Jeffrey K. Browne

Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial.

We administered recombinant human erythropoietin to 25 anemic patients with end-stage renal disease who were undergoing hemodialysis. The recombinant human erythropoietin was given intravenously three times weekly after dialysis, and transfusion requirements, hematocrit, ferrokinetics, and reticulocyte responses were monitored. Over a range of doses between 15 and 500 units per kilogram of body weight, dose-dependent increases in effective erythropoiesis were noted. At 500 units per kilogram, changes in the hematocrit of as much as 10 percentage points were seen within three weeks, and increases in ferrokinetics of three to four times basal values, as measured by erythron transferrin uptake, were observed. Of 18 patients receiving effective doses of recombinant human erythropoietin, 12 who had required transfusions no longer needed them, and in 11 the hematocrit increased to 35 percent or more. Along with the rise in hematocrit, four patients had an increase in blood pressure, and a majority had increases in serum creatinine and potassium levels. No organ dysfunction or other toxic effects were observed, and no antibodies to the recombinant hormone were formed. These results demonstrate that recombinant human erythropoietin is effective, can eliminate the need for transfusions with their risks of immunologic sensitization, infection, and iron overload, and can restore the hematocrit to normal in many patients with the anemia of end-stage renal disease.

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.

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.

Studies of TGEV Spike Protein Gp195 Expressed in E. Coli and by a TGE-Vaccinia Virus Recombinant

The gene coding for the surface spike protein gp195 of TGEV has been cloned and expressed in E. coli in the form of fusion proteins. These proteins were isolated and used to immunize laboratory animals. All animals developed antibodies cross-reacting with the TGEV virion but failed to neutralize the virus. The entire gp195 gene was also inserted into vaccinia to generate a TGEV-vaccinia recombinant virus (vTGE) that expressed TGEV gp195. Animals vaccinated with vTGE produced neutralizing antibodies against both TGEV and vaccinia. These results suggest the potential use of the recombinant vTGE as a vaccine against TGEV infection.

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 µg ≅ 200 U), we then calculated darbepoetin alfa activity in units. Activity varied with treatment regimen (1 µg darbepoetin alfa ≅ 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.