Genotoxicity and safety pharmacology of the rVSVInd(GML)-mspSGtc vaccine against SARS-CoV-2 in Sprague-Dawley rats and Beagle dogs.

The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a pandemic, prompting rapid vaccine development. Although vaccines are effective, the occurrence of rare adverse events following vaccination highlights the necessity of determining whether the benefits outweigh the risks posed by the infection itself. The recombinant Vesicular Stomatitis Virus (rVSV) platform is a promising vector for vaccines against emerging viruses. However, limited studies have evaluated the genotoxicity and safety pharmacology of this viral vector vaccine, which is crucial to ensure the safety of vaccines developed using this platform. Hence, the present study aimed to assess the genotoxicity and safety pharmacology of the rVSVInd(GML)-mspSGtc COVID-19 vaccine using micronucleus and comet assays, as well as neurobehavioral, body temperature, respiratory, and cardiovascular assessments in Sprague-Dawley rats and beagle dogs. The intramuscular administration of rVSVInd(GML)-mspSGtc at doses up to 1.5 × 109 PFU/animal did not increase the number of bone marrow micronucleated polychromatic erythrocytes or cause liver DNA damage. Additionally, it had no significant impact on neurobehavioral functions in rats and showed marginal temporary changes in body temperature, respiratory rate, heart rate, and electrocardiogram parameters in rats and dogs, all of which resolved within 24 h. Overall, following genotoxicity and pharmacological safety assessments, rVSVInd(GML)-mspSGtc displayed no notable systemic adverse effects in rats and dogs, suggesting its potential as a vaccine candidate for human clinical trials.

Evaluating the toxicity of the roots of Asarum heterotropoides var. mandshuricum extracted using the decoction method: Genotoxicity, single-dose toxicity, and 13-week repeated-dose toxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Asarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa (AR) is a traditional herbal medicine used across Asia, including Korea, China, and Japan. AR exhibits a range of biological activities, such as anti-inflammatory, anti-cancer, cold treatment, and anti-nociceptive effects. Various extraction methods, including decoction, which utilizes traditional knowledge and techniques. The AR decoction extract expected to contain fewer toxicants and have reduced toxicity due to the use of hot water in the extraction process. However, scientific evidence on the toxicity of AR decoction extracts is lacking, necessitating further studies for safe usage. AIM OF THE STUDY: This study aimed to evaluate the genotoxicity and toxicity of single and repeated administration of AR decoction extracts. MATERIALS AND METHODS: The genotoxicity was assessed using a bacterial reverse mutation (Ames test), an in vitro mammalian chromosome aberration test (CA test), and an in vivo micronucleus test (MN test) in Sprague-Dawley (SD) rats. The general toxicity was evaluated through single-dose and 13-week repeated-dose toxicity studies. In the single-dose toxicity study, 40 SD rats were orally administered AR decoction extract at doses of 1000, 2000, and 5000 mg/kg. In the 13-week repeated-dose toxicity study, 140 SD rats received daily oral doses of 0, 250, 500, 1000, 2000, and 5000 mg/kg of AR decoction extract. RESULTS: The genotoxicity tests revealed that AR decoction extract was not genotoxic. The single-dose toxicity study showed no changes in body weight, clinical pathology, or macroscopic findings, with the approximate lethal dose (ALD) exceeding 5000 mg/kg. The 13-week repeated-dose toxicity study demonstrated no treatment-related changes in body weight, general symptoms, hematology, clinical chemistry, or urinalysis. Histopathological findings revealed hyperplasia of squamous cells in the forestomach after AR decoction extract administration, a treatment-related effect that resolved during the recovery period. The no observed adverse effect level (NOAEL) for both male and female rats was estimated to be 2000 mg/kg. CONCLUSIONS: This study establishes the non-toxic dose of AR decoction extract, providing a foundation for further non-clinical and clinical evaluations AR safety.

Nonclinical safety assessment and immunogenicity of rVSVInd(GML)-mspSGtc vaccine for SARS-CoV-2 in rabbits.

