Chronic preclinical safety evaluation of EPO-018B, a pegylated peptidic erythropoiesis-stimulating agent in monkeys and rats

Toxicology and Applied Pharmacology 307 (2016) 45–61

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Chronic preclinical safety evaluation of EPO-018B, a pegylated peptidic erythropoiesis-stimulating agent in monkeys and rats Xue-Lian Gong a,1, Xiao-Lei Gu a,1, Yong-Chun Chen a,b,1, Hai Zhu a, Zhen-Na Xia a, Jian-Zhong Li c,⁎, Guo-Cai Lu a,⁎ a b c

Department of Hygiene and Toxicology, Second Military Medical University, Shanghai 200433, China Department of Pharmacy, No.422 Hospital, Zhanjiang 524005, China Department of Biochemical Pharmacy, Second Military Medical University, Shanghai 200433, China

a r t i c l e

i n f o

Article history: Received 29 May 2016 Revised 19 July 2016 Accepted 20 July 2016 Available online 22 July 2016 Keywords: EPO-018B Chronic toxicity Cynomolgus Monkeys Sprague-Dawley Rats

a b s t r a c t EPO-018B, a synthetic peptide-based erythropoiesis stimulating agent (ESA), is mainly designed for treatment of anemia caused by chronic renal failure and chemotherapy against cancer. It overcomes the deficiencies of currently approved ESA, including the frequent administration of temperature-sensitive recombinant protein and anti-EPO antibody-mediated pure red cell aplasia (PRCA). This study was designed to evaluate the potential chronic toxicity of EPO-018B. Subcutaneous administration doses were designed as 0, 0.2, 1 and 10 mg/kg for six months for 160 rats (20/gender/group) and 0, 0.3, 3 and 20 mg/kg for nine months for 32 monkeys (4/gender/group) once every three weeks. The vehicles received the same volume of physiological saline injection. All animals survived to the scheduled necropsies after six weeks (for rats) and fourteen weeks (for monkeys) recovery period, except for the two high-dose female rats and two high-dose male monkeys, which were considered related to the increased RBCs, chronic blood hyperviscosity and chronic cardiac injury. EPO-018B is supposed to be subcutaneously injected once every month and the intended human therapeutic dose is 0.025 mg/kg. The study findings at 0.2 mg/kg for rats and 0.3 mg/kg for monkeys were considered to be the study NOAEL (the no observed adverse effect level), which were more than ten times the intended human therapeutic dose. Higher doses caused adverse effects related to the liver toxicity, cardiotoxicity, appearance of neutralizing antibodies of EPO-018B and the decrease of serum glucose and cholesterol. Most treatment-induced effects were reversible or revealed ongoing recovery upon the discontinuation of treatment. The sequelae occurred in rats and monkeys were considered secondary to exaggerated pharmacology and would less likely occur in the intended patient population. As to the differences between human beings and animals, the safety of EPO-018B need to be further confirmed in the future clinical studies. © 2016 Published by Elsevier Inc.

1. Introduction Erythropoietin (EPO) is secreted when a signal of hypoxia is received by the oxygen sensor of the kidney, and stimulates the proliferation, survival and differentiation of erythroid progenitor cells (Haase, 2010). EPO is mainly produced by capillary endothelial cells and fibroblasts in cortical nephrons, which contain N85% of the total amount of EPO (Nagai et al., 2014). EPO is the most efficient drug for the treatment of anemia caused by chronic renal failure, chemotherapy against cancer, autoimmune disorders and malnutrition; which owes primarily to the hypoproliferation of red blood cell (RBC) precursors secondary to EPO deficiency. EPO-018B is a synthetic dimeric peptide-based PEGylated erythropoiesis-stimulating agent (ESA) that was designed to specifically bind ⁎ Corresponding authors. E-mail addresses: [email protected] (J.-Z. Li), [email protected] (G.-C. Lu). 1 These authors contributed equally to this paper.

http://dx.doi.org/10.1016/j.taap.2016.07.014 0041-008X/© 2016 Published by Elsevier Inc.

and activate the EPO receptor, resulting in the production of RBC; which is a structure analogue of Hematide™ (Moller et al., 2011). EPO-018B has been developed by the Research Laboratory of Hansoh Pharmaceutical Co., Ltd. (Jiangsu, China) for over 10 years. Since the amino acid sequence of the synthetic peptide is unrelated to human EPO, EPO-018B is not likely to induce a cross-reactive immune response against either recombinant or endogenous EPO; which is similar to Hematide™ (Del Vecchio et al., 2010). This overcomes the deficiencies of currently approved ESA, including recombinant human erythropoietin (rHuEPO); namely, the frequent administration of temperature-sensitive recombinant protein (must be stored at temperatures of approximately 4 °C) and anti-EPO antibody-mediated pure red cell aplasia (PRCA) (Woodburn et al., 2008a, 2008b; Leuenberger et al., 2011). According to pharmacodynamics studies, EPO-018B revealed relatively better erythropoiesis-stimulating efficacy and higher specific activity than Hematide™. The intended clinical dose of EPO-018B is 0.025 mg/kg once monthly, which will be administered subcutaneously to adult dialysis patients with anemia associated with chronic renal failure. The preclinical safety evaluation studies of EPO-018B were

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performed according to the guidelines issued by China Food and Drug Administration. As is prescribed in guidelines of repeated-dose toxicity studies (Edition 2007), two species and at least three dose levels must be designed in sub-chronic toxicity tests and the animals must be administered for at least two weeks. In chronic toxicity tests, the nonhuman primates should be dosed for nine months and the non-rodents dosed for six months for drug applied for at least six months in clinical treatment. The subchronic toxicity of EPO-018B has been evaluated in Cynomolgus monkeys and Sprague-Dawley rats by five-week multiple subcutaneous injections (Gong et al., 2013). These results revealed that most of the toxicological effects observed were related to exaggerated pharmacology and secondary sequelae of erythropoiesis-stimulating efficacy to healthy animals, such as the increased whole blood viscosity and blood pressure. Besides, the megakaryocytes and lymphocytes in administrative groups also proliferated actively after EPO-018B administration. In order to support chronic clinical therapy, a comprehensive non-clinical safety program was undertaken, including a six-month safety study in rats and a nine-month safety study in monkeys; providing further experimental evidences for its safe application in future clinical trials. 2. Materials and methods 2.1. Test substance EPO-018B is a synthetic peptide coupled to PEG (PEG molecules of 40 kDa), which is supplied by the Research Laboratory of Hansoh Pharmaceutical Co, Ltd., China. The purity of EPO-018B was higher than 97.3%, and was reserved in diaphanous glass ampoule bottles at −20 ° C. Before used, the appropriate amount of EPO-018B administration was calculated and dissolved in physiological saline. The concentration and four-hour stability of the tested articles were analyzed, which ranged from 95.7% to 104.6%, to ensure the proper administration of these reported doses. 2.2. Species and dose selection In pharmacodynamics studies, EPO-018B revealed relatively better erythropoiesis-stimulating efficacy and higher specific activity in human UT7/EPO cells than Hematide™. The activity of EPO-018B was also supported by in vivo rat studies, where EPO-018B increased reticulocytes, red blood cells, hemoglobin, and hematocrit levels in SpragueDawley rats. In pharmacokinetic studies, the subcutaneous bioavailability (subcutaneous vs intravenous injection dose) of EPO-018B was 73.1% in monkeys (exactly the same as in human beings) and 30.5% in rats, when 100 μg/kg of EPO-018B was single-dosed, respectively. Since these animals had the highest bioavailability for EPO-018B and were the same as animal species in preclinical studies of Hematide™, the monkeys and rats were dosed for preclinical evaluation in the toxicity tests of EPO-018B (Woodburn et al., 2009; Woodburn et al., 2010). For dose selection evidences that were primarily referred to the previous preclinical toxicological evaluation of Hematide™ (Woodburn et al., 2008a, 2008b; Woodburn et al., 2009), 280 rats received an intravenous injections of 0, 0.1, 1 and 10 mg/kg every three weeks for six months, followed by a six-week recovery period. In addition, 96 monkeys received intravenous injections of 0, 0.2, 2 and 20 mg/kg every three weeks for nine months, followed by a 14-week recovery period. After 90 days of study, unscheduled deaths were observed in 40 rats (40/70) and two rats (2/70) in the 10 mg/kg and 1 mg/kg groups, respectively. Three high-dose monkeys (3/32, 20 mg/kg) died on day 79, 104 and 207 during the dose period. Hematide™ was considered to be the cause of the mortality in the above accidental deaths. Furthermore, in the previous study of EPO-018B, subcutaneous administration doses for rats and monkeys were both designed as 0, 0.5, 5 and 50 mg/kg once a week for five weeks, followed by a six-week

recovery period for rats and a 12-week recovery period for monkeys (Gong et al., 2013). These results showed that most of the toxicological effects observed were related to the exaggerated pharmacology and secondary sequelae of erythropoiesis-stimulating efficacy to healthy animals, such as the increased whole blood viscosity and blood pressure. Besides, the megakaryocytes and lymphocytes in administrative groups also proliferated actively after EPO-018B administration. The raise of TP, LDH, TBIL and potassium and the decrease of glucose at hematology were deemed as relevant to the energy consumption of cells proliferation and improved metabolism rates of red blood cells. Based on the above data, subcutaneous administration doses were designed as 0, 0.2, 1 and 10 mg/kg for 30 weeks (six months) for rats, and 0, 0.3, 3 and 20 mg/kg for 42 weeks (nine months) for monkeys. Compared with the intended clinical dose, the doses of monkey studies covered from 12 to 800 times over the clinical dosing regimen, and 8 to 400 times for rats. Control animals received physiological saline injection of the same volume as that given to the administrative animals. All animals were dosed every three weeks, followed by a recovery period of 6 weeks for rats and 14 weeks for monkeys. 2.3. Animal care Animal care was in compliance with institutional animal care guidelines, and the applied protocols were approved by the Local Institutional Committee of the Second Military Medical University. Quarantine and acclimation periods of one week for rats and eight weeks for monkeys were carried out before the administration of subcutaneous injection. Animals judged to be suitable for assignment to the study were grouped by a computerized randomization procedure. Eighty male (weight: 137.4–168.2 g) and 80 female (weight: 124.0– 158.2 g) specific pathogen-free Sprague-Dawley rats (6–8 weeks old) were assigned in the experiment. These rats were obtained from B&K Universal Group Laboratorial Animal Co., Ltd. (Shanghai, China). Sixteen male (weight: 2.70–3.80 kg) and 16 female (weight: 2.60–3.25 kg) Cynomolgus monkeys (3–4 years old) were obtained from Xishan Zhongke Laboratorial Animal Co., Ltd. (Jiangsu, China). 2.4. Experimental design Non-clinical laboratory studies were performed in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification [S] GPT1-1, issued by the China Food and Drug Administration on January 2007) under Good Laboratory Practice Regulations. Eighty rats or 16 monkeys of each gender were randomized into four groups, and subcutaneously injected with EPO-018B (20/gender/group for rats and 4/gender/group for monkeys). Rats were dosed at 0, 0.2, 1 and 10 mg/kg once every three weeks for 27 weeks (a total of 10 times), and monkeys were dosed at 0, 0.3, 3 and 20 mg/kg once every three weeks for 39 weeks (a total of 14 times). After all administrations, the above experiments sustained a recovery period of six weeks for rats and 14 weeks for monkeys. Rats were anesthetized with sodium pentobarbital and sacrificed at day 91 (five animals/gender/group) and day 196 (10 animals/gender/ group, and eight females were in the 10 mg/kg group due to early death). The remaining rats (five animals/gender/group) were euthanized at day 232. In addition, half of these monkeys were anesthetized and sacrificed at day 281. The remaining monkeys (four animals/gender/group, and two male monkeys in the 20 mg/kg group due to early death) were euthanized at day 372. Clinical observations were recorded, which included ophthalmological examinations, as well as body weight, food consumption and daily clinical symptoms observation. Clinical pathological evaluations (hematology, serum chemistry detection, urinalysis and immune detection) were also performed. In addition, organ weight, histopathological observation and bone marrow cytomorphologic inspection were all

