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Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor.

ABSTRACT: The clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG₃-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for rhG-CSF modification with PAL-PEG₃-Ph-CHO were: reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH₃ in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PAL-PEG₃-Ph-CHO to rhG-CSF of 6:1, temperature of 20°C, and reaction time of 12 h, consequently, achieving a PAL-PEG₃-Ph-rhG-CSF product yield of 70.8%. The reaction mixture was purified via preparative liquid chromatography, yielding the single-modified product PAL-PEG₃-Ph-rhG-CSF with a HPLC purity exceeding 95%. The molecular weight of PAL-PEG₃-Ph-rhG-CSF was 19297 Da by MALDI-TOF-MS, which was consistent with the theoretical value. The circular dichroism analysis revealed no significant change in its secondary structure compared to unmodified rhG-CSF. The PAL-PEG₃-Ph-rhG-CSF retained 82.0% of the in vitro biological activity of unmodified rhG-CSF. The pharmacokinetic analyses showed that the serum half-life of PAL-PEG₃-Ph-rhG-CSF was 7.404 ± 0.777 h in mice, 4.08 times longer than unmodified rhG-CSF. Additionally, a single subcutaneous dose of PAL-PEG₃-Ph-rhG-CSF presented comparable in vivo efficacy to multiple doses of rhG-CSF. This study demonstrated an efficacious strategy for developing long-acting rhG-CSF drug candidates.

SUBMITTER: Wang XD

PROVIDER: S-EPMC10993259 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor.

Wang Xu-Dong XD Su Zhi-Hao ZH Du Jie J Yu Wei-Jia WJ Sun Wen-Long WL

Frontiers in bioengineering and biotechnology 20240318

The clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG3-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for rhG-CSF modification with PAL-PEG3-Ph-CHO were: reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH3 in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PA ...[more]

PMID: 38576447

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Project description:Currently, amino-terminal PEGylated human granulocyte colony stimulating factor (huG-CSF) is used to prevent and treat neutropenia. Although huG-CSF has been used as a drug for more than 20 years, it has three significant drawbacks: (i) it relies on PEG aldehyde for PEGylation of the alpha-amino group of the first amino acid, and this leads to non-specific PEGylation of the epsilon amino group of lysine residues within the G-CSF; (ii) longer-acting G-CSF variants are desirable to reduce the risk of chemotherapy-associated neutropenia; and (iii) G-CSF cannot be administered on the day of chemotherapy. In an attempt to overcome the above drawbacks, we engineered cysteine variants of G-CSF to facilitate the maleimide PEG-based site-specific PEGylation that leads to a highly homogenous PEGylated product. Importantly, we have demonstrated that 20 kDa thiol-reactive PEG conjugated by maleimide chemistry to the Cys2 G-CSF variant exhibits leukocyte proliferative activity similar to that of the commercially available G-CSF conjugated with aldehyde PEG in a neutropenia mice model. Moreover, we have demonstrated that PEGylation of the cysteine variant of huG-CSF with higher molecular weight PEGs, such as 30 kDa PEG and 40 kDa PEG, leads to significantly prolonged leukocyte proliferation activity compared to the variant conjugated with 20 kDa PEG. Importantly, even a half-dose of the engineered variant conjugated with 40 kDa PEG exhibited significantly longer biological activity than the commercially available 20 kDa PEGylated huG-CSF. Finally, we have demonstrated that administration of the engineered variant conjugated with 40 kDa PEG on the day of administration of cyclophosphamide for inducing neutropenia in mice can alleviate neutropenia through leukocyte proliferation. In summary, this study provides the design of site-specific PEGylated huG-CSF variants with improved therapeutic potential. It opens the possibility of long-acting and same-day prophylactic administration of G-CSF after chemotherapy drug regimens. These results may pave the way for the development of potential G-CSF derivatives possessing longer half-lives and favorable clinical attributes.

Project description:Despite recent advances in critical care and ventilator management, acute lung injury and acute respiratory distress syndrome continue to cause significant morbidity and mortality. Granulocyte-macrophage colony stimulating factor may be beneficial for patients with acute respiratory distress syndrome.To determine whether intravenous infusion of granulocyte-macrophage colony stimulating factor would improve clinical outcomes for patients with acute lung injury/acute respiratory distress syndrome.A randomized, double-blind, placebo-controlled clinical trial of human recombinant granulocyte-macrophage colony stimulating factor vs. placebo. The primary outcome was days alive and breathing without mechanical ventilatory support within the first 28 days after randomization. Secondary outcomes included mortality and organ failure-free days.Medical and surgical intensive care units at three academic medical centers.One hundred thirty individuals with acute lung injury of at least 3 days duration were enrolled, out of a planned cohort of 200 subjects.Patients were randomized to receive human recombinant granulocyte-macrophage colony stimulating factor (64 subjects, 250 ?g/M) or placebo (66 subjects) by intravenous infusion daily for 14 days. Patients received mechanical ventilation using a lung-protective protocol.There was no difference in ventilator-free days between groups (10.7 ± 10.3 days placebo vs. 10.8 ± 10.5 days granulocyte-macrophage colony stimulating factor, p = .82). Differences in 28-day mortality (23% in placebo vs. 17% in patients receiving granulocyte-macrophage colony stimulating factor (p = .31) and organ failure-free days (12.8 ± 11.3 days placebo vs. 15.7 ± 11.9 days granulocyte-macrophage colony stimulating factor, p = .16) were not statistically significant. There were similar numbers of serious adverse events in each group.In a randomized phase II trial, granulocyte-macrophage colony stimulating factor treatment did not increase the number of ventilator-free days in patients with acute lung injury/acute respiratory distress syndrome. A larger trial would be required to determine whether treatment with granulocyte-macrophage colony stimulating factor might alter important clinical outcomes, such as mortality or multiorgan failure. (ClinicalTrials.gov number, NCT00201409 [ClinicalTrials.gov]).

Dataset Information

Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor.

Publications

Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor.

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