship between aging and periodontal disease severity in gauge-raised cynomolgus monkeys (Macaca fascicularis).

The study aimed to evaluate the periodontal disease status in different age groups and clarify the relationship between aging and the severity of periodontal disease. The test animals were cynomolgus monkeys that were born and raised at the Tsukuba Primate Research Center of the National Institutes of Biomedical Innovation, Health, and Nutrition. The participants were divided into three groups: young (5-10 years old), middle (10-19 years old), and old (≥20 years old). The plaque Index (PLI), Gingival Index (GI), probing pocket depth (PPD), and Bleeding on probing (BOP) were used for the periodontal examination. Representative teeth were also examined. Polymerase chain reaction (PCR) was used to identify Porphyromonas macacae in dental plaque. Multiple comparisons and regression analyses were used to analyze the relationship between each age group and each oral examination index. Statistically significant differences were found between the age groups and periodontal examination index. Multiple regression analysis revealed that age was strongly correlated with each oral examination index. Based on these results, oral examinations of cynomolgus monkeys kept in the same environment confirmed an association between aging and periodontal disease severity. Monkeys at this facility are expected to serve as new experimental models for elucidating the mechanisms underlying the progression of age-related periodontal disease.

Wa-VP4* as a candidate rotavirus vaccine induced homologous and heterologous virus neutralizing antibody responses in mice, pigs, and cynomolgus monkeys.

Group A rotavirus (RVA) is the primary etiological agent of acute gastroenteritis (AGE) in children under 5 years of age. Despite the global implementation of vaccines, rotavirus infections continue to cause over 120,000 deaths annually, with a majority occurring in developing nations. Among infants, the P[8] rotavirus strain is the most prevalent and can be categorized into four distinct lineages. In this investigation, we expressed five VP4(aa26-476) proteins from different P[8] lineages of human rotavirus in E. coli and assessed their immunogenicity in rabbits. Among the different P[8] strains, the Wa-VP4 protein, derived from the MT025868.1 strain of the P[8]-1 lineage, exhibited successful purification in a highly homogeneous form and significantly elicited higher levels of neutralizing antibodies (nAbs) against both homologous and heterologous rotaviruses compared to other VP4 proteins derived from different P[8] lineages in rabbits. Furthermore, we assessed the immunogenicity of the Wa-VP4 protein in mice, pigs, and cynomolgus monkeys, observing that it induced robust production of nAbs in all animals. Interestingly, there was no significant difference between in nAb titers against homologous and heterologous rotaviruses in pigs and mankeys. Collectively, these findings suggest that the Wa-VP4* protein may serve as a potential candidate for a rotavirus vaccine.

Preclinical Pharmacokinetics and Pharmacology Study of RC98: A Programmed Cell Death Ligand 1 Monoclonal Antibody in Cynomolgus Monkeys.

INTRODUCTION: RC98 is the monoclonal antibody against Programmed Cell Death Ligand 1 (PD-L1). Relevant reports have confirmed that the influence of PD-L1 expressed by tumor cells on antitumor CD8+ T cell responses is well characterized, but the impact of PD-L1 expressed by immune cells has not been well defined. OBJECTIVE: This study aimed to design a Pharmacokinetics/Pharmacology (PK/PD) study of RC98 in normal cynomolgus monkeys to research the effect on the immune system. METHODS: RC98 and vehicle were administered to cynomolgus monkeys at 15 mg/kg via intravenous infusion once a week for 4 weeks to evaluate the relationship between PK and PD. The pharmacodynamic activity was measured by the PD-L1 receptor occupancy (RO) in CD3+ T cells, A T-cell-dependent antibody response (TDAR), and the concentration of soluble PD-L1. RESULTS: The pharmacokinetic result showed that the exposure from the last administration was lower than that of the first administration, probably due to immunogenicity production. There was a strong correlation between systemic exposure and RO in CD3+ T cells but decreased RO levels after the last dose, which indirectly reflected the activation of T cells. The keyhole limpet hemocyanin (KLH)-induced TDAR in the RC98 group was higher than in the vehicle group. The concentration of soluble PD-L1 had increased feedback with RC98, and the concentration of soluble PD-L1 was maintained at a higher level after multiple doses than before dosing. CONCLUSION: These data indicate that the immune system was clearly activated. In addition, the non-clinical data could provide a basis for its efficacy evaluation in clinical trials.