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performed. For monkeys, electrocardiographic examinations and blood pressure were additionally performed. 2.5. Clinical observation The observation for mortality and viability were recorded daily from the beginning of the quarantine period. Clinical symptoms, signs and food consumption in rats and monkeys were examined daily. In rats, body weights were recorded weekly throughout the treatment and recovery periods. In monkeys, body weights were recorded at pre-administration (twice, day one and day two), day 28, day 91, day 196, day 281, day 372 and every time before EPO-018B administration. Ocular fundus examinations, slit lamp examinations, and macroscopic observations were carried out during the pre-administration phase, at the end of the treatment, and after the recovery period. The conjunctiva, sclera, cornea, lens and iris of each eye of the rats and monkeys were also examined. For monkeys, rectal temperature, size of the pupil, and frequency of respiration were also recorded at pre-administration (twice, day one and day two), day 28, day 91, day 196, day 281 and day 372. 2.6. Clinical test parameters 2.6.1. ECG and blood pressure measurement. For monkeys, P wave, R wave, T wave, ST segment, P-R intervals, Q-T intervals, QRS duration and heart rate were recorded by the derivation DII of the ECG system. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP) were measured using an intelligent non-invasive sphygmomanometer. The indicators above were recorded at preadministration (twice, day one and day two), day 28, day 91, day154, day 196, day238, day281, day329 and day 372. 2.6.2. Hematology. Blood samples were collected in vacuum tube containers with sodium citrate. The following hematological parameters were examined: RBC count, hemoglobin (HB) concentration, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), RBC distribution width (RDW), platelet (PLT) count, mean platelet volume (MPV), white blood cell (WBC) count and WBC differential counts, including lymphocyte (LYMPH%), monocyte (MONO%), neutrophile granulocyte (NEUT%), eosinophils (EOS%), basophilic cell (BASO%) and large unstained cell (LUC%). In addition, reticulocyte (RETIC%), reticulocyte mean cellular volume (MCVr), reticulocyte mean cell hemoglobin concentration (CHCMr) and reticulocyte hemoglobin content (CHr) were measured. Coagulation indexes such as thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (FIB) were also detected in both monkeys and rats. 2.6.3. Serum biochemistry. Blood for clinical chemistry was collected in vacuum tubes devoid of anticoagulants, allowed to clot at room temperature, centrifuged, and the serum was separated. The detected serum biochemistry parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (CREA), creatine phosphokinase (CPK), MB isoenzyme of creatine phosphokinase (CPK-MB), uric acid (UA), total bilirubin (T-BIL), direct bilirubin (D-BIL), total protein (TP), albumin (ALB), globulin (Glo), total cholesterol (TCH), triglyceride (TG), glucose (GLU), γglutamyltranspeptidase (γ-GTP), ferrum (Fe), calcium (Ca) and phosphorus (P); which were determined by an automatic analyzer. Serum electrolytes including chloride (Cl), sodium (Na) and potassium (K) were measured as well. 2.6.4. Urinalysis. Urine samples from rats and monkeys were analyzed for specific gravity, pH, leukocytes, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, occult blood and hemoglobin.

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2.6.5. Immunotoxicity tests. Serum immune globulins including IgG, IgA, IgM (rat R&D, monkey Millipore Corp.) and serum complement (C3, C4) were detected by enzyme-linked immunosorbent assay (Life Science). Serum-circulating immune complexes (CIC) were analyzed by the UV-spectrophotometric method. Serum IL-1 and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (rat R&D, monkey eBioscience). Anti-EPO-018B antibodies were detected by enzyme-linked immunosorbent assay in the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (Beijing, China). CD3+/CD4+/CD8+ expression levels on peripheral blood lymphocytes were determined by flow cytometry using FITC-labeled anti-CD8 primary antibody (BD Pharmingen), PE-labeled anti-CD4 primary antibody (BD Pharmingen) and Cy5.5-labeled anti-CD3 primary antibody (BD Pharmingen). Rat or monkey mAb IgG1 were used as isotype controls. Relative antigen expression was reported as median fluorescence intensity (MFI). 2.6.6. Gross observation and organ weights. All surviving animals were sacrificed by exsanguination under anesthesia as scheduled and planned, and were examined carefully for macroscopic abnormalities. The absolute and relative (organ-to-body weight ratios) weights of major organs including the heart, liver, lungs, spleen, kidneys, brain, thymus, adrenal glands, thyroid gland (only for monkeys), testes, epididymis, uterus and ovaries were measured. 2.6.7. Histopathology and bone marrow cytomorphologic inspections. Histopathologic findings were recorded, and all results were compared with that in controls. The cellular medullares were obtained from the femoral bone. After film preparation and dyeing, bone marrow cytomorphologic examination was performed microscopically. 2.7. Statistical analysis 2.7.1. Rat study. For each gender, experimental data were analyzed by parametric one-way analysis using the F-test (ANOVA, two-sided) with the statistical software, Statistical Product and Service Solutions (SPSS, v11.5). If the resulting P-value was b0.05, a comparison of each group using the LSD test was performed for the hypothesis of equal means. When the data could not be assumed to follow a normal distribution test even after conversion, these would be analyzed by non-parametric one-way analysis using the Kruskal–Wallis test. Dunnett's T3 test was applied when the data could not be assumed to follow homogeneity of variance. Urinalysis data were analyzed using NPar Kruskal–Wallis test. 2.7.2. Monkey study. Experimental data were analyzed by repeated measures analysis of variance (ANOVA) with SPSS v16.0. If the resulting P-value was b 0.05, pairwise comparisons were performed to isolate the difference among the groups and testing periods. Urinalysis data were analyzed using NPar Kruskal–Wallis test. Since the number of monkeys per group was small (four/gender/group), male and female data were combined in monkey studies. More attention was focused on individual monkey data due to the small sample number. 3. Results 3.1. Clinical observation All animals survived to the scheduled necropsies, except for the two high-dose female rats (at day 183 and day 195) and two high-dose male monkeys (at day 309 and day 321) that died. There was no test articlerelated effect on the clinical observations.

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3.2. Blood pressure and ECG After nine months of administration, the SBP and DBP of monkeys in the 20 mg/kg, 3 mg/kg and 0.3 mg/kg groups were significantly elevated or had increased tendencies in different degrees from day 28 to day 281, followed by a significant decreased tendency in the 20 mg/kg group at the end of the recovery period (day 329 and day 372, Fig. 1). No significant change was found in the electrocardiographic measurements of monkeys in any of the dose groups, and ECG parameters were all within normal ranges, including QTc intervals.

3.3. Hematology EPO-018B administration for nine months on monkeys and six months on rats resulted in erythropoiesis, which led to pronounced and sustained polycythemia on all dose groups. Hematologic results were exactly same with the known erythropoietic pharmacological action of ESAs (Ribeiro et al., 2016; Zabaneh et al., 2015). In rats of all dose groups, RBC, HGB and RETIC% increased at day 91 and day 196; which was followed by subsequent decreases at the end of the recovery period (day 232, Fig. 2.1). Relevant erythrocytic parameters were also altered, including increased Hct and RDW, as well as decreased MCHC, in all dose groups at day 91 and day 196. The cellular hemoglobin concentration mean of reticulocytes (CHCMr) also decreased in all female dose groups at day 91 and day 196. In addition, MCV increased in the 0.2 and 1 mg/kg groups and decreased in the 10 mg/kg group, while MCVr decreased in the 10 mg/kg group at day 91 and day 196; which were consistent with the reported erythropoietic pharmacological action of Hematide™ (Woodburn et al., 2008a, 2008b). It was suspected that increased MCV in the low and middle dose groups may be the consequence of the excessive proliferation of RBC, which led to the rapid release of late erythroid precursors residing in the bone marrow (Chu et al., 2014). Conversely, reduced MCV in the high dose groups may be due to occurrence of functional iron deficiency following massive reticulocytosis and erythroid bone marrow exhaustion (Woodburn et al., 2008a, 2008b). Differently, reduced MCV in the 1 and 10 mg/kg groups at day 232 may be results of the sharp increase in serum ferrum caused by the rapid destruction of RBCs; which originated from the sudden withdrawal of EPO-018B during the recovery period. Accordingly, mean corpuscular hemoglobin (MCH) decreased in the 10 mg/kg group and reticulocyte hemoglobin content (CHr) decreased in all dose groups at day 91 and day 196 (Table 1). In addition, WBC increased in the 1 and 10 mg/kg groups at day 91, and in all dose groups at day 196. LYMPH% increased and NEU% decrease in all dose groups at day 91 and day 196. Furthermore, peripheral PLT also increased in the 10 mg/kg group and in the 1 and 0.2 mg/kg male groups at day 91 and day 196 (Fig. 2.1). Relevant thrombocytic parameters were also altered including increased MPV and APTT in all dose groups at day 91 and day 196. In the recovery period at day 232,

