Hemostimulating properties of the conjugates of granulocyte-macrophage colony stimulating factor with alendronic acid

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The aim of the work is to evaluate the hemostimulating activity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) conjugates with alendronic acid (ALN) in the model of cytostatic myelosuppression and the dynamics of rhGM-CSF accumulation as a part of the conjugate in the bone tissue and bone marrow of mice.

Materials and methods. The conjugates obtained by a solid-phase synthesis using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide or periodate oxidation, were used. A hemostimulating activity was evaluated in a model of a cytostatic myelosuppression induced by the administration of cyclophosphamide to CBA/Calac mice. RhGM-CSF preparations were injected subcutaneously for 4-5 days at the dose of 90 µg/kg. After the injections cycle had been completed, the total leukocyte and segmented neutrophil counts were carried out in the blood samples, and the total karyocyte count was carried out in the bone marrow samples.

The tissue distribution of rhGM-CSF preparations was assessed in outbred CD-1 mice after a single intravenous administration at the effective dose. The content of rhGM-CSF in blood, femoral tissue and bone marrow was determined by enzyme immunoassay.

Results. RhGM-CSF conjugates with ALN have been shown to retain the ability of the original protein to increase the number of leukocytes, segmented blood neutrophils, and bone marrow karyocytes under the action of conjugates. The stimulation of the neutrophil production used to be observed at earlier times than in the case of rhGM-CSF. The increase in the total number of bone marrow cells after the introduction of all three conjugates was more pronounced compared to the original protein (by 34%). The increased hemostimulatory effect of the AEG conjugate was accompanied by a more intense accumulation of rhGM-CSF in the bone tissue and bone marrow of mice. The rhGM-CSF introduced into the conjugate was detected in the bone tissue for 24 h and it circulated in the bloodstream for a longer time compared to the original protein.

Conclusion. The data obtained make it possible to conclude that further work on the development of effective hemostimulating drugs based on rhGM-CSF conjugates with ALN, is promising.

作者简介

Galina Shimina

Institute of Medical Biotechnology of State Research Center of Virology and Biotechnology “VECTOR”, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being

编辑信件的主要联系方式.
Email: shimina_gg@vector.nsc.ru
ORCID iD: 0000-0002-1078-7033

Researcher of the Department of Biological Research

俄罗斯联邦, 9, Khimzavodskaya St., Berdsk, 633004

Alena Bateneva

Institute of Medical Biotechnology of State Research Center of Virology and Biotechnology “VECTOR”, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being

Email: bateneva_av@vector.nsc.ru
ORCID iD: 0000-0002-3761-7798

Researcher of the Department of Biological Research

俄罗斯联邦, 9, Khimzavodskaya St., Berdsk, 633004

Elena Tsyplenkova

Institute of Medical Biotechnology of State Research Center of Virology and Biotechnology “VECTOR”, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being

Email: tsyplenkova_es@vector.nsc.ru
ORCID iD: 0000-0002-1277-6258

Junior Researcher, Department of Biological Research

俄罗斯联邦, 9, Khimzavodskaya St., Berdsk, 633004

Svetlana Gamaley

Institute of Medical Biotechnology of State Research Center of Virology and Biotechnology “VECTOR”, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being

Email: gamaley_sg@vector.nsc.ru
ORCID iD: 0000-0002-7441-333X

Head of the Department of Biological Research

俄罗斯联邦, 9, Khimzavodskaya St., Berdsk, 633004

Tatyana Esina

Institute of Medical Biotechnology of State Research Center of Virology and Biotechnology “VECTOR”, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being

Email: esina_ti@vector.nsc.ru
ORCID iD: 0000-0001-9006-8313

Junior Researcher at the Department of Technology Development and Pilot Production of Biological Products

俄罗斯联邦, 9, Khimzavodskaya St., Berdsk, 633004

Ekaterina Volosnikova

Institute of Medical Biotechnology of State Research Center of Virology and Biotechnology “VECTOR”, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being

Email: volosnikova_ea@vector.nsc.ru
ORCID iD: 0000-0001-5028-5647

Candidate of Sciences (Biology), Head of the Laboratory for Obtaining and Analyzing Biosubstances of the Department for Technology Development and Pilot Production of Biological Products

俄罗斯联邦, 9, Khimzavodskaya St., Berdsk, 633004

Elena Danilenko

Institute of Medical Biotechnology of State Research Center of Virology and Biotechnology “VECTOR”, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being

Email: danilenko_ed@vector.nsc.ru
ORCID iD: 0000-0001-5026-1602

Candidate of Sciences (Biology), Director

俄罗斯联邦, 9, Khimzavodskaya St., Berdsk, 633004

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2. Figure 1 – Electrophoregram of conjugates obtained by direct sequence of applying components, GEA (1) and reverse sequence, AEG (2). Note: Electrophoresis in 15% polyacrylamide gel under reducing conditions, R-250 Coomassie staining. Tracks: 3 – protein marker 10–250 kDa; 4 – rhGM-CSF protein, 20 µg.

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3. Figure 2 – Electropherogram of conjugate obtained using dextran (1). Note: Electrophoresis in 15% polyacrylamide gel under reducing conditions, R-250 Coomassie staining. Tracks: 2 – rhGM-CSF protein, 20 µg; 3 – protein marker 10–250 kDa.

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4. Figure 3 – Leukocyte count in peripheral CBA mice blood against the background of CP administration, rhGM-CSF drug and its conjugates with ALN. Note: abscissa shows study time (days); * – statistically significant difference in relation to the control (saline); ** – statistically significant difference in relation to rhGM-CSF at p ≤0.05. Area between dotted lines is confidence interval of indicator in intact mice.

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5. Figure 4 – Count of segmented neutrophils in peripheral CBA mice blood against the background of CP administration, rhGM-CSF drug and its conjugates with ALN. Note: abscissa shows study time (days); * – statistically significant difference in relation to the control (saline); ** – statistically significant difference in relation to rhGM-CSF at p ≤0.05. Area between dotted lines is confidence interval of indicator in intact mice.

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6. Figure 5 – Karyocyte counts in CBA mice bone marrow against the background of CP, rhGM-CSF its conjugates with ALN administration. Note: abscissa shows study time (days); * – statistically significant difference in relation to the control (saline); ** – statistically significant difference in relation to rhGM-CSF at p ≤0.05. Area between dotted lines is confidence interval of indicator in intact mice.

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