Characterization of preclinical Alzheimer's disease model: spontaneous type 2 diabetic cynomolgus monkeys with systemic pro-inflammation, positive biomarkers and developing AD-like pathology.

BACKGROUND: The key to the prevention and treatment of Alzheimer's disease (AD) is to be able to predict and diagnose AD at the preclinical or early stage, but the lack of a preclinical model of AD is the critical factor that causes this problem to remain unresolved. METHODS: We assessed 18 monkeys in vivo evaluation of pro-inflammatory cytokines and AD pathological biomarkers (n = 9 / type 2 diabetic mellitus (T2DM) group, age 20, fasting plasma glucose (FPG) ≥ 100 mg/dL, and n = 9 / negative control (NC) group, age 17, FPG < 100 mg/dL). Levels of pro-inflammatory cytokines and AD pathological biomarkers was measured by ELISA and Simoa Technology, respectively. 9 monkeys evaluated ex vivo for AD-like pathology (n = 6 / T2DM group, age 22.17, FPG ≥ 126 mg/dL, and n = 3 / NC group, age 14.67, FPG < 100 mg/dL). To evaluate the pathological features of AD in the brains of T2DM monkeys, we assessed the levels of Aß, phospho-tau, and neuroinflammation using immunohistochemistry, which further confirmed the deposition of Aß plaques by Bielschowsky's silver, Congo red, and Thioflavin S staining. Synaptic damage and neurodegeneration were assessed by immunofluorescence. RESULTS: We found not only increased levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) in peripheral blood (PB) and brain of T2DM monkeys but also changes in PB of AD pathological biomarkers such as decreased ß-amyloid (Aß) 42 and Aß40 levels. Most notably, we observed AD-like pathological features in the brain of T2DM monkeys, including Aß plaque deposition, p-tau from neuropil thread to pre-neurofibrillary tangles (NFTs), and even the appearance of extracellular NFT. Microglia were activated from a resting state to an amoeboid. Astrocytes showed marked hypertrophy and an increased number of cell bodies and protrusions. Finally, we observed impairment of the postsynaptic membrane but no neurodegeneration or neuronal death. CONCLUSIONS: Overall, T2DM monkeys showed elevated levels of peripheral and intracerebral inflammation, positive AD biomarkers in body fluids, and developing AD-like pathology in the brain, including Aß and tau pathology, glial cell activation, and partial synaptic damage, but no neuronal degeneration or death as compared to the healthy normal group. Hereby, we consider the T2DM monkeys with elevation of the peripheral pro-inflammatory factors and positive AD biomarkers can be potentially regarded as a preclinical AD model.

Human neural stem cells promote mitochondrial genesis to alleviate neuronal damage in MPTP-induced cynomolgus monkey models.

Currently, there is no effective treatment for Parkinson's disease (PD), and the regenerative treatment of neural stem cells (NSCs) is considered the most promising method. This study aimed to investigate the protective effect and mechanism of NSCs on neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced cynomolgus monkey (Macaca fascicularis) model of PD. We first found that injecting NSCs into the subarachnoid space relieved motor dysfunction in PD cynomolgus monkeys, as well as reduced dopaminergic neuron loss and neuronal damage in the substantia nigra (SN) and striatum. Besides, NSCs decreased 17-estradiol (E2) level, an estrogen, in the cerebrospinal fluid (CSF) of PD cynomolgus monkeys, which shows NSCs may provide neuro-protection by controlling estrogen levels in the CSF. Furthermore, NSCs elevated proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a), mitofusin 2 (MFN2), and optic atrophy 1 (OPA1) expression, three genes mediating mitochondrial biogenesis, in the SN and striatum of PD monkeys. In addition, NSCs suppress reactive oxygen species (ROS) production caused by MPTP, as well as mitochondrial autophagy, therefore preserving dopaminergic neurons. In summary, our findings show that NSCs may preserve dopaminergic and neuronal cells in an MPTP-induced PD cynomolgus monkey model. These protective benefits might be attributed to NSCs' ability of modulating estrogen balance, increasing mitochondrial biogenesis, and limiting oxidative stress and mitochondrial autophagy. These findings add to our understanding of the mechanism of NSC treatment and shed light on further clinical treatment options.