all indexes referred above were completely restored or had a significant recovery tendency compared to the control group (Table 1). In monkeys, RBC, HGB, RETIC% and PLT increased in all dose groups from day 28 to day 281, followed by subsequent declines at day 329 and day 372 (Fig. 2.2). Relevant erythrocytic and thrombocytic parameters were also altered including increased Hct, RDW, MPV and APTT, as well as decreased MCH, MCHC, MCVr, CHCMr and CHr, in all dose groups from day 28 to day 281. Simultaneously, MCV increased at day 28, which might reflect the rapid release of late erythroid precursors in the bone marrow due to the excessive proliferation of RBC in the early stages. Continuously, MCV decreased or had decreased tendencies from day 91 to day 281, which might be attributed to the occurrence of functional iron deficiency, following massive reticulocytosis and erythroid bone marrow exhaustion (Diekmann et al., 2012). All indexes referred above recovered completely or had no significant difference when compared to that of day zero or the control groups at day 329 and day 372 (Tables 2.1 and 2.2). Toxicologically relevant changes in PT, TT and FIB were not observed. 3.4. Clinical chemistry In rats, AST in the 10 mg/kg group was significantly elevated compared to that in the control group at day 91 and day 196. TBIL in the 10 and 1 mg/kg female groups at day 91 and in the 10 and 1 mg/kg groups at day 196 all significantly increased, compared to the control group. DBIL in the 10 and 1 mg/kg groups were also significantly elevated at day 91 and day 196. These indicators above were considered to be consequences of the toxicological effects of EPO-018B, reflecting liver toxicity. In addition, glucose, total cholesterol and ferrum significantly decreased in all dose groups, compared to of the control group at day 91 and day 196. After the six-week recovery period, all indicators referred above were completely restored. Furthermore, ferrum in the 10 and 1 mg/kg groups significantly increased, compared to the control group at day 232. These metabolic changes revealed the extended pharmacodynamic effects of EPO-018B, which might have been caused by the increased energy consumption spent in the production of RBCs (Jilani and Glaspy, 1998). Furthermore, it was found that potassium increased in the 10 mg/kg female group at day 91 and in the 10 mg/kg group at day 196. Chloride decreased in the 10 mg/kg group and the 1 mg/kg female group at day 91 and day 196. Calcium increased in the 10 mg/kg male group at day 91 and in the 10 mg/kg group at day 196. Phosphorus increased in the 10 mg/kg group and in the 1 mg/kg female group at day 91 and day 196 (Tables 3.1 and 3.2). In monkeys, TBil in the middle dose group significantly increased at day 91. Furthermore, TBil and DBil in the middle and high dose groups significantly increased from day 196 to day 281. Moreover, LDH in the middle and high dose groups significantly increased from day 91 to day 281. These indicators above were considered to be relevant to the heart and liver toxicity of EPO-018B to monkeys.

Fig. 1. Blood pressure curves (including SBP, DBP and MBP) for monkeys given EPO-018B for nine months and followed by 14-week recovery period. *P b 0.05, compared to the control group.

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Fig. 2.1. Selected hematology parameters of rats after 6-month administration of EPO-018B and 6 weeks recovery period.

In addition, glucose significantly decreased in all dose groups at day 196, day 238 and day 281. Ferrum significantly increased in all dose groups at day 28, day 91, day 196, day 238 and day 281 (Table 4). These indicators reflect the metabolic changes of EPO-018B to healthy monkeys.

3.5. Urinalysis No treatment-related change was observed in urinalysis parameters during the course of the study. All changes that attained statistical

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Fig. 2.1 (continued).

significance were considered to be incidental, and there was no clear dose-response relationship (data not shown). 3.6. Immunotoxicity In immunotoxicity tests, two monkeys (one in the 3 mg/kg group and one in the 0.3 mg/kg group) out of the 24 dosed animals were tested positive for EPO-018B specific antibodies, both after the fourth administration. Accordingly, there was no EPO-018B detected from plasma in the two antibody-positive monkeys since the fourth dosage, based on data of the toxicokinetics tests (data not shown). The presence of antibodies was accompanied by the reversion of erythroid-related indicators such as RBC, Hb, WBC and PLT. The alteration indicated that these antibodies might be neutralizing antibodies of EPO-018B, but the speculation need to be confirmed further in the clinical studies No anti-drug antibody was seen in any of the samples collected from rat studies. No treatment-related change was found on immunoglobulin, complement or classification assay of lymphocytes from peripheral blood of subjected animals. In addition, it was found that IL-1 and TNF-α level in rats in all dose groups increased or had tendencies to increase at day 91 and day 196,

followed by the subsequent decrease at day 232 (Table 5). In monkeys, serum IL-1 in all dose groups increased or had tendencies to increase at day 28, day 91, day 196, day 238 and day 281; followed by the subsequent decrease at day 372 (Table 6).

3.7. Necropsy and organ weights 3.7.1. Unscheduled deaths. Unscheduled deaths included two highdose female rats (10 mg/kg) at day 183 and day 195, and two highdose male monkeys (20 mg/kg) at day 309 and day 321. The animals were found dead early in the morning before feeding. There was no tested data or adverse clinical signs related to these two dead rats during the administration period. For the data demonstrated in monkeys, increased RBC count (8.65, 8.23 vs 5.46), Hct (67.1, 62.7 vs 40.8) and SBP (209, 197 vs 171) were present at day 281. In the autopsy, it was found that myocardial edema, the fibrosis of lesions and compensatory myocardial hypertrophy and nuclear hypertrophy existed in the monkey died at day 309. The other one did not show obvious lethal pathological changes macroscopically or microscopically. Therefore, we considered that increased RBCs, chronic blood hyperviscosity, elevated

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Table 1 Selected hematology parameters of rats after 6-month administration of EPO-018B and 6-week recovery.

Male D91

44.20 ± 2.19 52.06 ± 0.96 17.26 ± 0.48 331.40 ± 3.36 49.64 ± 55.04 ± 2.29 18.08 ± 0.91 328.20 ± 3.56 3.45⁎⁎

12.10 ± 0.29 64.18 ± 1.56 282.80 ± 1.92 18.10 ± 0.47 7.44 ± 0.32 18.76 ± 63.64 ± 2.91 279.20 ± 3.27 17.70 ± 0.76 10.14 ± 0.96⁎⁎ 0.93⁎⁎

16.38 ± 6.94 17.28 ± 3.29

1.0

51.90 ± 0.88⁎⁎

53.32 ± 3.79 17.08 ± 1.39 320.40 ± 3.29⁎⁎

21.40 ± 0.89⁎⁎

60.54 ± 4.48 279.00 ± 3.08 16.72 ± 1.13⁎

15.72 ± 3.07⁎⁎

16.62 ± 2.84

10.0

59.22 ± 2.35⁎⁎

49.92 ± 1.09 15.66 ± 0.27⁎⁎

22.00 ± 0.72⁎⁎

56.92 ± 2.13⁎⁎

21.18 ± 1.00⁎⁎

18.44 ± 4.90

10.0 D232 0 0.2 1.0 10.0

Female D91 0 0.2 1.0 10.0

313.40 ± 3.51⁎⁎

RDW (%)

MCVr (fL)

CHCMr (g·L−1)

286.20 ± 4.76 16.06 ± 0.38⁎⁎

44.27 ± 3.36 53.80 ± 1.60 17.21 ± 0.58 319.70 ± 6.09 57.43 ± 56.33 ± 2.47 18.00 ± 0.78 319.40 ± 3.17 3.73⁎⁎

11.39 ± 0.65 63.55 ± 1.39 281.50 ± 4.20 17.85 ± 0.53 7.06 ± 0.60 17.37 ± 62.51 ± 3.43 276.30 ± 3.37 17.19 ± 0.96 10.94 ± 0.74⁎⁎ 2.22⁎⁎

13.79 ± 3.42 19.23 ± 3.74⁎⁎

59.47 ± 7.80⁎⁎

19.68 ± 3.48⁎⁎

61.04 ± 4.64 280.90 ± 5.11 16.98 ± 1.38⁎

17.71 ± 4.54⁎⁎

20.29 ± 4.28⁎⁎

21.29 ± 0.70⁎⁎

57.76 ± 2.69⁎⁎

24.79 ± 1.20⁎⁎

22.28 ± 2.60⁎⁎

68.24 ± 3.04⁎⁎

56.36 ± 3.27 17.47 ± 1.73 309.90 ± 20.67 53.14 ± 1.41 16.22 ± 1.11 305.30 ± 20.05⁎⁎

283.00 ± 4.74 16.10 ± 0.49⁎⁎

44.84 ± 0.35 53.36 ± 0.49 15.74 ± 0.38 294.80 ± 5.89 43.48 ± 1.79 53.06 ± 2.28 15.84 ± 0.43 298.60 ± 6.15

12.30 ± 0.53 66.02 ± 1.01 300.80 ± 3.42 19.82 ± 0.28 6.40 ± 0.56 14.44 ± 65.52 ± 0.97 303.80 ± 3.83 19.86 ± 0.31 6.74 ± 0.34 0.34⁎⁎

15.74 ± 1.26 17.86 ± 3.28

43.64 ± 1.45 51.76 ± 2.67 15.52 ± 0.59 299.80 ±

17.54 ± 0.92⁎⁎

67.74 ± 2.52 305.20 ± 3.11 20.64 ± 0.67 6.64 ± 0.36

15.44 ± 2.29

17.16 ± 0.99⁎⁎

65.02 ± 2.07 310.00 ± 4.69⁎⁎

20.06 ± 0.70 7.02 ± 0.37

15.90 ± 2.86

10.68 ± 0.27 65.02 ± 2.19 294.40 ± 4.28 19.12 ± 0.74 7.38 ± 0.41 18.42 ± 68.50 ± 2.06 288.00 ± 19.70 ± 0.76 8.38 ± 0.35 0.60⁎⁎ 3.54⁎ 22.00 ± 68.74 ± 6.05 283.20 ± 19.44 ± 2.07 11.74 ± 0.16⁎⁎ 4.76⁎⁎ 2.94⁎⁎ 22.82 ± 62.78 ± 3.47 282.40 ± 17.56 ± 0.78 21.42 ± 1.34⁎⁎ 4.77⁎⁎ 3.44⁎⁎

16.50 ± 2.07 17.80 ± 2.02

10.73 295.80 ± 9.09

49.02 ± 3.23 48.78 ± 0.54⁎⁎

14.44 ± 0.56⁎⁎

41.02 ± 1.38 53.40 ± 2.44 52.32 ± 59.80 ± 1.84⁎⁎ 2.06⁎⁎ 56.50 ± 58.92 ± 2.09⁎⁎ 3.66⁎⁎

18.26 ± 1.00 342.40 ± 4.62 19.92 ± 333.80 ± 8.53⁎ 0.72⁎

61.72 ± 4.12⁎⁎

D196 0 0.2

MCHC (g/L)

APTT (s)

0 0.2

1.0

MCH (pg)

MPV (fL)

Hct (%)

D196 0 0.2

MCV (fL)

CHr (pg)

Dose (mg/kg)

