Safety Studies In Rats And Beagle Dogs Nephrotoxicity Biomarkers And EV71 Inactivated Vaccine Preclinical

Kidney is an important organ for metabolism and elimination of drug. Due to its special structure and function, it is susceptible to drug-induced injury. Renal toxicity induced by drugs has become the main cause of end of preclinical drug development and adverse reactions. Drug-induced nephrotoxicity has been reported to contribute to approximately19-25%of cases of acute kidney injury (AKI). Thus, early rapid and accurate evaluation of nephrotoxicity is important in the drug development. Currently, blood urea nitrogen (BUN) and creatinine (Cr) are the most commonly used biomarkers of nephrotoxicity. Owing to strong compensatory function of kidney, BUN and Cr will not increase immediately during early renal damage, which have low sensitivity of detection. Therefore, in order to explore more sensitive and reliable biomarkers of nephrotoxicity, performances of a series of novel candidate biomarkers suggested by international organization or clinical practice were evaluated in gentamicin-induced AKI in Wistar rats and Beagle dogs in this study. Furthermore, microRNA (miRNA) expression profiles in rat urine were analyzed for assessing the utility of miRNA as early biomarker of renal injury.A rat model of renal damage was induced successfully by daily intraperitoneal injection of60or120mg/kg gentamicin sulfate for consecutive10days. The results of weight test revealed that the dose-related decreasing trend were found in animals in the high-and low-dose groups on day10following continuous administration. Compared with the control group, kidney weight of animals treated with120mg/kg gentamicin began to increase from day8onward and a increase trend was also observed in the low-dose group on day11, suggesting the changes in kidney weight had associated with the time and dosage of administration. Histopathological analysis demonstrated that the number of animals with tubular cell necrosis injury appeared to increase and the severity of renal damage had been aggravated over time, which were closely related to the treatment with gentamicin. Urine biomarker analysis showed that the urinary concentrations of kidney injury molecule (Kim-1), clusterin, albumin, β2-microglobulin (B2M) and neutrophil gelatinase-associated lipocalin (NGAL) were markedly elevated following the administration with gentamicin, and the magnitude of these increase was much higher than that of BUN and Cr. According to the results from individual animal, elevations of these urinary biomarker paralleled severity of kidney lesions. Receiver operator characteristics (ROC) analysis also demonstrated that AUCs of these five biomarkers were higher than that of BUN and Cr, further supporting that performance of Kim-1, clusterin, Albumin, B2M and NGAL were superior to BUN and Cr for detection of AKI. Due to high sensitivity and specificity, these urinary biomarkers will have good prospects in the preclinical drug safety evaluation studies in rats. Inclusion and exclusion ROC analysis indicated that AUCs of N-acetyl-beta-D-glucosaminidase (NAG), Cystatin C (CysC) and epidermal growth factor (EGF) were higher than BUN and Cr, suggesting their performances were superior to BUN and Cr in the diagnosis of renal injury, but less obvious than the above five biomarkers, such as Kim-1. It was found that AUC values of vascular endothelial growth factor (VEGF), acid glycoprotein (AGP), alkaline phosphatase (ALP), tissue inhibitor of metalloproteinase-1(Timp-1), Vanin-1, calbindin and lactate dehydrogenase (LDH) were equivalent to or slightly lower than BUN and Cr, which had certain diagnostic efficacy for AKI, but no obvious advantage compared with BUN and Cr. IL-18and osteopontin (OPN) did not show diagnostic value in rat AKI.MiRNA expression profiles of urines from different time points and dosage groups were analyzed using TaqMan (?) Array Rodent MicroRNA Cards. Compared with difference of miRNA between60mg/kg and120mg/kg groups on day8, and between120mg/kg group on day4and8,32miRNAs were found to be up-regulated, and14miRNAs were down-regulated, which were related with time and dosage of administration, respectively. According the ROC analysis based on the tubular degeneration, necrosis histopathological damage as a diagnostic gold standard,8up-regulated miRNAs including miR-16-1-3p,345-5p,539,324-3p,466k,99b-3p,489and140-3p, and6down-regulated miRNAs including miR-1961,363,367,590-5p,215and451were indentified to serve as the prediction of renal injury potential biomarkers. Further analysis found that these miRNA changes may be occur earlier than tissue damage by histopathological assessment. Therfore, further studies need be performed to investigate the diagnostic value of these miRNA and its possible role in the pathway associated with mechanisms of kidney damage in the future.We also evaluated the performance of4urinary