Immunohistochemical characterization of integrin alfa 6 in uterus and cervix of the cynomolgus monkeys (Macaca fascicularis) with MfPV3 infection.

BACKGROUND: Cervical cancer is an abnormal growth of cervical tissue epithelial cells due to persistent human papilloma virus (HPV) infection. Cynomolgus monkeys (Macaca fascicularis) can be naturally and spontaneously infected with M. fascicularis Papillomavirus Type 3 (MfPV3), a virus that is phylogenetically closely related to human oncogenic HPV (HPV-16 and HPV-34), and therefore a potentially beneficial for modeling HPV disease. This study aims to evaluate the expression of the integrin alpha 6 (ITGα6) receptor in cynomolgus monkeys spontaneously infected with MfPV3, which this receptor also found in human infected with HPV. METHODS: The study was done on archived Formalin-fixed Paraffin-Embedded (FFPE) samples of uterine and cervix tissue of cynomolgus monkeys. Immunohistochemistry was also performed to quantify the expression levels of ITGα6. RESULTS: The results showed 80% of the samples positive Cervical Intraepithelial Neoplasia (CIN) and increased expression of ITGα6 significantly in Positive-MfPV3 group than negative-MfPV3 group. CONCLUSIONS: This indicated the potential of cynomolgus monkeys as a spontaneous oncogenesis model of PV infection type.

A multi-tissue metabolome atlas of primate pregnancy.

Pregnancy induces dramatic metabolic changes in females; yet, the intricacies of this metabolic reprogramming remain poorly understood, especially in primates. Using cynomolgus monkeys, we constructed a comprehensive multi-tissue metabolome atlas, analyzing 273 samples from 23 maternal tissues during pregnancy. We discovered a decline in metabolic coupling between tissues as pregnancy progressed. Core metabolic pathways that were rewired during primate pregnancy included steroidogenesis, fatty acid metabolism, and arachidonic acid metabolism. Our atlas revealed 91 pregnancy-adaptive metabolites changing consistently across 23 tissues, whose roles we verified in human cell models and patient samples. Corticosterone and palmitoyl-carnitine regulated placental maturation and maternal tissue progenitors, respectively, with implications for maternal preeclampsia, diabetes, cardiac hypertrophy, and muscle and liver regeneration. Moreover, we found that corticosterone deficiency induced preeclampsia-like inflammation, indicating the atlas's potential clinical value. Overall, our multi-tissue metabolome atlas serves as a framework for elucidating the role of metabolic regulation in female health during pregnancy.

Acquisition of cocaine reinforcement using fixed-ratio and concurrent choice schedules in socially housed female and male monkeys.

RATIONALE: Previous studies in socially housed monkeys examining acquisition of cocaine self-administration under fixed-ratio (FR) schedules of reinforcement found that subordinate males and dominant females were more vulnerable than their counterparts. OBJECTIVES: The present studies extended these findings in two ways: (1) to replicate the earlier study, in which female monkeys were studied after a relatively short period of social housing (~ 3 months) using cocaine-naïve female monkeys (n = 9; 4 dominant and 5 subordinate) living in well-established social groups (~ 18 months); and (2) in male monkeys (n = 3/social rank), we studied cocaine acquisition under a concurrent schedule, with an alternative, non-drug reinforcer available. RESULTS: In contrast to earlier findings, subordinate female monkeys acquired cocaine reinforcement (i.e., > saline reinforcement) at significantly lower cocaine doses compared with dominant monkeys. In the socially housed males, no dominant monkey acquired a cocaine preference (i.e., > 80% cocaine choice) over food, while two of three subordinate monkeys acquired cocaine reinforcement. In monkeys that did not acquire, the conditions were changed to an FR schedule with only cocaine available and after acquisition, returned to the concurrent schedule. In all monkeys, high doses of cocaine were chosen over food reinforcement. CONCLUSIONS: The behavioral data in females suggests that duration of social enrichment and stress can differentially impact vulnerability to cocaine reinforcement. The findings in socially housed male monkeys, using concurrent food vs. cocaine choice schedules of reinforcement, confirmed earlier social-rank differences using an FR schedule and showed that vulnerability could be modified by exposure to cocaine.

