Page 64 - ITPS-8-2

INNOSC Theranostics and
Pharmacological Sciences Preclinical study of GBpoietin biosimilar

2.5. Repeated-dose toxicity assay 3. Results

A total number of 24 rats were separated into four different 3.1. Body weight assessment
groups consisting of 6 rats (3 males and 3 females) in each
group. There were four different treatment groups (1 and Body weight increased steadily and significantly during
2), one placebo group, and one control group. Each rat of the single-dose toxicity study, as shown in Figure 1A. The
the two treatment groups was subcutaneously (SC) injected differences in body weight for all study groups at different
with sterile 125 IU/kg, 250 IU/kg, 500 IU/kg, 750 IU/kg, evaluation time points (day 0, 7, and 14) suggested that
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1000 IU/kg, 1250 IU/kg, and 1500 IU/kg of GBpoietin the body weight gains were significant (P < 0.05) for
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(Group 1) and Eprex (Group 2) on 7 consecutive days. Treatment-2 (P = 0.01), Treatment-4 (P = 0.006), Control
The placebo and the control groups were similar to those (P = 0.001), and Placebo (P = 0.017) groups but were not
in the single-dose toxicity assay. For CBC analysis, whole significant in Treatment-1 (P = 0.09) and Treatment-3
blood was collected 2 days before dosing as well as after (P = 0.07) groups. On the other hand, during the repeated-
the last dosing on day 7 using similar protocols described dose toxicity study, body weight increased steadily but not
in Section 2.4. PD endpoints including RBC, WBC, HGB, significantly, as shown in Figure 1C; the differences in body
HCT, MCV, MCH, MCHC, and PLT count were measured weight across all study groups at different evaluation time
for all test subjects before the first dose injection and the points (day 0, 3, and 7) were not significant (P > 0.05). The
day after the last dose. ALT, AST, and BUN assays as well results remained consistent when the data were analyzed
as necropsy tests and histopathological evaluations were independently per gender or evaluation time points. An
performed as described in Section 2.4. Body temperature increase of body weight for all the groups suggests a normal
and weight were also measured during the whole study evolution of body weight which is an indicator of sound
period. health for the animals, substantiating the non-toxicity
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of the GBpoietin under both single- and repeated-dose
2.6. Data evaluation and statistical analysis toxicity studies. The absence of negative effects on body
The variables used for statistical processing are body weight gain is favorable for the evaluation of the substance
weight, body temperature, hematological and biochemical under the study, as a decrease in body weight is one of
parameters, as well as microscopy findings. Central the primary clinical symptoms of stress or illness in this
tendency and dispersion statistics, including mean, rat strain. Therefore, the increase in body weight serves
standard deviation, maximum, and minimum values, were as indirect evidence of the non-toxicity of the substance
calculated. The variable FD (difference between the final being analyzed.
and initial value) was calculated to assess the treatment 3.2. Body temperature assessment
effects on hematological and biochemical parameters. The
assumptions of normal distribution and homogeneity of During the single-dose toxicity test, the changes in
variance were verified using the Kolmogorov-Smirnov and body temperature were not significant (P > 0.05) for
Shapiro-Wilk tests, and the Levene test, respectively, before all four treatment groups (Treatment-1, Treatment-2,
the analysis of body weight and body temperature variables Treatment-3, Treatment-4) and Placebo group (P = 0.009),
for each evaluation time point. A parametric analysis while nearly significant in Control group (P = 0.07), as
of variance (ANOVA) or a non-parametric alternative shown in Figure 1B. On the other hand, the changes in
(Kruskall–Wallis test) was used, depending on whether the body temperature were not significant (P > 0.05) for all
data followed a normal distribution. Paired comparisons study groups (Treatment-1, Treatment-2, Placebo, and
were performed in consecutive intervals, using either the Control) in the repeated-dose toxicity study, as shown in
paired t-test or the Wilcoxon test, depending on whether Figure 1D. These results remained consistent when the
the data followed a normal distribution. The results from data were analyzed independently per gender or evaluation
the histopathological studies were analyzed using cross- time points, indicating normal health for the animals
tabulated classification tables and the test for association and substantiating the non-toxicity of the GBpoietin .
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of independence (Fisher’s exact test). The data were In addition, the absence of changes in body temperature
processed with Microsoft Excel, 2010, running on the outside the normal range serves as an indirect indicator of
Windows operating system. The mean difference between non-toxicity for the test animals.
the test and the comparator product was calculated using 3.3. Hematological assessments
linear mixed-effect analysis of variance, along with the
corresponding P-value. Statistical significance was defined When analyzing the results of the hematological tests,
as a P < 0.05. A P > 0.05 for the PD parameters of the sample differences were detected among treatment groups of both
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formulations was considered similar and non-significant. doses of GBpoietin and the reference drug, Eprex .
Volume 8 Issue 2 (2025) 58 doi: 10.36922/itps.5797