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Design Rationale and Development Approach for Pegfilgrastim as a Long-Acting Granulocyte Colony-Stimulating Factor.

Arvedson T, O'Kelly J, Yang BB - BioDrugs (2015)

Bottom Line: Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim.Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy.This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim.

View Article: PubMed Central - PubMed

Affiliation: Amgen Inc., 14-1-B, One Amgen Center Drive, Thousand Oaks, CA, 91320-1799, USA, tara.arvedson@amgen.com.

ABSTRACT
Filgrastim, a recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF), is efficacious in stimulating neutrophil production and maturation to prevent febrile neutropenia (FN) in response to chemotherapy. Because of its relatively short circulating half-life, daily filgrastim injections are required to stimulate neutrophil recovery. In an effort to develop a long-acting form of filgrastim that was as safe and efficacious as filgrastim but had a longer in vivo residence time, a number of strategies were considered. Ultimately, fusion of filgrastim to polyethylene glycol (PEG) was selected. Following extensive analysis of conjugation chemistries as well as in vitro and in vivo characterization of a panel of PEGylated proteins, a construct containing a 20 kDa PEG moiety covalently conjugated to the N-terminus of filgrastim was chosen for advancement as pegfilgrastim. Pegfilgrastim is primarily cleared by neutrophils and neutrophil precursors (rather than the kidneys), meaning that clearance from the circulation is self-regulating and pegfilgrastim is eliminated only after neutrophils start to recover. Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim. Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy. This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim. Approaches that may provide advances for therapeutic agonists of G-CSF receptor are also discussed.

No MeSH data available.


Related in: MedlinePlus

PEGylation-mediated increases in vivo activity were associated with decreases in vitro activity. The in vitro and in vivo activity of various polyethylene glycol (PEG)–recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF) constructs were assessed and plotted according to the amount of PEG added per molecule. For candidates that were pegylated at multiple sites, the amount of PEG reflects the total molecular weight of the PEG additions. Triangles indicate the proliferation induced in cell line 32D clone 3 stably expressing human G-CSF receptor. Squares indicate the weighted area under the concentration–time curve (AUC) obtained from the daily average absolute neutrophil count (ANC) from mice (n = 5/timepoint) weighted by multiplying by the number of days after injection, then summed. The red triangle and the red square mark the in vitro and in vivo activity of the pegfilgrastim construct selected for further development. Reproduced from Molineux [13] with permission of Springer Science + Business Media (© Birkauser Verlag/Switzerland)
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Fig2: PEGylation-mediated increases in vivo activity were associated with decreases in vitro activity. The in vitro and in vivo activity of various polyethylene glycol (PEG)–recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF) constructs were assessed and plotted according to the amount of PEG added per molecule. For candidates that were pegylated at multiple sites, the amount of PEG reflects the total molecular weight of the PEG additions. Triangles indicate the proliferation induced in cell line 32D clone 3 stably expressing human G-CSF receptor. Squares indicate the weighted area under the concentration–time curve (AUC) obtained from the daily average absolute neutrophil count (ANC) from mice (n = 5/timepoint) weighted by multiplying by the number of days after injection, then summed. The red triangle and the red square mark the in vitro and in vivo activity of the pegfilgrastim construct selected for further development. Reproduced from Molineux [13] with permission of Springer Science + Business Media (© Birkauser Verlag/Switzerland)

Mentions: As shown in Fig. 2, there was a positive relationship between the molecular weight of the added PEG and the in vivo activity of the modified filgrastim. Similar results were obtained by other researchers with PEGylated filgrastim or nartograstim [41, 44], whereby white blood cell counts in mice were found to increase as the size or number of PEG units was increased [55]. In those studies, like our own, it was also observed that molecules with increased in vivo activity demonstrated decreased in vitro activity. In general, these characteristics became more magnified as the molecular weight of the PEG molecule increased. The decreased in vitro activity is likely a consequence of the PEG molecule interfering directly and/or indirectly with G-CSF binding to its receptor. This is likely a consequence of the PEG molecule’s bulk and mobility which could cause periodic occlusion of the binding interface, slowing the on-rate of binding. A similar relationship between enhanced in vivo activity and decreased in vitro activity has been observed for PEG conjugates of human growth hormone [29] and asiolofetuin [45]. The result of our studies was the selection of an r-metHuG-CSF–PEG conjugate containing a single linear 20 kDa PEG attached to the N-terminal methionine. This construct would become pegfilgrastim.Fig. 2


Design Rationale and Development Approach for Pegfilgrastim as a Long-Acting Granulocyte Colony-Stimulating Factor.

