Abstract
The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia. The State Research Center of Virology and Biotechnology “Vector” of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing has developed a high-performance technology for production of recombinant human GM-CSF (rhGM-CSF) based on a recombinant E. coli strain.The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration.Materials and methods: in vitro evaluation of rhGM-CSF hemostimulating activity was performed by MTT assay in the commercial HL-60 promyelocytic leukemia cell culture with preliminary suppression of cell growth rate by adding a low concentration of dimethyl sulfoxide to the medium. In vivo studies were carried out in CBA/CaLac mice with cyclophosphamide-induced myelosuppression. The hemostimulating properties of the drug were evaluated after subcutaneous administration of 1–175 µg/kg doses for 4–5 days, following administration of a cytostatic agent. The total number of leukocytes and the content of their morphological forms were determined in blood samples taken at different time points after the drug administration. The statistical processing of the experimental data was based on analysis of variance using Statgraphics v. 5.0 software.Results: the proliferative activity of HL-60 cells incubated with the rhGM-CSF preparation for 72 hours was shown to be dose-dependent. The highest values of the increase in proliferative activity associated with an increase in the drug dose were observed in the concentration range from 0.04 to 0.64 ng/mL (proliferative activity increased by 11–18% when the dose was increased twofold). The experiments in mice demonstrated a two-phase pattern of the dose-dependent effect. The drug showed the highest hemostimulating effect at the dose of 90 µg/kg.Conclusions: the rhGM-CSF preparation obtained using the new developed technology has a pronounced hemostimulating activity confirmed by both in vitro and in vivo test systems.
Highlights
Институт медицинской биотехнологии Федерального бюджетного учреждения науки «Государственный научный центр вирусологии и биотехнологии «Вектор» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека, ул
The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia
The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration
Summary
В экспериментах использовали препарат рчГМ-КСФ, полученный в Институте медицинской биотехнологии ФБУН ГНЦ ВБ «Вектор» Роспотребнадзора [10, 11]. Для этого в лунки вносили по 25 мкл раствора красителя 3-(4,5-диметилтиазол-2-ил)-2,5-дифенил-2Н-тетразолиум бромида (МТТ) в концентрации 5 мг/мл, планшеты инкубировали при температуре 37 °С в течение 4 ч. После удаления среды в лунки вносили по 100 мкл ДМСО для растворения кристаллов образовавшегося формазана, встряхивали на шейкере при 600 об/мин в течение 5 мин. В первой серии исследовали динамику восстановления показателей крови под действием препарата рчГМ-КСФ в стандартной дозе (100 мкг/кг), определенной ранее в предварительных экспериментах. С этой целью препарат вводили мышам опытных групп подкожно в объеме 0,2 мл на 20 г массы тела один раз в сутки в течение 4, 5 и 6 суток, первое введение — через 24 ч после инъекции ЦФ, забор крови на анализ — на 5, 6, 7 и 8-е сутки после введения ЦФ. Критический уровень значимости при проверке статистических гипотез (р) принимали равным 0,05 [18]
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