19.14 ± 1.39 325.00 ± 5.15⁎⁎

54.34 ± 2.45 17.42 ± 0.56 321.00 ± 4.58⁎⁎

20.00 ± 2.45 20.72 ± 9.31

41.05 ± 1.75 56.43 ± 1.78 18.57 ± 0.48 329.20 ± 4.39 56.47 ± 62.72 ± 20.60 ± 328.80 ± 6.70 3.68⁎⁎ 2.09⁎⁎ 0.71⁎⁎

10.47 ± 1.08 66.12 ± 2.27 291.40 ± 5.80 19.22 ± 0.59 6.68 ± 0.37 17.76 ± 70.26 ± 281.90 ± 19.76 ± 0.83 7.60 ± 1.06⁎⁎ 3.05⁎ 5.02⁎⁎ 0.43⁎⁎

14.03 ± 2.11 15.87 ± 3.16

1.0

66.81 ± 2.82⁎⁎

61.39 ± 3.30⁎⁎

19.10 ± 2.18 310.50 ± 27.17⁎⁎

20.11 ± 0.85⁎⁎

67.41 ± 3.97 272.40 ± 2.80⁎⁎

18.29 ± 1.11 12.62 ± 2.80⁎⁎

20.41 ± 2.53⁎⁎

10.0

72.98 ± 2.36⁎⁎

55.65 ± 2.84 17.60 ± 1.06 316.00 ± 6.82⁎⁎

21.66 ± 1.06⁎⁎

62.93 ± 5.78 277.88 ± 4.42⁎⁎

17.24 ± 1.49⁎⁎

25.84 ± 4.97⁎⁎

D232 0 0.2 1.0 10.0

24.44 ± 6.62⁎⁎

44.26 ± 0.98 57.58 ± 1.78 17.56 ± 0.69 304.80 ± 4.82 42.00 ± 56.10 ± 2.01 16.90 ± 0.53 301.20 ± 8.29 1.18⁎

10.80 ± 0.69 68.94 ± 2.88 315.40 ± 2.30 21.66 ± 0.99 6.68 ± 0.50 12.90 ± 67.56 ± 2.08 310.80 ± 20.94 ± 0.63 6.72 ± 0.41 0.46⁎⁎ 1.48⁎

13.88 ± 2.00 15.32 ± 0.43

39.90 ± 1.42⁎⁎

14.98 ± 0.74⁎⁎

67.48 ± 1.71 315.00 ± 5.48 21.22 ± 0.81 6.74 ± 0.51

14.80 ± 1.98

15.68 ± 0.83⁎⁎

64.96 ± 2.72 311.80 ± 4.82 20.18 ± 0.66 7.14 ± 0.46

15.72 ± 2.89

53.92 ± 2.04⁎⁎

16.60 ± 0.96⁎

44.32 ± 2.57 51.00 ± 1.74⁎⁎

15.36 ± 0.48⁎⁎

307.80 ± 10.06 301.40 ± 9.89

⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group.

blood pressure and chronic cardiac injury might be the major causes of death of the monkeys. 3.7.2. Organ weight. Rats were sacrificed at day 91, day 196 and day 232; except for two high-dose female rats that died at day 183 and day 195. For male rats in the 10 mg/kg group, higher absolute and relative weight of the liver was observed at day 196. For female rats, the absolute and relative weight of the liver increased in the 10 mg/kg group at day 91 and day 196. Relative weight of the liver in the 1 mg/kg female group also increased at day 91. In addition, apparent increases in absolute and relative weights of spleens were found in all dosed groups at day 91 and day 196, compared to controls; and there was an obvious recovery trend at day 232 (Table 7). Monkeys were sacrificed at day 281 and day 372, except for two high-dose male monkeys that died at day 309 and day 321. The relative weight of the liver increased in the 3 mg/kg and 20 mg/kg groups at day 281. The absolute and relative weight of the spleen increased in all dose

groups at day 281, and there was an obvious recovery trend after the 13week recovery period. No other treatment-related effects on the organ weight of monkeys were found (Table 8). 3.7.3. Gross findings and histopathologic findings. After 91 and 196 days of administration for rats and 281 days for monkeys, most of the sacrificed animals revealed enlarged spleens and organ congestion, including the lung, kidneys, liver and adrenal glands. Few other isolated findings were observed, which were within the range of background lesions or did not show a dose relationship. After the recovery period, the altered phenomenon above disappeared or showed significant retrieval tendencies. Upon histopathological examination, it was found that the red pulp region of the spleens augmented significantly, the spleen trabecular thickened, and the number of hematopoietic cells increased, when compared with that in the control groups, for both rats and monkeys. Accordingly, the white pulp dispersed away from small follicles, and

52

X.-L. Gong et al. / Toxicology and Applied Pharmacology 307 (2016) 45–61

Fig. 2.2. Selected hematological parameters of monkeys during 9-month administration of EPO-018B and 14-week recovery period.

most of these lack germinal centers; which was consistent with gross findings. In addition, hemosiderin deposition in spleen stromal cells was significantly reduced in dosed rats, when compared with that of the control groups (Fig. 4.1). Furthermore, in some of the dosed

monkeys, lymphocytes were sparse and red dye materials deposited in lymphoid follicle center, which might be the residual materials of the destruction of RBCs in the spleen (Dogan et al., 2003; Prado et al., 2003) (Fig. 5A and B).

Table 2.1 Selected hematology parameters of monkeys after 9-month administration of EPO-018B and 14-week recovery. Items

Dose (mg/kg)

D0

D28

D91

D154

D196

D238

D281

D329

D372

Hct (%)

0 0.3 3.0 20.0 0 0.3 3.0 20.0

41.4 ± 2.1 43.0 ± 2.3 42.6 ± 4.1 42.0 ± 3.1 74.8 ± 0.6 74.2 ± 2.0 75.4 ± 4.4 74.3 ± 3.6

42.9 ± 2.2 47.7 ± 4.0#,⁎ 54.5 ± 6.0##,⁎⁎ 50.7 ± 7.7#,⁎⁎ 74.3 ± 1.8 77.3 ± 3.8##,⁎ 80.0 ± 4.6##,⁎⁎ 77.9 ± 5.9#,⁎

42.5 ± 2.1 58.2 ± 7.0##,⁎⁎ 62.9 ± 10.2##,⁎⁎ 59.8 ± 8.9##,⁎⁎ 74.8 ± 2.5 74.0 ± 5.1 73.9 ± 7.2 68.6 ± 7.9##,⁎⁎

42.3 ± 2.9 56.5 ± 7.0##,⁎⁎ 60.6 ± 9.0##,⁎⁎ 59.7 ± 10.5##,⁎⁎ 75.1 ± 1.8 74.7 ± 4.6 75.1 ± 7.6 71.0 ± 6.5#,⁎

43.6 ± 3.4 50.4 ± 7.1# 52.4 ± 6.8#,⁎ 56.5 ± 10.3##,⁎⁎ 74.5 ± 1.4 74.7 ± 4.5 73.8 ± 7.0 71.8 ± 5.9#,⁎

43.1 ± 1.8 52.1 ± 5.9##,⁎ 54.1 ± 5.8##,⁎⁎ 57.9 ± 11.9#,⁎⁎ 74.9 ± 1.4 74.7 ± 3.7 72.5 ± 6.0# 71.4 ± 7.2#,⁎

40.8 ± 1.9 48.9 ± 5.0##,⁎ 51.1 ± 3.5##,⁎⁎ 55.8 ± 11.9#,⁎⁎ 74.8 ± 1.2 74.3 ± 3.4 72.3 ± 6.1# 71.1 ± 7.2#,⁎

43.1 ± 1.1 43.1 ± 2.2 45.2 ± 2.5 45.9 ± 6.2 74.6 ± 2.2 74.6 ± 2.7 73.9 ± 3.8 71.1 ±

42.5 ± 2.0 45.7 ± 2.8 44.1 ± 3.2 41.6 ± 0.1 74.9 ± 1.7 75.1 ± 2.9 75.5 ± 0.9 75.8 ± 2.3

0 0.3 3.0 20.0 0 0.3

23.9 ± 0.6 24.4 ± 0.5 24.4 ± 1.2 24.7 ± 1.4 312.0 ± 8.8 310.1 ±

24.0 ± 1.0 23.6 ± 1.2 23.6 ± 1.6 22.8 ± 1.7## 310.5 ± 11.3 305.8 ±

24.3 ± 0.7 20.8 ± 1.8##,⁎ 20.3 ± 2.2##,⁎ 18.1 ± 2.1##,⁎⁎ 309.4 ± 6.9 281.0 ±

23.7 ± 0.4 21.2 ± 1.5##,⁎ 20.8 ± 2.4##,⁎ 19.4 ± 2.3##,⁎⁎ 312.8 ± 4.3 284.0 ± 9.5##,⁎⁎

23.5 ± 0.6 21.4 ± 1.6##,⁎ 20.8 ± 1.9##,⁎ 19.6 ± 2.3##,⁎⁎ 314.8 ± 9.4 286.5 ± 7.1##,⁎⁎

24.4 ± 0.6 21.8 ± 1.5##,⁎ 20.9 ± 2.0##,⁎ 19.9 ± 2.4##,⁎⁎ 309.5 ± 7.4 291.5 ±

23.6 ± 0.5 20.9 ± 1.5##,⁎ 20.6 ± 1.7##,⁎ 19.7 ± 2.6##,⁎⁎ 311.9 ± 5.6 289.1 ±

6.2##,⁎ 23.4 ± 0.3 22.9 ± 1.2# 21.7 ± 1.8 20.5 ± 2.9 309.0 ± 8.4 307.4 ± 6.7

23.1 ± 0.4 22.9 ± 1.6 22.3 ± 1.0 23.6 ± 0.4 308.6 ± 6.0 304.9 ±

3.0

10.9 309.3 ±

16.5##,⁎ 294.9 ±

14.3##,⁎⁎ 274.4 ± 8.8##,⁎

276.6 ±

282.5 ±

8.8##,⁎ 288.0 ±

13.0##,⁎ 284.9 ± 5.6##,⁎⁎ 293.0 ± 10.3

20.0

14.6 311.7 ± 6.4

10.5##,⁎ 292.5 ±

263.8 ±

14.0##,⁎⁎ 272.6 ±

11.1##,⁎ 272.7 ±

6.5##,⁎⁎ 278.1 ±

11.1 294.5 ± 9.7⁎

276.4 ±

287.1 ± 17.2

311.3 ± 4.0

11.4 ± 0.6 11.8 ± 0.6 11.8 ± 0.5 12.0 ± 0.4

11.4##,⁎⁎ 11.0 ± 0.5 12.7 ± 1.3##,⁎⁎ 13.1 ± 1.0##,⁎⁎ 13.5 ± 0.8##,⁎⁎

10.2##,⁎⁎ 12.2 ± 0.8 16.8 ± 1.9##,⁎⁎ 17.4 ± 2.0##,⁎⁎ 19.1 ± 2.3##,⁎⁎

12.2##,⁎⁎ 12.7 ± 0.7 17.1 ± 2.0##,⁎⁎ 17.0 ± 2.3##,⁎⁎ 18.2 ± 2.0##,⁎⁎

12.9##,⁎⁎ 12.2 ± 1.0 17.7 ± 1.8##,⁎⁎ 17.7 ± 2.3##,⁎⁎ 18.6 ± 1.5##,⁎⁎

9.0##,⁎⁎ 12.4 ± 1.2 17.3 ± 1.7##,⁎⁎ 17.5 ± 2.6##,⁎⁎ 17.9 ± 1.3##,⁎⁎

10.0##,⁎⁎ 12.3 ± 0.8 16.7 ± 1.8##,⁎⁎ 17.7 ± 2.5##,⁎⁎ 18.6 ± 1.8##,⁎⁎

12.8 ± 0.7 14.5 ± 1.1 16.9 ± 3.1#,⁎ 17.9 ±

12.0 ± 1.1 13.1 ± 1.1 13.5 ± 0.6 13.6 ± 1.5

MCV (fL)

MCH (pg)

MCHC (g/L)

RDW (%)

0 0.3 3.0 20.0

2.4##,⁎⁎ ⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group. # P b 0.05. ## P b 0.01 vs d0.