Combined untargeted and targeted lipidomics approaches reveal potential biomarkers in type 2 diabetes mellitus cynomolgus monkeys.

BACKGROUND: The significantly increasing incidence of type 2 diabetes mellitus (T2DM) over the last few decades triggers the demands of T2DM animal models to explore the pathogenesis, prevention, and therapy of the disease. The altered lipid metabolism may play an important role in the pathogenesis and progression of T2DM. However, the characterization of molecular lipid species in fasting serum related to T2DM cynomolgus monkeys is still underrecognized. METHODS: Untargeted and targeted LC-mass spectrometry (MS)/MS-based lipidomics approaches were applied to characterize and compare the fasting serum lipidomic profiles of T2DM cynomolgus monkeys and the healthy controls. RESULTS: Multivariate analysis revealed that 196 and 64 lipid molecules differentially expressed in serum samples using untargeted and targeted lipidomics as the comparison between the disease group and healthy group, respectively. Furthermore, the comparative analysis of differential serum lipid metabolites obtained by untargeted and targeted lipidomics approaches, four common serum lipid species (phosphatidylcholine [18:0_22:4], lysophosphatidylcholine [14:0], phosphatidylethanolamine [PE] [16:1_18:2], and PE [18:0_22:4]) were identified as potential biomarkers and all of which were found to be downregulated. By analyzing the metabolic pathway, glycerophospholipid metabolism was associated with the pathogenesis of T2DM cynomolgus monkeys. CONCLUSION: The study found that four downregulated serum lipid species could serve as novel potential biomarkers of T2DM cynomolgus monkeys. Glycerophospholipid metabolism was filtered out as the potential therapeutic target pathway of T2DM progression. Our results showed that the identified biomarkers may offer a novel tool for tracking disease progression and response to therapeutic interventions.

Effects of two weeks of food restriction on toxicological parameters in cynomolgus monkeys.

Animals frequently eat less after a test-article treatment in nonclinical toxicological studies, and it can be difficult to distinguish test article-derived toxicities from secondary changes related to this reduced food intake. Therefore, in this study, we restricted the food intake of cynomolgus monkeys (Cambodian, male, n=2 or 3, 48 ± 3 months old) to 25% of the control for two weeks and evaluated the effects on toxicological parameters (general conditions, body weight, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, pathological examination). After 2 weeks, the monkeys exhibited decreases in bone marrow erythropoiesis (e.g., decreases in reticulocytes and bone marrow erythrocytes), as well as glycogenesis induction (e.g., increase in aspartate aminotransferase (AST)) and malnutrition (e.g., decrease in triglyceride and systemic adipocytes atrophy). Additionally, histopathological analysis revealed granuloma and inflammatory cell infiltration in coronary fat, which had never been found in previous food restriction studies using other animal species. These findings will enable researchers to more accurately evaluate the toxicological risks of test articles that simultaneously induce food intake reduction.

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

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.

