Comparison of epoetin alfa and darbepoetin alfa biological activity under different administration schedules in normal mice.

The unit of erythropoietic activity has long been the standard by which erythropoietic agents are judged, but the development of long-acting agents such as darbepoetin alfa has highlighted the shortcomings of this approach. To this point, we compared the in vivo activity of Epoetin alfa and darbepoetin alfa per microgram of protein core. Using the established mass-to-unit conversion for Epoetin alfa (1 microg congruent with 200 U), we then calculated darbepoetin alfa activity in units. Activity varied with treatment regimen (1 microg darbepoetin alfa congruent with 800 U for 3 times weekly dosing to 8,000 U for a single injection). This analysis reveals the inadequacy of evaluating darbepoetin alfa activity in terms of standard erythropoietic units. We therefore propose that for molecules with heightened biological activity, a more legitimate basis for comparison is the protein mass.

Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.

OBJECTIVE: 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. MATERIALS AND METHODS: Comparative pharmacokinetic and pharmacodynamic studies and biochemical analyses of darbepoetin alfa and rHuEPO were performed to define the consequences of the increased carbohydrate content. RESULTS: 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. CONCLUSIONS: 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.

Development and characterization of darbepoetin alfa.

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogs were developed to test this hypothesis. Darbepoetin alfa (Aranesp), which was engineered to contain five N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, darbepoetin alfa is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximate threefold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. Darbepoetin alfa is currently being evaluated in human clinical trials for treatment of anemia and reduction in its incidence.