The worldwide health, economic, and societal consequences of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have been devastating. The primary strategy to prevent new infectious diseases is to vaccinate the majority of people worldwide. However, the significant hurdles that are faced include vaccine safety concerns and vaccine reluctance. Among the various types of vaccines, the recombinant vesicular stomatitis virus (rVSV) is a promising candidate owing to its safety and efficacy. Therefore, we investigated the toxicity, immunogenicity, and local tolerance of the rVSVInd(GML)-mspSGtc vaccine against SARS-CoV-2. New Zealand White (NZW) rabbits were administered single or three repeated intramuscular injections of rVSVInd(GML)-mspSGtc every 2 weeks, followed by a 4-week recovery period. Male and female rabbits were randomly assigned into three groups: a control group and two dose-level groups (1 × 109 and 4 × 109 PFU/mL). Treatment-related changes included a temporary increase in body temperature and local inflammation at the injection site. These findings indicated recovery or a trend toward recovery, with no overt systemic toxicity. Immunogenicity analysis results suggested that rVSVInd(GML)-mspSGtc elicited a robust dose-dependent immune response in terms of neutralizing antibodies and IgG antibodies against the SARS-CoV-2 spike protein. In addition, the immune response intensity was increased by repeated vaccine administration. In conclusion, both the approximate lethal dose and the no observed adverse effect level for rVSVInd(GML)-mspSGtc exceeded 4 × 109 PFU/mL in NZW rabbits. Overall, rVSVInd(GML)-mspSGtc induced no adverse effects at the maximum dosage tested; however, its efficacy warrants further clinical evaluation.

Preclinical evaluation of general toxicity and safety pharmacology of a receptor-binding domain-based COVID-19 subunit vaccine in various animal models.

The coronavirus disease 2019 pandemic has resulted in the introduction of several naïve methods of vaccine development, which have been used to prepare novel viral vectors and mRNA-based vaccines. However, reluctance to receive vaccines owing to the uncertainty regarding their safety is prevalent. Therefore, rigorous safety evaluation of vaccines through preclinical toxicity studies is critical to determine the safety profiles of vaccine candidates. This study aimed to evaluate the toxicity profile of HuVac-19, a subunit vaccine of SARS-CoV-2 utilizing the receptor-binding domain as an antigen, in rats, rabbits, and dogs using single- and repeat-dose study designs. Repeat-dose toxicity studies in rats and rabbits showed transient changes in hematological and serum biochemical parameters in the adjuvant and/or vaccine groups; however, these changes were reversed or potentially reversible after the recovery period. Moreover, temporary reversible changes in absolute and relative organ weights were observed in the prostate of rats and the thymus of rabbits. Gross examination of the injection sites in rats and rabbits treated with the adjuvant- and HuVac-19 showed discoloration and foci, whereas histopathological examination showed granulomatous inflammation, inflammatory cell infiltration, and myofiber degeneration/necrosis. This inflammatory response was local, unassociated with other toxicological changes, and resolved. In a pharmacological safety study, no toxicological or physiological changes associated with HuVac-19 administration were observed. In conclusion, HuVac-19 was not associated with any major systemic adverse effects in the general toxicity and safety pharmacology evaluation, demonstrating that HuVac-19 is a vaccine candidate with sufficient capacity to be used in human clinical trials.

Preclinical Evaluation of interferon-gamma primed human Wharton's jelly-derived mesenchymal stem cells.

The potential of human mesenchymal stem cells (MSCs) for cell therapy has been investigated in numerous immune-mediated conditions; MSCs are considered one of the most promising cellular therapeutics to treat intractable diseases. Recently, approaches to prime MSCs have been investigated, thereby generating cellular products with enhanced potential for a variety of clinical applications. Interferon-gamma (IFN-γ) priming is a current approach used to increase the therapeutic efficacy of MSCs. In this study, we determined the systemic toxicity, tumorigenicity and biodistribution of IFN-γ-primed Wharton's jelly-derived (WJ)-MSCs in male and female BALB/c-nu/nu mice. There were no deaths or pathologic lesions in the mice treated with 5 × 106 cells/kg IFN-γ-primed MSCs in the repeated dose study. In the tumorigenicity study, one of the subcutaneously treated mice showed bronchioloalveolar adenoma in the lung but tested negative for human-specific anti-mitochondrial antibody, suggesting the spontaneous murine origin of the adenoma. A biodistribution study using real-time quantitative polymerase chain reaction demonstrated the systemic IFN-γ-primed MSC clearance by day 28. Based on the toxicity, biodistribution, and tumorigenicity studies, we concluded that IFN-γ-primed MSCs at 5 × 106 cells/kg do not induce tumor formation and adverse changes.