Repeat-dose toxicity study of human umbilical cord mesenchymal stem cells in cynomolgus monkeys by intravenous and subcutaneous injection.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are proposed for the treatment of acute lung injury and atopic dermatitis. To advance hUC-MSC entry into clinical trials, the effects of hUC-MSCs on the general toxicity, immune perturbation and toxicokinetic study of hUC-MSCs in cynomolgus monkeys were assessed. hUC-MSCs were administered to cynomolgus monkeys by intravenous infusion of 3.0 × 106 or 3.0 × 107cells/kg or by subcutaneous injection of 3.0 × 107cells/kg twice a week for 3 weeks followed by withdrawal and observation for 6 weeks. Toxicity was assessed by clinical observation, clinical pathology, ophthalmology, immunotoxicology and histopathology. Moreover, toxicokinetic study was performed using a validated qPCR method after the first and last dose. After 3rd or 4th dosing, one or three the monkeys in the intravenous high-dose group exhibited transient coma, which was eliminated by slow-speed infusion after 5th or 6th dosing. In all dose groups, hUC-MSCs significantly increased NEUT levels and decreased LYMPH and CD3+ levels, which are related to the immunosuppressive effect of hUC-MSCs. Subcutaneous nodules and granulomatous foci were found at the site of administration in all monkeys in the subcutaneous injection group. Other than above abnormalities, no obvious systemic toxicity was observed in any group. The hUC-MSCs was detectable in blood only within 1 h after intravenous and subcutaneous administration. The present study declared the preliminary safety of hUC-MSCs, but close monitoring of hUC-MSCs for adverse effects, such as coma induced by intravenous infusion, is warranted in future clinical trials.

Resting-state functional connectivity in a non-human primate model of cortical ischemic stroke in area F1.

BACKGROUND: The application of functional MRI to non-human primates after stroke has not yet been undertaken. This is the first study to explore the functional connectivity changes in non-human primate models during acute stages after stroke onset. METHODS: Nineteen healthy male cynomolgus monkeys (4-5 years) were used in this study. The photothrombosis model was employed to induce focal ischemic stroke in F1 area in the monkey's left hemisphere. T1-weighted structural images and resting-state functional magnetic resonance imaging (rs-fMRI) of all subjects were obtained using a 3.0 Tesla MRI system on the third day following stroke. Based on the D99 atlas, the structural and functional changes of bilateral F1 areas in monkeys were analyzed using region of interest (ROI)-based functional connectivity (FC). The bilateral F1 areas were selected as the seed regions due to their crucial role in motor control and their potential to unveil the comprehensive functional reorganization of the motor system at a whole-brain level following stroke. RESULTS: Ischemic lesions were observed after the stroke, with larger lesion volumes associated with poorer neurological dysfunction. Compared with baseline condition, left area F1 demonstrated decreased FC with the left cerebellum, left ventral pons and left 5_(PEa). When the ROI was located in the right area F1, ischemic monkeys showed decreased FC in left ventral pons, left cerebellum, left primary visual cortex and left 5_(PEa), accompanied by increased FC in the right orbitofrontal cortex. Importantly, the degree of altered FC between left area F1 and left cerebellum was associated with upper limb tone. CONCLUSIONS: These results provide valuable insights into the early-stage functional connectivity changes in the F1 areas of monkeys under ischemic conditions, highlighting the potential involvement of specific brain regions in the pathophysiology of ischemic injury.

Reduced Fasting Duration in Cynomolgus Monkeys Enhances Animal Welfare During Toxicology Studies.

During toxicology studies, fasting animals prior to clinical pathology blood collection is believed to reduce variability in some clinical chemistry analytes. However, fasting adds stress to animals that are already stressed from the administration of potentially toxic doses of the test article. The purpose of this study was to assess the impacts of different fasting durations on cynomolgus monkeys' welfare during toxicology studies. To this end, we assessed the cynomolgus monkeys traditional and ancillary clinical pathology endpoints at different fasting times. We showed that most clinical pathology endpoints were largely comparable between different fasting times suggesting that cynomolgus monkeys could be fasted for as little as 4 hours for toxicology studies, as longer fasting times (up to 20 hours) resulted in stress, dehydration, and significant decreases in blood glucose- changes that impacts animal welfare. Shorter fasting times were associated with higher triglycerides variability among individual animals. Therefore, we propose that shorter fasting time (i.e., 4 hours) should be adequate for most toxicology studies except when: (1) parameters that could be affected by non-fasting conditions are important for safety and pharmacodynamic assessments (i.e., glucose and lipids) and (2) fasting would be needed for the bioavailability of an orally administered test article.

Performance and feasibility of three different approaches for computer based semi-automated analysis of ventricular arrhythmias in telemetric long-term ECG in cynomolgus monkeys.

