Experiments on human erythropoietin (EPO) demonstrated that there is a direct relationship
between the sialic acid-containing carbohydrate content of EPO, its circulating half-life,
and in vivo bioactivity. This led to the hypothesis that an EPO analogue engineered
to contain additional oligosaccharide chains would have enhanced biological activity.
Darbepoetin alfa, a hyperglycosylated recombinant human EPO (rHuEPO) analogue with
two extra carbohydrate chains, was designed and developed to test this hypothesis.
Comparative pharmacokinetic and pharmacodynamic studies and biochemical analyses of
darbepoetin alfa and rHuEPO were performed to define the consequences of the increased
Due to its increased sialic acid-containing carbohydrate content, darbepoetin alfa
has a higher molecular weight, a greater negative charge, and a approximately fourfold
lower EPO receptor binding activity than rHuEPO. It also has a threefold longer circulating
half-life than rHuEPO in rats and dogs. In spite of its lower receptor binding, and
perhaps counterintuitively, darbepoetin alfa is significantly more potent in vivo
than rHuEPO. Due to the pharmacokinetic differences, the relative potency of the two
molecules varies as a function of the dosing frequency. Darbepoetin alfa is 3.6-fold
more potent than rHuEPO in increasing the hematocrit of normal mice when each is administered
thrice weekly, but when the administration frequency is reduced to once weekly, darbepoetin
alfa is approximately 13-fold to 14-fold more potent than rHuEPO.
Increasing the sialic acid-containing carbohydrate content beyond the maximum found
in EPO leads to a molecule with a longer circulating half-life and thereby an increased
in vivo potency that can be administered less frequently.