Specific activity and toxicity of lyophilized recombinant human granulocyte-macrophage colony-stimulating factor (E. coli BL21/pET-GST6His-GM): in vitro and in vivo study

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Abstract
INTRODUCTION. The development of new effective drugs based on granulocyte-macrophage colony-stimulating factor (GM-CSF) remains a relevant objective: the study of the drug’s biological effects opens new prospects for its clinical use in the treatment of a wide range of diseases – from oncological and hematological disorders to neurodegenerative conditions. Developing a new lyophilized recombinant human GM-CSF (rhGM-CSF) based on Es с herichia coli BL21/pET-GST-6His-GM producer strain warrants an assessment of its specific activity and toxicity. AIM. This study aimed to examine the specific proliferative activity ( in vitro ) and hematopoietic stimulatory effect ( in vivo ), as well as acute and subchronic toxicity of a new lyophilized rhGM-CSF preparation for subcutaneous administration. MATERIALS AND METHODS. Lyophilized rhGM-CSF (strain E. coli BL21/pET-GST-6His-GM) produced by State Research Center of Virology and Biotechnology “Vector” (Russia) was used in the study. The proliferative activity was assessed in the human erythroleukemia TF-1 cell culture by XTT assay. Hematopoietic stimulatory activity was assessed in male CBA/Calac mice subjected to cyclophosphamide-induced myelosuppression (200 mg/kg, intraperitoneally). The rhGM-CSF preparation was administered at a dose of 90 μg/kg subcutaneously daily for 4 days. A leukogram was calculated on Day 5. The acute toxicity of the preparation was assessed by a single subcutaneous administration to male and female ICR mice at 500 and 1,000 μg/kg, subchronic toxicity – by four subcutaneous administrations at 90 μg/kg. On Days 1 and 7, body weight, temperature, complete blood count, biochemical markers and internal organ histology (heart, lungs, liver, kidneys, etc.) were evaluated. RESULTS. The rhGM-CSF preparation demonstrated pronounced proliferative activity on TF-1 cells (ED 50 =0.48 ng/mL). In mice with myelosuppression, the preparation resulted in a 2.1-fold increase in the number of segmented neutrophils compared to the control group. Single and repeated administration did not cause death of animals, did not affect body weight or temperature, nor did it result in significant changes of hematological or biochemical parameters or macro- and microscopic anatomy of organs. CONCLUSIONS. The new lyophilized rhGM-CSF preparation has demonstrated high proliferative and hematopoietic stimulatory activity, as well as insignificant acute and subchronic toxicity. The obtained results substantiate further pharmaceutical development of the preparation as an effective and safe hematopoietic stimulatory agent.

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