X.-L. Gong et al. / Toxicology and Applied Pharmacology 307 (2016) 45–61

53

Table 2.2 Selected hematology parameters of monkeys after 9-month administration of EPO-018B and 14-week recovery. Items

Dose (mg/kg)

D0

D28

D91

D154

D196

D238

D281

D329

D372

MCVr (fL)

0 0.3 3.0 20.0 0

89.6 ± 2.6 92.1 ± 2.9 91.3 ± 2.8 92.5 ± 3.1 281.8 ±

89.2 ± 3.3 82.4 ± 7.0#,⁎ 85.2 ± 8.6#,⁎ 82.0 ± 6.0##,⁎⁎ 279.0 ± 8.9

90.9 ± 2.6 85.7 ± 9.5 87.8 ± 8.7 83.8 ± 7.7##,⁎⁎ 284.3 ± 9.5

90.6 ± 3.7 83.5 ± 8.7#,⁎ 83.4 ± 6.9#,⁎ 80.2 ± 6.5##,⁎⁎ 287.0 ± 8.3

91.3 ± 3.0 88.2 ± 9.0 86.7 ± 7.8 84.6 ± 8.3##,⁎⁎ 287.3 ± 11.0

92.0 ± 3.3 85.6 ± 9.7 84.7 ± 8.5##,⁎⁎ 85.3 ± 8.1 280.3 ± 9.8

90.8 ± 3.8 85.2 ± 7.0 83.8 ± 8.0##,⁎⁎ 84.5 ± 9.4 284.0 ± 12.3

90.5 ± 2.2 91.5 ± 2.4 89.1 ± 5.0 88.5 ± 5.6 280.8 ± 7.4

90.4 ± 2.9 91.3 ± 0.8 90.2 ± 2.6 90.9 ± 0.1 284.5 ±

0.3

9.9 281.2 ±

251.9 ±

260.4 ±

259.1 ±

258.0 ± 9.4##,⁎⁎ 260.0 ±

256.5 ±

3.0

6.3 281.7 ±

14.6##,⁎⁎ 18.9##,⁎⁎ 244.4 ± 7.0##,⁎⁎ 259.8 ±

15.9##,⁎⁎ 254.6 ±

275.3 ± 9.6⁎

252.8 ±

20.0

7.8 281.1 ±

15.0##,⁎⁎ 15.4##,⁎⁎ 12.1##,⁎⁎ 13.7##,⁎⁎ 236.5 ± 4.7##,⁎⁎ 250.9 ± 6.1##,⁎⁎ 246.0 ± 8.0##,⁎⁎ 244.3 ± 7.8##,⁎⁎ 245.8 ± 7.9##,⁎⁎ 256.6 ±

0 0.3 3.0 20.0 0 0.3 3.0 20.0 0 0.3 3.0 20.0

6.6 25.2 ± 0.8 25.9 ± 0.7 25.7 ± 1.4 26.0 ± 1.0 9.7 ± 0.9 9.2 ± 1.0 9.8 ± 1.2 9.7 ± 1.6 24.3 ± 2.0 24.1 ± 3.1 23.4 ± 1.9 23.9 ± 1.6

25.3 ± 0.9 20.7 ± 2.7##,⁎⁎ 20.7 ± 2.7##,⁎⁎ 19.3 ± 1.8##,⁎⁎ 9.7 ± 1.2 17.2 ± 8.3#,⁎ 16.0 ± 5.4#,⁎ 23.7 ± 6.7##,⁎⁎ 24.7 ± 1.7 25.9 ± 5.0 25.4 ± 2.2 25.5 ± 2.6

CHCMr (g·L−1)

CHr (pg)

MPV (fL)

APTT (s)

25.6 ± 0.7 22.3 ± 3.5#,⁎⁎ 22.8 ± 3.4#,⁎⁎ 21.0 ± 2.2##,⁎⁎ 9.4 ± 0.9 16.7 ± 7.6#,⁎ 15.5 ± 6.5#,⁎ 18.3 ± 4.6##,⁎⁎ 25.2 ± 2.3 27.2 ± 4.1##,⁎ 27.9 ± 3.4##,⁎⁎ 29.1 ± 3.1##,⁎⁎

26.0 ± 1.0 21.7 ± 3.4##,⁎⁎ 21.2 ± 2.8##,⁎⁎ 19.7 ± 2.2##,⁎⁎ 9.6 ± 0.9 13.6 ± 5.7#,⁎ 13.4 ± 4.8#,⁎ 16.5 ± 3.2##,⁎⁎ 24.8 ± 3.2 27.4 ± 3.5##,⁎ 28.6 ± 4.0##,⁎⁎ 29.7 ± 2.9##,⁎⁎

13.7##,⁎⁎ 255.6 ±

26.1 ± 0.8 22.8 ± 3.0#,⁎ 21.9 ± 2.7##,⁎⁎ 20.6 ± 2.7##,⁎⁎ 9.2 ± 1.3 12.7 ± 4.8⁎ 12.0 ± 4.8 14.9 ± 3.0##,⁎⁎ 25.1 ± 3.6 27.6 ± 5.8##,⁎ 29.9 ± 3.8##,⁎⁎ 30.9 ± 4.3##,⁎⁎

26.2 ± 0.8 22.3 ± 3.6#,⁎⁎ 21.6 ± 3.1##,⁎⁎ 20.9 ± 2.6##,⁎⁎ 9.7 ± 1.1 12.6 ± 4.3#,⁎ 11.3 ± 2.7 12.5 ± 2.3##,⁎ 24.4 ± 2.5 27.6 ± 6.6## 28.4 ± 5.6##,⁎⁎ 31.0 ± 2.8##,⁎⁎

10.8##,⁎⁎ 252.3 ± 5.5##,⁎⁎ 277.0 ± 7.8⁎

18.2##,⁎⁎ 25.7 ± 0.9 21.8 ± 2.4##,⁎⁎ 21.1 ± 2.3##,⁎⁎ 21.7 ± 3.5#,⁎⁎ 9.3 ± 1.0 11.2 ± 3.4 12.2 ± 3.8⁎ 13.7 ± 2.8##,⁎⁎ 25.2 ± 3.4 28.7 ± 4.0##,⁎⁎ 32.6 ± 3.4##,⁎⁎ 35.6 ± 2.8##,⁎⁎

6.4 288.0 ± 8.3 279.7 ±

279.7 ±

6.5 289.0 ±

18.8 26.3 ± 0.6 25.2 ± 0.9 24.6 ± 1.7 24.8 ± 3.1 9.3 ± 1.1 9.9 ± 4.5 9.3 ± 1.4 10.5 ± 1.7 24.3 ± 2.8 25.4 ± 5.9 25.2 ± 3.9 30.4 ± 9.0

5.7 26.0 ± 1.2 26.5 ± 0.7 25.6 ± 1.6 26.2 ± 0.4 9.1 ± 0.6 8.8 ± 1.1 9.1 ± 0.4 9.7 ± 1.0 25.0 ± 1.8 25.4 ± 5.9 25.2 ± 3.9 30.4 ± 9.0

⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group. # P b 0.05. ## P b 0.01 vs d0.

In bone marrow cytomorphologic inspections, all dose groups of rats and monkeys revealed significant hyperplasia of erythroblasts, especially for intermediate erythroblasts and late erythroblasts. Granulocytes in the bone marrow markedly decreased either in administered rats or dosed monkeys. Accordingly, the myeloid erythroid ratio decreased or was even inverted. In addition, hyperplasia of megakaryocytes was also distinguished, including both granular and mature

megakaryocytes. Remarkably, morphological characteristics and the cellular subset of each type of cell were absolutely normal. After the recovery periods, all differences above were restored completely or had obvious recovery tendencies (Figs. 3.1 and 3.2). In histopathological observations, it was found that in the bone marrow of the dose groups, including rats and monkeys, immature hematopoietic cells in the erythrocyte series significantly increased, and megakaryocyte in all

Table 3.1 Selected serum chemistry parameters of rats after 6-month administration of EPO-018B and 6-week recovery.