Genotoxicity assessment of root extracts of Paeonia lactiflora Pall.

The roots of Paeonia lactiflora Pall., (Paeoniae Radix, PL) are a well-known herbal remedy used to treat fever, rheumatoid arthritis, systemic lupus erythematosus, hepatitis, and gynecological disorders in East Asia. Here we evaluated the genetic toxicity of PL extracts (as a powder [PL-P] and hot-water extract [PL-W]) in accordance with the Organization for Economic Co-operation and Development guidelines. The Ames test revealed that PL-W was not toxic to S. typhimurium strains and E. coli in absence and presence of the S9 metabolic activation system at concentrations up to 5000 µg/plate, but PL-P produced a mutagenic response to TA100 in the absence of S9 mix. PL-P was cytotoxic in in vitro chromosomal aberrations (more than a 50 % decrease in cell population doubling time), and it increased the frequency of structural and numerical aberrations in absence and presence of S9 mix in a concentration-dependent manner. PL-W was cytotoxic in the in vitro chromosomal aberration tests (more than a 50 % decrease in cell population doubling time) only in the absence of S9 mix, and it induced structural aberrations only in the presence of S9 mix. PL-P and PL-W did not produce toxic response during the in vivo micronucleus test after oral administration to ICR mice and did not induce positive results in the in vivo Pig-a gene mutation and comet assays after oral administration to SD rats. Although PL-P showed genotoxic in two in vitro tests, the results from physiologically relevant in vivo Pig-a gene mutation and comet assays illustrated that PL-P and PL-W does not cause genotoxic effects in rodents.

Implementation of Systematic Bioanalysis of Antibody-Drug Conjugates for Preclinical Pharmacokinetic Study of Ado-Trastuzumab Emtansine (T-DM1) in Rats.

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies covalently bound to cytotoxic drugs by a linker. They are designed to selectively bind target antigens and present a promising cancer treatment without the debilitating side effects of conventional chemotherapies. Ado-trastuzumab emtansine (T-DM1) is an ADC that received US FDA approval for the treatment of HER2-positive breast cancer. The purpose of this study was to optimize methods for the quantification of T-DM1 in rats. We optimized four analytical methods: (1) an enzyme-linked immunosorbent assay (ELISA) to quantify the total trastuzumab levels in all drug-to-antibody ratios (DARs), including DAR 0; (2) an ELISA to quantify the conjugated trastuzumab levels in all DARs except DAR 0; (3) an LC-MS/MS analysis to quantify the levels of released DM1; and (4) a bridging ELISA to quantify the level of anti-drug antibodies (ADAs) of T-DM1. We analyzed serum and plasma samples from rats injected intravenously with T-DM1 (20 mg/kg, single dose) using these optimized methods. Based on these applied analytical methods, we evaluated the quantification, pharmacokinetics, and immunogenicity of T-DM1. This study establishes the systematic bioanalysis of ADCs with validated assays, including drug stability in matrix and ADA assay, for future investigation on the efficacy and safety of ADC development.

Preclinical safety assessment of the suction-assisted intradermal injection of the SARS-CoV-2 DNA vaccine candidate pGO-1002 in white rabbit.

pGO-1002, a non-viral DNA vaccine that expresses both spike and ORF3a antigens of SARS-CoV-2, is undergoing phase 1 and phase 2a clinical trials in Korea and the US. A preclinical repeated-dose toxicity study in New Zealand white rabbits in compliance with Good Laboratory Practice (GLP) was conducted to assess the potential toxicity, local tolerance, and immunogenicity of the vaccine and GeneDerm suction device. The dose rate was 1.2 mg/head pGO-1002, and this was administered intradermally to a group of animals (eight animals/sex/group) three times at 2-week intervals, followed by a 4-week recovery period. After each administration, suction was applied to the injection site using the GeneDerm device. Mortality, clinical signs, body weight, food consumption, skin irritation, ophthalmology, body temperature, urinalysis, and clinical pathology were also monitored. Gross observations and histopathological evaluation were performed. Overall, pGO-1002 administration-related changes were confined to minor damage or changes at the injection site, increased spleen weight and minimal increased cellularity in white pulp. All changes of injection site were considered local inflammatory changes or pharmacological actions due to the vaccine with the changes in spleen considered consistent with vaccine-induced immune activation. All findings showed reversibility during the 4-week recovery period. Animals vaccinated with pGO-1002, administered by intradermal injection and followed by application of suction with GeneDerm, developed humoral and cellular responses against the SARS-CoV-2 antigens consistent with prior studies in rats. Collectively, it was concluded that the pGO-1002 vaccine was safe and effective under these experimental conditions and these data supported future human study of the vaccine, now known as GLS-5310, for clinical trial use.