Computer-based analysis of long-term electrocardiogram (ECG) monitoring in animal models represents a cost and time-consuming process as manual supervision is often performed to ensure accuracy in arrhythmia detection. Here, we investigate the performance and feasibility of three ECG interval analysis approaches A) attribute-based, B) attribute- and pattern recognition-based and C) combined approach with additional manual beat-to-beat analysis (gold standard) with regard to subsequent detection of ventricular arrhythmias (VA) and time consumption. ECG analysis was performed on ECG raw data of 5 male cynomolgus monkeys (1000 h total, 2 × 100 h per animal). Both approaches A and B overestimated the total number of arrhythmias compared to gold standard (+8.92% vs. +6.47%). With regard to correct classification of detected VA event numbers (accelerated idioventricular rhythms [AIVR], ventricular tachycardia [VT]) approach B revealed higher accuracy compared to approach A. Importantly, VA burden (% of time) was precisely depicted when using approach B (-1.13%), whereas approach A resulted in relevant undersensing of ventricular arrhythmias (-11.76%). Of note, approach A and B could be performed with significant less working time (-95% and - 91% working time) compared to gold standard. In sum, we show that a combination of attribute-based and pattern recognition analysis (approach B) can reproduce VA burden with acceptable accuracy without using manual supervision. Since this approach allowed analyses to be performed with distinct time saving it represents a valuable approach for cost and time efficient analysis of large preclinical ECG datasets.

Klebsiella pneumoniae infection in cynomolgus monkeys at primate research center facility in Indonesia.

BACKGROUND: Klebsiella pneumoniae infection in nonhuman primates has been widely reported and causes significant morbidity and mortality. Animal deaths occur routinely at the Primate Research Center of IPB University. The results of necropsy and culture suggested a K. pneumoniae infection. METHODS: A mass health assessment of Cynomolgus monkeys (n = 429) was carried out by physical examination and molecular targeting K. pneumoniae (n = 96), family of Coronaviridae (n = 148) and Paramyxoviridae (n = 148). RESULTS: A total of 49.18% of the animals had clinical symptoms of respiratory disorders, abscesses, trauma, and others. PCR results indicated that 28.57% were positive for K. pneumoniae with 35.71% mortality, while all samples were negative for both virus families. CONCLUSIONS: There have been outbreaks caused by K. pneumoniae and/or K. pneumoniae subsp. pneumoniae. This disease is chronic, infects all of the buildings, and no tendency for disease transmission according to gender and age class.

Effective nose-to-brain drug delivery using a combination system targeting the olfactory region in monkeys.

The nose-to-brain (N2B) pathway has garnered attention because it transports drugs directly into the brain. Although recent studies have suggested the necessity of selective drug administration to the olfactory region for effective N2B drug delivery, the importance of delivering the formulation to the olfactory region and the detailed pathway involved in drug uptake in primates brain remain unclear. Here, we developed a combination system for N2B drug delivery comprising a proprietary mucoadhesive powder formulation and a dedicated nasal device (N2B-system) and evaluated it for nasal drug delivery to the brain in cynomolgus monkeys. This N2B-system demonstrated a much greater formulation distribution ratio in the olfactory region in an in vitro experiment using a 3D-printed nasal cast and in vivo experiment using cynomolgus monkeys, as compared to that in other nasal drug delivery systems that comprise of a proprietary nasal powder device developed for nasal absorption and vaccination and a commercially available liquid spray. Additionally, Texas Red-labeled dextran (TR-DEX, 3 kDa) was administered using the N2B-system to estimate the drug transition pathway from the nasal cavity to the brain. TR-DEX preferentially localized to the olfactory epithelium and reached the olfactory bulb through the cribriform foramina. Moreover, domperidone, a model drug with poor blood-brain barrier permeability, was administered to assess the brain uptake of medicine after olfactory region-selective administration by using the N2B-system. Domperidone accumulation in the brain was evaluated using positron emission tomography with intravenously administered [18F]fallypride based on competitive inhibition of the dopamine D2 receptor (D2R). Compared to other systems, the N2B-system significantly increased D2R occupancy and domperidone uptake in the D2R-expressing brain regions. The current study reveals that the olfactory region of the nasal cavity is a suitable target for efficient nasal drug delivery to the brain in cynomolgus monkeys. Thus, the N2B-system, which targets the olfactory region, provides an efficient approach for developing effective technology for nasal drug delivery to the brain in humans.