Male D91

D196

D232

Female D91

D196

D232

Dose (mg/kg)

ALT (nmol·s−1·L−1)

AST (nmol·s−1·L−1)

TBIL (μmol·L−1)

DBIL (μmol·L−1)

GLU (mmol·L−1)

Tch (mmol·L−1)

0 0.2 1.0 10.0 0 0.2 1.0 10.0 0 0.2 1.0 10.0

633.80 ± 63.35 782.20 ± 76.06 757.40 ± 74.47 633.40 ± 68.59 656.30 ± 59.52 707.60 ± 66.47 690.30 ± 67.39 675.70 ± 66.86 672.60 ± 68.79 675.00 ± 62.51 608.80 ± 47.65 753.40 ± 60.00

2458.00 ± 229.80 2359.20 ± 281.30 2846.80 ± 572.75 2752.00 ± 273.38⁎ 2550.70 ± 363.76 2570.00 ± 533.42 2620.50 ± 895.54 3262.10 ± 496.47⁎⁎

2.03 ± 0.48 2.13 ± 1.38 2.20 ± 0.57 2.77 ± 0.95 1.94 ± 0.54 2.10 ± 0.37 3.55 ± 0.48⁎⁎ 4.34 ± 0.50⁎⁎

1.78 ± 0.18 1.93 ± 0.43 2.07 ± 0.36⁎ 2.09 ± 0.49⁎ 1.73 ± 0.21 1.61 ± 0.21 2.07 ± 0.25⁎ 2.24 ± 0.22⁎⁎

8.52 ± 1.24 7.31 ± 2.65 6.37 ± 1.14 5.22 ± 0.74⁎ 8.69 ± 1.79 6.72 ± 1.10⁎⁎ 6.36 ± 1.34⁎⁎ 3.88 ± 0.60⁎⁎

1.75 ± 0.22 1.39 ± 0.15⁎ 1.35 ± 0.12⁎ 1.44 ± 0.24⁎

2697.00 ± 71.40 2751.60 ± 521.19 2552.00 ± 523.31 2382.40 ± 418.77

1.98 ± 0.38 1.78 ± 1.05 2.10 ± 0.51 1.76 ± 0.46

1.74 ± 0.14 1.53 ± 0.35 1.86 ± 0.20 1.61 ± 0.17

8.31 ± 1.23 7.58 ± 0.52 7.64 ± 0.82 7.04 ± 0.73

1.52 ± 0.35 1.28 ± 0.12 1.76 ± 0.35 1.49 ± 0.23

0 0.2 1.0 10.0 0 0.2 1.0 10.0 0 0.2 1.0 10.0

587.60 ± 42.42 545.40 ± 61.48 640.20 ± 76.57 551.40 ± 65.54 589.30 ± 60.51 684.90 ± 53.71 679.40 ± 67.69 466.13 ± 45.15 611.40 ± 67.15 643.80 ± 58.82 611.60 ± 75.60 538.80 ± 45.33

2395.60 ± 315.76 2696.80 ± 589.92 2875.20 ± 846.92 2936.00 ± 487.02 2327.10 ± 974.15 2843.10 ± 378.91 3770.40 ± 747.55 4345.63 ± 1265.96 2577.20 ± 355.07 2865.20 ± 596.33 2594.20 ± 578.78 2075.60 ± 367.59

2.10 ± 0.20 2.24 ± 1.05 2.72 ± 0.40⁎⁎ 2.52 ± 0.55⁎⁎ 1.97 ± 0.60 2.05 ± 0.81 2.67 ± 0.56⁎⁎ 2.85 ± 0.94⁎

1.81 ± 0.09 2.03 ± 0.48 2.36 ± 0.18⁎⁎ 2.28 ± 0.23⁎⁎ 1.79 ± 0.24 1.65 ± 0.36 2.28 ± 0.28⁎⁎ 2.33 ± 0.38⁎⁎

8.52 ± 0.52 6.99 ± 0.95 6.26 ± 0.35⁎ 5.54 ± 0.98⁎ 7.93 ± 0.79 5.23 ± 0.65⁎⁎ 4.46 ± 0.78⁎⁎ 3.35 ± 0.81⁎⁎

1.89 ± 0.55 1.79 ± 0.53 1.35 ± 0.28⁎ 1.22 ± 0.20⁎ 1.82 ± 0.29 1.57 ± 0.46 1.35 ± 0.20⁎ 1.28 ± 0.22⁎

2.04 ± 0.89 2.10 ± 0.57 2.02 ± 0.21 2.41 ± 0.96

1.74 ± 0.36 1.70 ± 0.25 1.69 ± 0.07 1.89 ± 0.41

7.96 ± 0.48 7.31 ± 0.61 7.62 ± 0.59 7.43 ± 0.52

1.68 ± 0.57 1.67 ± 0.60 1.54 ± 0.36 1.62 ± 0.58

⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group.

1.68 ± 0.22 1.41 ± 0.33⁎ 1.26 ± 0.32⁎⁎ 1.04 ± 0.27⁎⁎

54

X.-L. Gong et al. / Toxicology and Applied Pharmacology 307 (2016) 45–61

Table 3.2 Selected serum chemistry parameters of rats after 6-month administration of EPO-018B and 6-week recovery.

Male D91

D196

D232

Female D91

D196

D232

Dose (mg/kg)

Fe (mmol·L−1)

K (mmol·L−1)

Cl (mmol·L−1)

Ca (mmol·L−1)

P (mmol·L−1)

0 0.2 1.0 10.0 0 0.2 1.0 10.0 0 0.2 1.0 10.0

124.01 ± 25.30 114.59 ± 66.40 26.24 ± 14.11⁎⁎ 5.73 ± 1.77⁎⁎ 126.18 ± 21.99 86.07 ± 21.18⁎⁎

5.49 ± 0.89 5.52 ± 0.85 5.46 ± 0.42 5.50 ± 0.68 5.67 ± 0.55 5.46 ± 0.45 5.88 ± 0.50 6.51 ± 0.46⁎⁎ 5.03 ± 0.22 4.96 ± 0.12 4.91 ± 0.17 4.85 ± 0.27

103.34 ± 0.87 103.38 ± 1.65 102.26 ± 0.62 101.86 ± 0.78⁎ 104.22 ± 1.65 104.12 ± 1.97 104.33 ± 2.57 102.07 ± 1.58⁎

2.49 ± 0.06 2.49 ± 0.06 2.49 ± 0.06 2.63 ± 0.13⁎ 2.44 ± 0.05 2.46 ± 0.07 2.48 ± 0.06 2.56 ± 0.11⁎⁎

2.16 ± 0.23 2.14 ± 0.10 2.18 ± 0.18 2.54 ± 0.25⁎ 2.10 ± 0.16 2.15 ± 0.26 2.09 ± 0.34 2.59 ± 0.16⁎⁎

106.46 ± 1.33 106.96 ± 2.03 106.88 ± 1.05 105.60 ± 1.27

2.46 ± 0.07 2.42 ± 0.03 2.44 ± 0.04 2.46 ± 0.04

1.84 ± 0.19 1.81 ± 0.11 1.79 ± 0.11 1.94 ± 0.16

0 0.2 1.0 10.0 0 0.2 1.0 10.0 0 0.2 1.0 10.0

321.69 ± 56.96 170.04 ± 78.89⁎⁎ 142.59 ± 120.02⁎⁎ 5.50 ± 1.97⁎⁎

4.97 ± 0.54 5.08 ± 0.18 4.92 ± 0.40 5.98 ± 0.96⁎ 5.01 ± 0.88 4.92 ± 0.39 5.00 ± 0.59 5.74 ± 0.58⁎ 4.97 ± 0.35 4.92 ± 0.27 4.89 ± 0.37 5.03 ± 0.33

104.96 ± 2.24 104.04 ± 0.53 102.66 ± 0.61⁎ 102.08 ± 1.93⁎⁎

2.58 ± 0.06 2.52 ± 0.07 2.60 ± 0.07 2.60 ± 0.16 2.57 ± 0.10 2.65 ± 0.09 2.61 ± 0.09 2.73 ± 0.12⁎⁎ 2.47 ± 0.05 2.50 ± 0.07 2.54 ± 0.06 2.52 ± 0.09

1.77 ± 0.15 1.79 ± 0.33 2.26 ± 0.31⁎⁎ 2.56 ± 0.20⁎⁎

60.63 ± 102.02 6.94 ± 1.54⁎⁎ 118.81 ± 22.77 112.21 ± 29.30 168.97 ± 30.40 342.19 ± 75.93⁎⁎

316.02 ± 65.56 166.03 ± 41.68 171.19 ± 34.86⁎⁎ 7.44 ± 1.68⁎⁎ 306.69 ± 26.03 293.36 ± 47.09 369.75 ± 25.54⁎⁎ 380.33 ± 76.38⁎⁎

104.97 ± 1.57 104.34 ± 1.25 102.90 ± 0.75⁎⁎ 102.79 ± 1.59⁎⁎ 105.82 ± 1.30 106.22 ± 1.58 107.80 ± 1.30 106.48 ± 0.77

1.76 ± 0.12 1.90 ± 0.35 2.17 ± 0.14⁎⁎ 2.74 ± 0.35⁎⁎ 1.71 ± 0.16 1.67 ± 0.14 1.66 ± 0.20 1.86 ± 0.23

⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group.

stages proliferated actively. Furthermore, fat vacuoles in the bone marrow significantly reduced in the high and middle dose groups, when compared with the control group (Fig. 4.2; Fig.5C and D). Additionally, in rats, the number of hepatic extramedullary hematopoiesis foci significantly increased in the dose groups, when compared with the control group, especially in the medium and high dose groups. Furthermore, the vacuolar degeneration of liver cells was found in the middle- and high-dose groups during the interim and withdrawal periods. These lesions are clustered around the portal area of liver cells, and these female rats were more severe than the male rats (Fig. 4.3). In monkeys, some animals in the 3 mg/kg group (2/8) and 20 mg/kg group (3/8) revealed myocardial edema and fibrosis lesions in a large

area. There were small pieces of myocardial hypertrophy and nuclear hypertrophy around the pathologic tissues in two animals (one in the 3 mg/kg group and one in the 20 mg/kg group), which manifested as compensatory pathological changes (Fig. 5E and F). Finally, it was found that organ congestion was widespread in rats and monkeys, including the lungs, kidneys, liver and adrenal glands. 4. Discussion EPO-018B is a branched conjugate that originated from two-linear peptides, which experience oxidative cyclization, dimerization and pegylation (PEG). Furthermore, it can specifically bind to EPO receptors

Table 4 Selected serum chemistry parameters of monkeys after 9-month administration of EPO-018B and 14-week recovery. Items

Dose (mg/kg) D0

TBIL (μmol·L−1)

0 0.3 3.0 20.0 DBIL (μmol·L−1) 0 0.3 3.0 20.0 −1 GLU (mmol·L ) 0 0.3 3.0 20.0 LDH (μmol·s−1·L−1) 0 0.3 3.0 20.0 0 Fe (mmol·L−1) 0.3 3.0 20.0 ⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group. # P b 0.05. ## P b 0.01 vs d0.