biomarkers, including NGAL, clusterin, total protein, and NAG in AKI induced by80mg/kg gentamicin-(daily up to9days) in beagle dogs. The results showed that urinary NGAL and clusterin levels were significantly elevated in a time-dependent manner dogs on days1and3after administration of gentamicin, respectively. The individual animal data revealed that urinary NGAL and clusterin changes closely related with severity of renal injury. The high area (both AUCs=1.000) under ROC curve indicated that these2urinary biomarkers had perfect specificity and sensitivity for detection of gentamicin-induced renal toxicity in dog, which were superior to BUN and Cr. Real time PCR assay also demonstrated that NGAL and clusterin gene expression were up-regulated following renal injury, further provided evidence to support the specificity of NGAL and clusterin as biomarkers for the early assessment of drug-induced renal damage. NAG also exhibited high sensitivity of detection, together with a time-dependent increase in urinary concentrations, although the magnitude of its increase was less than that of NGAL and clusterin after administration with gentamicin. ROC analysis indicated that performance of NAG was better than BUN. Moreover, urinary total protein (uTP) was found to be less sensitive as it was not superior to BUN or Cr and inferior to NGAL, clusterin and NAG in pairwise comparisons, suggesting that uTP had some diagnostic evalue for detection of renal injury. The present data provided insights into the preclinical use of these biomarkers for detection of drug-induced AKI in nonrodent species. Enterovirus71(EV71), a non-enveloped single positive stranded RNA virus, is one of the major causative agents for hand, footand mouth disease (HFMD) in childhood. In recent years, regular epidemics of HFMD occurred every several years in many countries. Nowadays, HFMD or EV71infections have already become an important public health issue throughout the world, especially in the Asia Pacific region. Unfortunately, no effective antiviral drug is available for EV71infection now. Vaccination may be the most effective measure to control the transmission of the virus. Therefore, to pave EV71vaccine into human clinical trial, in the present study a comprehensive preclinical safety assessment of inactivated EV71vaccine were conducted in rats and cynomolgus monkeys.Single and repeat-dose toxicity studies were performed in two species animals. For the repeat-dose toxicity study in rats, animals were administrated intramuscularly with low (160U per rat) and high (320U per rat) dosages of EV71vaccines on weeks0,2,4,6. The two dose levels used in the rat repeat-dose studies were equivalent to800U/kg and1600U/kg according to the calculation of rat weight (200g per rat), which were25～50and50～100times higher than16～32U/kg of the proposed clinical dose (according to10～20kg of children average weight), respectively. Furthermore, additional adjuvant group was also included in rat repeat-dose toxicity study. In the cynomolgus monkeys repeat-dose toxicity study, animal were injected intramuscularly on weeks0,2,4,6with low (320U per monkey) and high dosage (1280U per monkey) EV71vaccines. The high dosage was4times higher than the proposed single human dose (320U). Based on the calculation of weight, the two dose levels were3.3～6.7and13.3～26.8times higher than the proposed clinical dose, respectively.Following single intramuscular administration with EV71vaccine (640U), no obvious signs of abnormalities observed clinically and increase of animal weight had not been affected. No abnormal findings were observed at necropsy examination on day15after administration. These results suggested that the maximum tolerated dose was greater than640U per rat following single intramuscular administration with EV71vaccine. The results also showed no obvious systemic toxicities related with immunization after four repetitive intramuscular injections of high and low dosages of EV71vaccine in the two animal species except for slight changes of neutrophil or monocyte counting and ratio within the background data. Antinuclear antibody response was not detected after the repeated administrations. Histopathological examination demonstrated the minimal to severe inflammatory changes in muscle tissues of the injection sites in EV71vaccine-immunized animals, which decreased over time. Furthermore, the results of neutralizing antibody analysis showed that all rats and monkeys treated with low-and high-dose EV71vaccines were seroconverted following the second administration, suggesting that inactivated EV71vaccine developed in this study have good immunogenicity. In summary, our study suggested a favorable safety profile for inactivated EV71vaccine and supported this product to enter human phase I clinical trial. These data will contribute to the definition of dosage and immunization procedure used in the clinical trial in the future.