Evaluation of genotoxicity and 13-week subchronic toxicity of root of Asarum heterotropoides var. seoulense (Nakai) Kitag.

ETHNOPHARMACOLOGICAL RELEVANCE: Asarum heterotropoides var. seoulense (Nakai) Kitag is a traditional herbal medicine used in Korea and China. It is effective in aphthous stomatitis, local anesthesia, headache, toothache, gingivitis, and inflammatory diseases. However, information on the toxicity of the root of Asarum heterotropoides var. seoulense (Nakai) Kitag (AR) is limited. Therefore, preclinical toxicity studies on AR are needed to reduce the risk of excessive intake. AIM OF THE STUDY: We aimed to evaluate genotoxicity and the potential toxicity due to repeated administration of AR powder. MATERIALS AND METHODS: In vitro bacterial reverse mutation assay (Ames), in vitro chromosomal aberration assay (CA), and in vivo micronucleus (MN) assay in ICR mice were conducted. As positive results were obtained in Ames and CA assays, alkaline comet assay and pig-a gene mutation test were conducted for confirmation. For evaluating the general toxicity of AR powder, a 13-week subchronic toxicity test was conducted, after determining the dose by performing a single and a 4-week dose range finding (DRF) test. A total of 152 Sprague-Dawley (SD) rats were orally administered AR powder at doses of 0, 150, 350, 500, 1000, and 2000 mg/kg/day in the 13-week subchronic toxicity test. Hematology, clinical chemistry, urinalysis, organ weight, macro-, and microscopic examination were conducted after rat necropsy. RESULTS: AR powder induced genotoxicity evidenced in the Ames test at 187.5, 750, 375, and 1500 µg/plate of TA100, TA98, TA1537, and E. coli WP2uvrA in the presence and absence of S9, respectively; CA test at 790 µg/mL for 6 h in the presence of S-9; 75 µg/mL for 6 h in the absence of S-9, and 70 µg/mL for 22 h in the absence of S-9 in the stomach in the comet assay but not in MN and pig-a assays. In the 13-week subchronic toxicity study, clinical signs including irregular respiration, noisy respiration, salivation, and decreased body weight or food consumption were observed in males and females in the 2000 mg/kg/day group. In hematology tests, clinical chemistry, urinalysis, organ weight, and macroscopic examination, changes were observed in the dose groups of 500 mg/kg/day and above. Microscopic examination revealed hyperplasia of the stomach as a test-related change. Hepatocellular adenoma and changes in liver-related clinical chemistry parameters were observed. The rat No Observed Adverse Effect Level (NOAEL) was 150 mg/kg/day in males and <150 mg/kg/day in females. CONCLUSIONS: AR powder is potentially toxic to the liver and stomach and should be used with caution in humans. A long-term study on carcinogenicity is necessitated because DNA damage or changes in tissue lesions were observed in SD rats.

Tumorigenicity Assessment of Human Cancer Cell Lines Xenografted on Immunodeficient Mice as Positive Controls of Tumorigenicity Testing.