3.32 ± 0.45 3.23 ± 0.61 3.44 ± 0.53 3.40 ± 0.83 1.91 ± 0.22 1.72 ± 0.30 1.79 ± 0.21 1.90 ± 0.33 3.78 ± 0.64 3.88 ± 0.60 3.79 ± 1.02 3.83 ± 0.94 7.91 ± 2.13 8.02 ± 1.99 7.73 ± 1.53 8.25 ± 2.81 160.24 ± 15.11 141.97 ± 20.46 168.30 ± 36.12 165.16 ± 25.34

D28

D91

D196

D238

D281

D372

3.35 ± 0.80 3.46 ± 0.37 4.04 ± 1.07 3.98 ± 1.07 1.88 ± 0.26 1.86 ± 0.14 1.96 ± 0.27 1.91 ± 0.23 3.61 ± 0.59 3.72 ± 0.68 3.99 ± 0.76 4.04 ± 0.90 7.97 ± 1.86 8.46 ± 2.74 9.89 ± 3.17 8.93 ± 4.24 167.98 ± 71.64 78.01 ± 71.44⁎⁎

3.30 ± 0.97 3.56 ± 0.76 4.75 ± 1.10#,⁎ 3.73 ± 1.15 1.78 ± 0.34 1.64 ± 0.32 1.95 ± 0.74 1.80 ± 0.30 3.64 ± 0.84 3.80 ± 0.68 4.01 ± 1.21 3.53 ± 0.53 7.81 ± 3.19 9.69 ± 5.01 10.64 ± 3.82#,⁎ 13.46 ± 2.27##,⁎⁎ 169.31 ± 31.14 77.58 ± 48.43##,⁎⁎ 65.31 ± 66.09##,⁎⁎ 36.96 ± 13.38##,⁎⁎

3.64 ± 0.98 3.84 ± 0.87 5.53 ± 1.50#,⁎ 5.79 ± 1.48#,⁎ 1.84 ± 0.44 1.92 ± 0.64 2.48 ± 0.32##,⁎⁎ 2.36 ± 0.73#,⁎ 3.61 ± 1.04 3.22 ± 0.72#,⁎ 2.43 ± 0.70##,⁎⁎ 3.03 ± 1.07##,⁎ 7.49 ± 2.73 10.01 ± 5.72 16.15 ± 8.09#,⁎ 22.26 ± 12.35##,⁎⁎ 157.82 ± 44.12 93.62 ± 37.11#,⁎ 64.90 ± 33.18##,⁎⁎ 36.47 ± 11.43##,⁎⁎

3.47 ± 0.60 3.78 ± 0.73 5.82 ± 1.64#,⁎ 6.06 ± 1.44##,⁎⁎ 1.98 ± 0.55 1.90 ± 0.30 2.54 ± 1.98#,⁎ 3.01 ± 1.42##,⁎⁎ 3.58 ± 0.94 2.82 ± 0.77##,⁎⁎ 2.73 ± 0.93##,⁎⁎ 2.64 ± 0.95##,⁎⁎ 8.07 ± 2.62 9.17 ± 5.41 15.37 ± 2.96##,⁎⁎ 19.73 ± 10.01##,⁎⁎ 147.06 ± 25.31 71.66 ± 44.66##,⁎⁎ 59.52 ± 40.34##,⁎⁎ 26.13 ± 9.51##,⁎⁎

3.53 ± 0.44 3.72 ± 1.44 6.26 ± 1.72##,⁎⁎ 7.41 ± 1.41##,⁎⁎ 1.87 ± 0.53 1.86 ± 0.45 3.05 ± 1.18#,⁎ 3.28 ± 0.80##,⁎⁎ 3.76 ± 0.65 2.31 ± 1.18##,⁎⁎ 2.58 ± 1.45##,⁎⁎ 3.02 ± 1.16##,⁎⁎ 7.36 ± 1.79 10.12 ± 6.24 15.58 ± 4.06#,⁎ 14.43 ± 6.26##,⁎⁎ 158.33 ± 27.12 84.28 ± 51.35#,⁎⁎ 82.99 ± 69.83##,⁎⁎ 37.18 ± 23.51##,⁎⁎

3.39 ± 0.73 3.81 ± 0.83 5.25 ± 1.86#,⁎ 4.77 ± 0.64#,⁎ 1.82 ± 0.32 1.88 ± 0.22 2.85 ± 0.57#,⁎ 2.21 ± 0.81#,⁎ 3.75 ± 1.53 3.59 ± 0.37 3.60 ± 0.95 3.57 ± 1.28 7.80 ± 1.26 8.94 ± 2.84 7.10 ± 2.57 8.41 ± 2.89 169.65 ± 58.17 168.59 ± 45.73 161.58 ± 31.06 146.29 ± 103.54

50.43 ± 30.76##,⁎⁎ 30.59 ± 14.33##,⁎⁎

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Table 5 Selected serum immune parameters of rats after 6-month administration of EPO-018B and 6-week recovery.

Male

Items

Dose (mg/kg)

D91

D196

D232

IL-1 (pg/ml)

0 0.2 1 10 0 0.2 1 10 0 0.2 1 10 0 0.2 1 10

6.47 ± 0.67 13.39 ± 1.69⁎ 16.05 ± 2.12⁎ 35.86 ± 3.90⁎⁎ 32.68 ± 2.66 51.67 ± 5.42 94.69 ± 14.83⁎⁎ 124.83 ± 16.70⁎⁎

8.61 ± 1.81 10.12 ± 1.52 13.20 ± 2.44 36.40 ± 4.89⁎⁎ 40.23 ± 5.25 66.95 ± 7.02 145.46 ± 12.45⁎⁎ 201.20 ± 17.33⁎⁎

4.27 ± 0.74 6.20 ± 0.67 11.27 ± 1.34⁎ 19.55 ± 2.47⁎ 28.59 ± 2.72 36.30 ± 4.16 37.04 ± 4.76 63.42 ± 5.95⁎

7.44 ± 0.98 26.63 ± 3.96⁎⁎ 30.95 ± 2.98⁎⁎ 61.24 ± 7.56⁎⁎ 44.07 ± 3.31 88.08 ± 7.39⁎⁎

6.52 ± 0.73 4.97 ± 0.97 4.05 ± 0.72 5.69 ± 0.68 40.79 ± 3.32 46.72 ± 5.29 51.71 ± 6.05 106.53 ± 10.67⁎⁎ 5.31 ± 0.71 6.51 ± 0.88 5.51 ± 0.94 5.09 ± 0.55 48.66 ± 6.20 50.08 ± 6.15 64.20 ± 5.55 87.40 ± 9.28⁎⁎

TNF-α (pg/ml)

Female

IL-1 (pg/ml)

TNF-α (pg/ml)

146.03 ± 16.52⁎⁎ 172.00 ± 16.79⁎⁎

⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group.

with an affinity equivalent to that of the natural ligand, and has the superiority of decreasing immunogenicity and antigenicity. The amino acid sequence of the synthetic peptide is unrelated to human EPO, which overcomes the deficiencies of anti-EPO antibody-mediated PRCA. The molecular weight of PEG moiety is 40 kDa, which means that increasing the solubility and stability of peptides and reducing renal clearance would thereby increase the plasma persistence of EPO018B. Potential indications for EPO-018B include the treatment of anemia associated with chronic kidney disease (CKD), anemia associated with chemotherapy in cancer patients, as well as the treatment of anemia in patients with erythropoietin antibody-mediated PRCA. EPO-018B administration for nine months in monkeys and six months in rats induces the proliferation of reticulocytes with subsequent maturation into RBCs, enhances naïve erythroid hematopoietic cells in the bone marrow, and increment extramedullary hematopoiesis foci in the liver and spleen. Increased RBCs, Hgb and Hct promoted increased blood pressure and cardiac stress in the tested animals. Altered erythrocytic parameters such as RDW, MCHC, MCV, CHCMr and MCVr revealed the excessive proliferation of RBCs and exhaustion of the erythroid bone marrow. Enhanced hematopoietic cells and organ congestion were considered as causes of the weight increment and enlargement of the liver and spleen, which were macroscopically observed at the interim and withdrawal time. In addition, increased PLT and MPV were consistent with the proliferated megakaryocytes in the bone marrow of the administrative groups. The prolongation of APTT revealed the effects of EPO-018B to the coagulation system. Increases in WBC and LYMPH%, as well as decreases in NEU%, were consistent with data in a report (Macdougall et al., 2009; Woodburn et al., 2009); in which Hematide stimulates the proliferation of WBC in rats. The pharmacological effects induced by EPO-018B were reversed after drug withdrawal during the recovery period. Alterations of hematology indexes were substantially similar to that in five-week evaluation studies

of EPO-018B, which were mainly considered to be the extension of pharmacological action. The toxicological effects of EPO-018B were mainly reflected as hepatotoxicity to rats and monkeys. Liver toxicity manifested as increased serum AST, T-BIL and conjugated bilirubin. Additionally, in rats, the vacuolar degeneration of the liver cells was found in the middle- and highdose groups during the interim and withdrawal periods. Lesions are clustered around the portal area of liver cells, and female rats were more severe than in male rats. It was reported that the repeated parenteral administration of PEGylated proteins to animals has been associated with cellular vacuolation in macrophages and/or histiocytes in various organs in some cases, which is considered a normal physiological response to remove foreign bodies (Mohammadpour et al., 2014; Wang et al., 2016). Since the tested substance contained a PEG moiety of 40 kDa, the PEG moiety was deemed as the main cause of the vacuolar degeneration in the liver. Furthermore, the cardiotoxicity of EPO-018B mainly revealed as increased serum lactate dehydrogenase in the middle- and high-dose groups of monkeys. Some animals revealed that myocardial edema, the fibrosis of lesions, compensatory myocardial hypertrophy and nuclear hypertrophy surrounded pathologic tissues. The causes may be summarized as drug-induced perturbations in hemodynamics, including chronic blood hyperviscosity, increased peripheral resistance and hypertension; which have been reported and are considered to be secondary to the exaggerated pharmacology associated with ESAs (Piloto et al., 2009; Wagner et al., 2001; Semenza et al., 1989). Widespread organ congestions in rats and monkeys were also the evidences of high pressure in peripheral circulation systems. In addition, decreases in serum ferrum, glucose and cholesterol levels were relevant to the energy consumption of cellular proliferation of erythron in vivo, which improved the oxygen content or metabolic rates of RBCs. Elevated potassium concentrations in serum of rats

Table 6 Selected serum immune parameters of monkeys after 9-month administration of EPO-018B and 14-week recovery. Items

Dose (mg/kg)

D0

D28

D91

D196

D238

D281

D372

IL-1 (pg·mL−1)

0 0.3 3.0 20.0

3.65 ± 0.63 3.39 ± 0.35 3.59 ± 0.33 4.34 ± 0.59

3.81 ± 0.55 4.07 ± 0.38 4.40 ± 0.28#,⁎ 5.83 ± 0.39#,⁎

4.09 ± 0.74 5.40 ± 0.58##,⁎ 5.87 ± 0.62##,⁎ 8.72 ± 0.96##,⁎⁎

3.71 ± 0.83 5.59 ± 1.19#,⁎⁎ 6.54 ± 0.77##,⁎⁎ 7.63 ± 0.52#,⁎⁎

4.46 ± 0.41 6.10 ± 0.51##,⁎ 6.89 ± 0.36##,⁎⁎ 8.73 ± 0.46#,⁎⁎

3.92 ± 0.74 6.88 ± 1.05##,⁎ 7.64 ± 1.78##,⁎⁎ 9.08 ± 1.47##,⁎⁎

3.64 ± 0.45 4.43 ± 0.86 4.33 ± 1.86 5.81 ± 2.12

⁎ P b 0.05. ⁎⁎ P b 0.01 vs control group. # P b 0.05. ## P b 0.01 vs d0.