Arvedson T, O'Kelly J, Yang BB - BioDrugs (2015)

PEGylation-mediated increases in vivo activity were associated with decreases in vitro activity. The in vitro and in vivo activity of various polyethylene glycol (PEG)–recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF) constructs were assessed and plotted according to the amount of PEG added per molecule. For candidates that were pegylated at multiple sites, the amount of PEG reflects the total molecular weight of the PEG additions. Triangles indicate the proliferation induced in cell line 32D clone 3 stably expressing human G-CSF receptor. Squares indicate the weighted area under the concentration–time curve (AUC) obtained from the daily average absolute neutrophil count (ANC) from mice (n = 5/timepoint) weighted by multiplying by the number of days after injection, then summed. The red triangle and the red square mark the in vitro and in vivo activity of the pegfilgrastim construct selected for further development. Reproduced from Molineux [13] with permission of Springer Science + Business Media (© Birkauser Verlag/Switzerland)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4488452&req=5

Fig2: PEGylation-mediated increases in vivo activity were associated with decreases in vitro activity. The in vitro and in vivo activity of various polyethylene glycol (PEG)–recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF) constructs were assessed and plotted according to the amount of PEG added per molecule. For candidates that were pegylated at multiple sites, the amount of PEG reflects the total molecular weight of the PEG additions. Triangles indicate the proliferation induced in cell line 32D clone 3 stably expressing human G-CSF receptor. Squares indicate the weighted area under the concentration–time curve (AUC) obtained from the daily average absolute neutrophil count (ANC) from mice (n = 5/timepoint) weighted by multiplying by the number of days after injection, then summed. The red triangle and the red square mark the in vitro and in vivo activity of the pegfilgrastim construct selected for further development. Reproduced from Molineux [13] with permission of Springer Science + Business Media (© Birkauser Verlag/Switzerland)
Mentions: As shown in Fig. 2, there was a positive relationship between the molecular weight of the added PEG and the in vivo activity of the modified filgrastim. Similar results were obtained by other researchers with PEGylated filgrastim or nartograstim [41, 44], whereby white blood cell counts in mice were found to increase as the size or number of PEG units was increased [55]. In those studies, like our own, it was also observed that molecules with increased in vivo activity demonstrated decreased in vitro activity. In general, these characteristics became more magnified as the molecular weight of the PEG molecule increased. The decreased in vitro activity is likely a consequence of the PEG molecule interfering directly and/or indirectly with G-CSF binding to its receptor. This is likely a consequence of the PEG molecule’s bulk and mobility which could cause periodic occlusion of the binding interface, slowing the on-rate of binding. A similar relationship between enhanced in vivo activity and decreased in vitro activity has been observed for PEG conjugates of human growth hormone [29] and asiolofetuin [45]. The result of our studies was the selection of an r-metHuG-CSF–PEG conjugate containing a single linear 20 kDa PEG attached to the N-terminal methionine. This construct would become pegfilgrastim.Fig. 2

Bottom Line: Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim.Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy.This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim.

View Article: PubMed Central - PubMed

Affiliation: Amgen Inc., 14-1-B, One Amgen Center Drive, Thousand Oaks, CA, 91320-1799, USA, tara.arvedson@amgen.com.

ABSTRACT
Filgrastim, a recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF), is efficacious in stimulating neutrophil production and maturation to prevent febrile neutropenia (FN) in response to chemotherapy. Because of its relatively short circulating half-life, daily filgrastim injections are required to stimulate neutrophil recovery. In an effort to develop a long-acting form of filgrastim that was as safe and efficacious as filgrastim but had a longer in vivo residence time, a number of strategies were considered. Ultimately, fusion of filgrastim to polyethylene glycol (PEG) was selected. Following extensive analysis of conjugation chemistries as well as in vitro and in vivo characterization of a panel of PEGylated proteins, a construct containing a 20 kDa PEG moiety covalently conjugated to the N-terminus of filgrastim was chosen for advancement as pegfilgrastim. Pegfilgrastim is primarily cleared by neutrophils and neutrophil precursors (rather than the kidneys), meaning that clearance from the circulation is self-regulating and pegfilgrastim is eliminated only after neutrophils start to recover. Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim. Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy. This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim. Approaches that may provide advances for therapeutic agonists of G-CSF receptor are also discussed.

No MeSH data available.


Related in: MedlinePlus