Recent advances in human pluripotent stem cell (hPSC)-derived cell therapies and genome editing technologies such as CRISPR/Cas9 make regenerative medicines promising for curing diseases previously thought to be incurable. However, the possibility of off-target effects during genome editing and the nature of hPSCs, which can differentiate into any cell type and infinitely proliferate, inevitably raises concerns about tumorigenicity. Tumorigenicity acts as a major obstacle to the application of hPSC-derived and gene therapy products in clinical practice. Thus, regulatory authorities demand mandatory tumorigenicity testing as a key pre-clinical safety step for the products. In the tumorigenicity testing, regulatory guidelines request to include human cancer cell line injected positive control group (PC) animals, which must form tumors. As the validity of the whole test is determined by the tumor-forming rates (typically above 90%) of PC animals, establishing the stable tumorigenic condition of PC animals is critical for successful testing. We conducted several studies to establish the proper positive control conditions, including dose, administration routes, and the selection of cell lines, in compliance with Good Laboratory Practice (GLP) regulations and/or guidelines, which are essential for pre-clinical safety tests of therapeutic materials. We expect that our findings provide insights and practical information to create a successful tumorigenicity test and its guidelines.

Anti-Inflammatory Lignans from the Roots of Asarum heterotropoides var. mandshuricum and Their Mechanism of Action.

Bioassay-guided fractionation of Asarum heterotropoides var. mandshuricum F. Maekawa (Aristolochiaceae) root extract led to the isolation and characterization of one new ferulic acid glucose ester (1) and nine known lignans (2-10). Their structures were elucidated using extensive spectroscopic methods, including 1D and 2D NMR, and MS spectra. The anti-inflammatory effects of the isolated compounds were investigated via their inhibition against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. Among them, compound 7 ((1R,2S,5R,6R)-5'-O-methylpluviatilol) showed the most effective inhibitory activity against NO production and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein in an exceedingly dose-dependent manner. In addition, further study revealed that the mechanism of anti-inflammatory activity of the most active lignan (7) might be associated with the inhibition of extracellular-signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) phosphorylation.

An International Collaborative Animal Study of the Carcinogenicity of Mobile Phone Radiofrequency Radiation: Considerations for Preparation of a Global Project.

Radiofrequency radiation (RFR) was classified as a "possible" human carcinogen in 2011, which caused great public concern. A carcinogenicity study by the National Toxicology Program (NTP) found Code Division Multiple Access-and Global System for Mobile Communications-modulated mobile phone RFR to be carcinogenic to the brain and heart of male rats. As part of an investigation of mobile phone carcinogenesis, and to verify the NTP study results, a 5-year collaborative animal project was started in Korea and Japan in 2019. An international animal study of this type has two prerequisites: use of the same study protocol and the same RF-exposure system. This article discusses our experience in the design of this global study on radiofrequency electromagnetic fields (RF-EMFs).© 2022 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

Safety evaluation of fermented Platycodon grandiflorus (Jacq.) A.DC. extract: Genotoxicity, acute toxicity, and 13-week subchronic toxicity study in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Platycodon grandiflorus (Jacq.) A.DC. is a well-known traditional herbal medicine administered for bronchitis and inflammatory diseases. Especially, anti-inflammatory effect of fermented P. grandiflorus (Jacq.) A.DC. extract (FPGE) was higher than that of P. grandiflorus (Jacq.) A.DC. extract. However, toxicological information for FPGE is lacking. AIM OF THE STUDY: In this study, we establish a toxicological profile for FPGE by testing genotoxicity, acute and 13-week subchronic toxicity. MATERIALS AND METHODS: FPGE was evaluated with bacterial reverse mutation, chromosome aberration, and micronucleus test. For the acute- and 13-week subchronic toxicity tests, FPGE was administered orally at doses of 0, 750, 1500, and 3000 mg/kg in SD rats. RESULTS: The results of the genotoxic assays indicated that FPGE induced neither mutagenicity nor clastogenicity. The acute toxicity test showed that FPGE did not affect animal mortality, clinical signs, body weight changes, or microscopic findings at ≤ 3000 mg/kg. The approximate lethal dose (ALD) of FPGE in SD rats was >3000 mg/kg. For the 13-week subchronic toxicity assay, no FPGE dose induced any significant change in mortality, clinical signs, body or organ weight, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings and histopathologic examination in either SD rat sex. The rat no observed adverse effects level (NOAEL) for FPGE was set to 3000 mg/kg. CONCLUSIONS: The present study empirically demonstrated that FPGE has a safe preclinical profile and indicated that it could be safely integrated into health products for atopic dermatitis treatment.

Preclinical safety assessment of a therapeutic human papillomavirus DNA vaccine combined with intravaginal interleukin-7 fused with hybrid Fc in female rats.