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Table 7 Selected absolute organ weights (AW) and relative organ weights (RW) of rats after 6-month administration of EPO-018B and 6-week recovery. Sex

Organ

Time

Index

0 mg/kg

0.2 mg/kg

1.0 mg/kg

10.0 mg/kg

Male

Liver

D91 D196 D232 D91 D196 D232 D91 D196 D232 D91 D196 D232 D91 D196 D232 D91 D196 D232 D91 D196 D232 D91 D196 D232

AW (g)

13.203 ± 1.127 13.088 ± 1.926 12.979 ± 0.929 2.493 ± 0.076 2.234 ± 0.185 2.139 ± 0.120 0.818 ± 0.075 0.803 ± 0.098 0.995 ± 0.166 0.161 ± 0.013 0.138 ± 0.013 0.158 ± 0.022 6.861 ± 0.250 7.177 ± 0.892 6.736 ± 0.727 2.562 ± 0.111 2.380 ± 0.251 2.188 ± 0.165 0.552 ± 0.053 0.529 ± 0.065 0.520 ± 0.063 0.206 ± 0.017 0.176 ± 0.023 0.169 ± 0.016

12.590 ± 1.529 13.171 ± 1.878 13.470 ± 0.982 2.477 ± 0.149 2.310 ± 0.153 2.206 ± 0.148 1.500 ± 0.198** 1.401 ± 0.259** 0.976 ± 0.134 0.310 ± 0.049** 0.246 ± 0.037** 0.156 ± 0.013 6.740 ± 0.223 7.083 ± 0.408 6.730 ± 0.659 2.558 ± 0.060 2.425 ± 0.121 2.259 ± 0.177 0.990 ± 0.072** 1.121 ± 0.130** 0.638 ± 0.084 0.376 ± 0.021** 0.385 ± 0.057** 0.214 ± 0.021*

12.942 ± 1.223 13.160 ± 1.092 12.981 ± 1.551 2.487 ± 0.108 2.354 ± 0.124 2.134 ± 0.161 2.333 ± 0.109** 2.856 ± 0.537** 1.260 ± 0.185 0.468 ± 0.033** 0.502 ± 0.083** 0.196 ± 0.023 7.204 ± 0.410 6.902 ± 0.484 6.917 ± 0.278 2.758 ± 0.137* 2.451 ± 0.162 2.302 ± 0.082 1.663 ± 0.103** 2.019 ± 0.208** 0.726 ± 0.079* 0.637 ± 0.035** 0.717 ± 0.066** 0.241 ± 0.024**

13.120 ± 1.892 15.524 ± 4.352* 12.844 ± 1.436 2.445 ± 0.177 2.623 ± 0.768* 2.127 ± 0.111 3.867 ± 0.318** 5.143 ± 0.980** 1.514 ± 0.298** 0.754 ± 0.055** 0.869 ± 0.128** 0.261 ± 0.062* 7.362 ± 0.537* 7.821 ± 0.542* 7.336 ± 0.639 2.767 ± 0.255* 2.564 ± 0.091* 2.454 ± 0.118 3.104 ± 0.211** 4.487 ± 1.710** 1.096 ± 0.212** 1.167 ± 0.112** 1.452 ± 0.482** 0.365 ± 0.054**

Spleen

Female

Liver

Spleen

RW (g*100/g)

AW (g)

RW (g*100/g)

AW (g)

RW (g*100/g)

AW (g)

RW (g*100/g)

* P b 0.05, ** P b 0.01 compared with control group.

suggests the accelerated metabolism of RBCs and increased RBC destruction, which released more potassium from the cytoplasm (Wald et al., 1985; Yamada et al., 2010). The alteration of chloride, phosphorus and calcium might be the appropriate regulating mechanisms after the increment of serum potassium. The toxicological effects observed with EPO-018B in this study are considered to be secondary to the exaggerated pharmacology that occurs with its administration to normocythemic animals in the high-dose and/ or middle-dose groups. As previously mentioned, EPO-018B is non-immunogenic in rats; and two monkeys in the low- and middle-dose groups were tested positive for EPO-018B specific antibodies, both after the fourth administration. As data of the toxicokinetics tests revealed, no EPO-018B was detected from the plasma of these two antibody-positive monkeys since the fourth dosage. Accordingly, the presence of antibodies was accompanied by the reversion of erythroid-related indicators. This alteration indicated that these antibodies might be neutralizing antibodies of EPO-018B, and had partial blockade effects to the erythropoiesis of EPO-018B, but the speculation need to be confirmed in the future clinical studies. (Lee et al., 2010; Shin et al., 2011). Meanwhile, IL-1 and TNF-α in all dose groups of rats increased or had increased tendencies at day 91 and day 196. Serum IL-1 increased or had increased tendencies in all dose groups of monkeys during the administrative period. In monkeys, serum IL-1 in all dose groups increased during the administrative period and decrease after withdrawal. It is known that pro-inflammatory cytokines such as interferon-gamma (IFN-γ), TNF-α and interleukin-1 (IL-1) suppress erythropoiesis, resulting in an inadequate response to recombinant human erythropoietin

(Mohammadpour et al., 2014). The increase in IL-1 and TNF-α might be associated with organism adaption after the dose of EPO-018B. Based on the toxicological changes above, we came to the following conclusions: The study findings at 0.2 mg/kg for rats and 0.3 mg/kg for monkeys were expected pharmacological changes of the exuberant haematogenesis of erythroid cells and so these dose levels were considered to be the study NOAEL (the no observed adverse effect level). Higher doses caused adverse effects related to the liver toxicity, cardiotoxicity, appearance of neutralizing antibodies of EPO-018B and the decrease of glucose and cholesterol at hematology. EPO-018B has been evaluated in phase II clinical trials some years ago. The studies described herein were performed five years ago, and were designed to support the phase III clinical trials of EPO-018B in the clinical setting. The toxicological effects of EPO-018B observed after subcutaneous administration are considered to be related to the exaggerated pharmacology and secondary sequelae that resulted from ESA treatment to healthy animals. The anticipated patient population for EPO-018B treatment is targeted to be anemia patients caused by chronic renal failure or chemotherapy against cancer, in order to return RBC parameters towards normal values. Therefore, the sequelae occurred in rats and monkeys in toxic studies, which were considered secondary to exaggerated pharmacology, and would less likely occur in the intended patient population. Conflict of interest The authors declare that there are no conflicts of interest.

Table 8 Selected absolute organ weights (AW) and relative organ weights (RW) of monkeys after 9-month administration of EPO-018B and 14-week recovery. Organ

Time

Index

0 mg/kg

0.3 mg/kg

3.0 mg/kg

20.0 mg/kg

Liver

D281 D372 D281 D372 D281 D372 D281 D372

AW (g)

56.53 ± 6.35 64.20 ± 6.52 18.598 ± 2.937 22.632 ± 2.978 3.85 ± 0.51 3.80 ± 0.32 1.236 ± 0.176 1.351 ± 0.118

60.15 ± 6.18 67.98 ± 7.06 18.617 ± 3.175 23.048 ± 1.841 4.88 ± 0.52* 3.50 ± 0.52 1.531 ± 0.163* 1.197 ± 0.184

67.63 ± 7.21 68.55 ± 7.12 21.892 ± 2.656* 22.951 ± 3.024 4.85 ± 0.55* 4.20 ± 0.88 1.519 ± 0.180* 1.405 ± 0.294

62.18 ± 5.06 64.03 ± 7.51 20.200 ± 2.075* 22.443 ± 3.020 4.95 ± 0.61* 3.60 ± 0.46 1.596 ± 0.196* 1.262 ± 0.163

Spleen

* P b 0.05 compared with control group.

RW (g/kg) AW (g) RW (g/kg)

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Fig. 3.1. Giemsa-stained bone marrow cytomorphologic sections of rats (A–D) (1000×). (A) Bone marrow cytomorphologic section from control rats. (100 ×) (B) Bone marrow cytomorphologic section from high dose rats. (100×) (C) Bone marrow cytomorphologic section from control rats. (1000×) (D) Bone marrow cytomorphologic section from high dose rats. (1000×) Images captured from sections prepared from rats euthanized at day196 necropsy.

Fig. 3.2. Giemsa-stained bone marrow cytomorphologic sections of monkeys (A–D) (1000×). (A) Bone marrow cytomorphologic section from control monkeys. (100×) (B) Bone marrow cytomorphologic section from high dose monkeys. (100×) (C) Bone marrow cytomorphologic section from control monkeys. (1000×) (D) Bone marrow cytomorphologic section from high dose monkeys. (1000×) Images captured from sections prepared from monkeys euthanized at day281 necropsy.

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Fig. 4.1. Hematoxylin and eosin-stained histologic sections of rat spleen. (A) Spleen section from control rats at day91. (40×) (B) Spleen section from control rats at day91. (400×) (C) Spleen section from high dose rats at day91. (40×) (D) Spleen section from high dose rats at day91. (400×) (E) Spleen section from high dose rats at day196. (40×) (F) Spleen section from high dose rats at day196. (400×) (G) Spleen section from high dose rats at day232. (40×) (H) Spleen section from high dose rats at day232 (400×).

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Fig. 4.2. Hematoxylin and eosin-stained histologic sections of rat bone marrow. (A) Bone marrow section from control rats at day91. (100×) (B) Bone marrow section from high dose rats at day91. (100×) (C) Bone marrow section from high dose rats at day196. (100×) (D) Bone marrow section from high dose rats at day232 (100×).

Fig. 4.3. Hematoxylin and eosin-stained histologic sections of rat liver. (A) Liver section from control rats at day91. (100×) (B) Liver section from high dose rats at day91. (100×) (C) Liver section from high dose rats at day196. (100×) (D) Liver section from high dose rats at day232 (100×).

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Fig. 5. Hematoxylin and eosin-stained histologic sections of monkey spleen, bone marrow and myocardium. (A) Spleen section from control monkeys. (100×) (B) Spleen section from high dose monkeys. (100×) (C) Bone marrow section from control monkeys. (200×) (D) Bone marrow section from high dose monkeys. (200×) (E) Myocardium section from control monkeys. (200×) (F) Myocardium section from high dose monkeys. (200×) Images captured from sections prepared from monkeys euthanized at day281 necropsy.

Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgment This work was supported by Major Project of National Science and Technology (No. 2014ZX09J14106-06C, No. 13CXZ005), National Natural Science Foundation of China (No. 81473291). References Chu, H.C., Lee, H.Y., Huang, Y.S., Tseng, W.L., Yen, C.J., Cheng, J.C., Tseng, C.P., 2014. Erythroid differentiation is augmented in Reelin-deficient K562 cells and homozygous reeler mice. FEBS Lett. 588, 58–64.

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