C9orf72 poly(PR) aggregation in nucleus induces ALS/FTD-related neurodegeneration in cynomolgus monkeys.

Poly(PR) is a dipeptide repeat protein comprising proline and arginine residues. It is one of the translational product of expanded G4C2 repeats in the C9orf72 gene, and its accumulation is contributing to the neuropathogenesis of C9orf72-associated amyotrophic lateral sclerosis and/or frontotemporal dementia (C9-ALS/FTD). In this study, we demonstrate that poly(PR) protein alone is sufficient to induce neurodegeneration related to ALS/FTD in cynomolgus monkeys. By delivering poly(PR) via AAV, we observed that the PR proteins were located within the nucleus of infected cells. The expression of (PR)50 protein, consisting of 50 PR repeats, led to increased loss of cortical neurons, cytoplasmic lipofuscin, and gliosis in the brain, as well as demyelination and loss of ChAT positive neurons in the spinal cord of monkeys. While, these pathologies were not observed in monkeys expressing (PR)5, a protein comprising only 5 PR repeats. Furthermore, the (PR)50-expressing monkeys exhibited progressive motor deficits, cognitive impairment, muscle atrophy, and abnormal electromyography (EMG) potentials, which closely resemble clinical symptoms seen in C9-ALS/FTD patients. By longitudinally tracking these monkeys, we found that changes in cystatin C and chitinase-1 (CHIT1) levels in the cerebrospinal fluid (CSF) corresponded to the phenotypic progression of (PR)50-induced disease. Proteomic analysis revealed that the major clusters of dysregulated proteins were nuclear-localized, and downregulation of the MECP2 protein was implicated in the toxic process of poly(PR). This research indicates that poly(PR) expression alone induces neurodegeneration and core phenotypes associated with C9-ALS/FTD in monkeys, which may provide insights into the mechanisms of disease pathogenesis.

Development of a New Method to Evaluate the Biodistribution of Antibodies Using Non-Radioactive Metal Labeling and Inductively Coupled Plasma Mass Spectrometry.

PURPOSE: The use of radiolabeled compounds is associated with a number of limitations. Therefore, a new method for the radioisotope-free evaluation of antibody distribution using metal labeling and inductively coupled plasma-mass spectrometry (ICP-MS) was developed herein. METHODS: Indium-labeled monoclonal antibodies were administrated intravenously to tumor-bearing mice and cynomolgus monkeys, and antibody concentrations in plasma and tissues were measured by ICP-MS. The results were compared with those obtained using a ligand binding assay (LBA) and radioisotope-labeled antibody administration. Indium-, terbium-, holmium-, and yttrium-labeled cetuximab were co-administered to one C57BL/6 J mouse for simultaneous PK and tissue distribution evaluations. RESULTS: The administration of a radioactive or non-radioactive indium-labeled anti-human interleukin-6 receptor (hIL-6R) antibody to tumor-bearing hIL-6R transgenic mice resulted in similar plasma antibody concentration-time profiles by ICP-MS, a ligand binding assay (LBA), and gamma-ray detector. Liver, kidney, brain, spleen, and tumor concentrations of antibodies measured by ICP-MS were similar to those after the administration of radiolabeled anti-hIL-6R antibodies. Following the administration of indium-labeled cetuximab to cynomolgus monkeys, plasma antibody concentrations measured by ICP-MS were similar to those measured by LBA, and antibody concentrations in organs were evaluable by ICP-MS. The PK of all metals were similar to antibody PK evaluated by LBA, and concentrations in each tissue were equivalent among metals. CONCLUSIONS: The assessment of antibody distribution using ICP-MS is a novel alternative to the traditional radiolabeled approach. It facilitates the assessment of antibody distribution in the early stages of drug discovery and accelerates the assessment of target engagement.