This study was conducted to establish the toxicological profile of combination treatment with therapeutic HPV DNA vaccines (GX-188E) and the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (IL-7hyFc). GX-188E was administered intramuscularly by electroporation with or without IL-7hyFc intravaginally once per 2 weeks for 8 weeks (five times) in female Sprague-Dawley rats. Because up-regulation of immune responses and migration of antigen-specific T cells in cervicoviginal tissue were predicted as therapeutic effects, we distinguished adverse effects from therapeutic effects based on the severity of the systemic immune response, reversibility of lymphoid tissue changes, target tissue damage, and off-target immune responses. We observed that the number of neutrophils was increased, and the number of lymphocytes was decreased in the blood. Further, myofiber degeneration, necrosis, fibroplasia, and cell infiltration were observed at the GX-188E administration site. These changes were fully or partially recovered over a 4-week period. Analysis of lymphocytes in spleen revealed that CD4+ T cells and total T cells decreased in rats treated with GX-188E in combination with a high dose of IL-7hyFc (1.25 mg/animal). However, these changes were not considered adverse because they were transient and may have been related to electroporation-mediated DNA delivery or the local migration of lymphocytes induced by IL-7. Therefore, the potential toxicity of the combination of GX-188E and IL-7hyFc treatment was comparable to that of GX-188E treatment alone, and the no observed adverse effect level for GX-188E with IL-7hyFc was considered as 320 µg/animal for GX-188E and 1.25 mg/animal for IL-7hyFc.

Safety evaluation of a novel variant of consensus bacterial phytase.

A 90-day subchronic oral toxicity study was conducted to evaluate the safety of a consensus bacterial phytase variant 6-phytase (PhyG) for use as an animal feed additive. This phytase is produced by fermentation with a fungal (Trichoderma reesei) production strain expressing a biosynthetic variant of a consensus bacterial phytase gene assembled via ancestral reconstruction with sequence bias for the phytase from Buttiauxella sp. Rats were administered PhyG daily via oral gavage at dose-levels of 0 (distilled water), 250, 500 or 1000 mg total organic solids (TOS)/kg bodyweight (bw)/day (equivalent to 0, 112,500, 225,000 and 450,000 phytase units (FTU)/kg bw/day, respectively). No test article-related adverse effects were observed. A no-observed-adverse-effect level (NOAEL) for PhyG was established as 1000 mg TOS/kg bw/day, the highest test concentration. Based on this NOAEL and an estimate of broiler consumption determined from the proposed inclusion of the phytase in feed at the maximum recommended level (4000 FTU/kg), a margin of safety value of 1613 was calculated. Results of in vitro genotoxicity testing and in silico protein toxin evaluation further confirmed PhyG to be non-genotoxic and not likely to be a protein toxin upon consumption. These data support the safety of PhyG as an animal feed additive.

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

Toxicity Screening of Single Dose of Inorganic and Organic Arsenics on Hematological and Serum Biochemical Parameters in Male Cynomolgus Monkeys.

A screening study of the acute toxicity of organic arsenics such as arsenobetaine and arsenocholine, a product of arsenic methylation metabolite, and inorganic arsenic was carried out to examine hematological and serum biochemical parameters in cynomolgus monkeys (Macaca fascicularis). We found soft and liquid feces, and vomiting in all treated groups with inorganic and organic arsenics. The monkeys in inorganic arsenic-treated group showed a significant increase in vomiting frequency compared with those in three organic arsenics-treated groups. These results suggest that inorganic arsenic might be more toxic than three other organic arsenics tested. The monkeys in inorganic arsenic-treated group showed a decrease in platelet and an increase in monocyte on day 4 and the monkeys in arsenocholine-treated group showed an increase in reticulocyte percentage on day 8. The monkeys in inorganic-treated group also showed decreases in AST and ALT values and the monkeys in arsenobetaine-treated group showed a decrease in AST value and an increase in T-CHO value. However, these hematological and biochemical changes were within the physiological ranges, showing that the single dose of inorganic and organic arsenics did not affect at least hematological and serum biochemical parameters. The present study of toxicity with single dose of arsenics provides valuable indicators for longer term study of toxicity of repeated doses of arsenics in primates.