ABSTRACT: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. [ONCOLOGY 16(Suppl 11):13-22, 2002]
Erythropoietin (EPO) is a glycoprotein hormone that is the primary regulator of erythropoiesis, maintaining the body’s red blood cell mass at an optimum level.[1,2] In response to a decrease in tissue oxygenation, EPO synthesis increases in the kidney. The secreted hormone binds to specific receptors on the surface of red blood cell precursors in the bone marrow, leading to their survival, proliferation and differentiation, and ultimately to an increase in hematocrit.[3,4]
Since its introduction more than a decade ago, recombinant human EPO (rHuEPO) has become the standard of care in treating the anemia associated with chronic renal failure (CRF). It is highly effective at correcting the anemia, restoring energy levels, and increasing patient well-being and quality of life.[5-8] It has also been approved for the treatment of anemia associated with cancer, HIV infection, and use in the surgical setting to decrease the need for allogeneic blood transfusions. For all indications, it has proven to be remarkably well tolerated and highly efficacious.[9-11]
The recommended and usual therapy with rHuEPO is two to three times per week by subcutaneous or intravenous injection. For CRF patients, the duration of therapy is for the life of the patient, or until a successful kidney transplant restores kidney function, including the production of the natural hormone. For cancer patients, rHuEPO therapy is indicated for as long as the anemia persists, generally through the entire course of chemotherapy.
It can be a hardship to administer rHuEPO two to three times per week, particularly for those patients who do not otherwise need to be seen in the clinic this frequently. In these cases, the patient needs to make a special trip to the clinic for his or her rHuEPO therapy. As is true for all growth factors, reduction in dose frequency results in a significant loss in efficiency. That is, the total weekly dose required when rHuEPO is administered one time per week is greater than when it is administered as two to three divided doses. It was anticipated that this clinical need could be addressed by creating a molecule with enhanced in vivo bioactivity to allow for less frequent dosing of patients.
To create a molecule with enhanced activity, research was initially directed towards elucidating those factors and structural features that control the in vivo activity of EPO. This research led to the discovery and development of darbepoetin alfa, a novel erythropoiesis stimulating protein (Aranesp), that can be administered less frequently than epoetin.
Structure of EPO
Human EPO is a 30,400-dalton heavily glycosylated protein hormone.[14,15] Sixty percent (by weight) of the molecule is an invariant 165 amino acid single polypeptide chain containing two disulfide bonds.[16,17] The remaining 40% of the mass of the molecule is carbohydrate. Carbohydrate addition (glycosylation) is a posttranslational event that results in the addition of sugar chains to specific asparagine (N-linked) or serine/threonine (O-linked) amino acids in the polypeptide. The carbohydrate portion of natural and recombinant human EPO consists of three N-linked sugar chains at Asn 24, 38, and 83, and one O-linked (mucin-type) sugar chain at Ser 126.[18,19]
Structure determinations using nuclear magnetic resonance spectroscopy and x-ray crystallography have indicated that human EPO is an elongated molecule with an overall topology of a left-handed four-helix bundle, typical of members of the hematopoietic growth factor family. In addition, these studies have identified the amino acids at the receptor-binding sites. The carbohydrate addition sites are clustered at one end of the molecule, distal from the receptor-binding site. While the four carbohydrate chains contribute approximately 40% of the mass of the hormone, they probably cover much of the surface of the molecule since they have an extended and flexible molecular structure.
In contrast to the invariant amino acid sequence of the protein portion of glycoproteins, the carbohydrate structures are variable, a feature referred to as microheterogeneity. For example, N-glycosylation sites on the same protein may contain different carbohydrate structures. Furthermore, even at the same glycosylation site on a given glycoprotein, different structures may be found. This heterogeneity is a consequence of the non-template-directed synthesis of carbohydrates.
The carbohydrate structures of EPO have been determined and the extent of the microheterogeneity defined for both rHuEPO and the natural hormone.[22-25] One of the most prominent examples of microheterogeneity for EPO is seen on the N-linked carbohydrate chains, where the oligosaccharides may contain two, three, or four branches (or antennae), each of which is typically terminated with the negatively-charged sugar molecule, sialic acid (Figure 1). With the exception of sialic acid, all of the other sugar molecules on EPO are neutral. Similarly, the single O-linked carbohydrate may contain zero to two sialic acid molecules. Since each of the three N-linked oligosaccharides can contain up to four sialic acid residues, and the single O-linked chain can contain two, the EPO molecule can have a maximum of 14 sialic acid residues.
Therefore, because of the variability in sugar structure, the number of sialic acid molecules on EPO varies, and as a consequence, so does the molecule’s net negative charge. As indicated in Figure 1, an isoform of EPO is defined as a subset of the EPO molecules that has a defined charge due to its sialic acid content. For reference, epoetin alfa (Epogen, Procrit), the source of the purified rHuEPO used for these studies, has been purified so as to contain isoforms 9 to 14.
Role of Carbohydrate in Biological Activity
The carbohydrate portions of different glycoprotein molecules have been shown to have many diverse functions, including effects on the biosynthesis and secretion, immune protection, conformation, stability, solubility, and biological activity of molecules.[26,27] For rHuEPO, in particular, it has been shown that the addition of carbohydrate is required for secretion from the cell, and for increasing the solubility of the molecule.[28-30] Early research on EPO from natural sources indicated that the sialic acid residues were necessary for biological activity in vivo.[31-33] Removal of the sialic acid from either native EPO or rHuEPO resulted in molecules having an increased activity in vitro, but very low activity in vivo, presumably due to removal from circulation by the asialoglycoprotein receptor in the liver.[34,35] Similarly, it was shown that EPO molecules, which have been deglycosylated to remove carbohydrate (or produced in Escherichia coli to allow expression of only the EPO polypeptide), are active in vitro, but have very low in vivo activity.[36,37]
In order to define further the role of carbohydrate in biological activity, the approach taken was to purify EPO carbohydrate isoforms, measure their in vivo activity and determine how the different carbohydrate structures affect activity.
rHuEPO, produced by Chinese hamster ovary cells, was purified to contain the entire complement of isoforms 4 to 14, and then further fractionated by ion exchange chromatography to isolate the individual isoforms. The in vivo efficacy of each of the individual isoforms was tested in normal mice to determine the effect of repetitive dosing on the hematocrit. In this assay, CD-1 mice were injected with either a vehicle control or an equimolar dose (2.5 µg/kg of peptide) of each of the individual isoforms by intraperitoneal injection three times per week for 1 month. The results of this experiment demonstrated a striking difference in the biological activity of the individual isoforms, with those isoforms having a higher sialic acid content exhibiting a progressively higher in vivo efficacy (Figure 2). By day 30, the group mean hematocrit of isoform 14-treated animals increased by 26.2 ± 2.7 points (to a hematocrit of 76.2%), compared with an increase of only 6.3 ± 3.5 points for the isoform 8-treated group, a 4.2-fold increase in efficacy. In contrast, animals receiving vehicle control showed no hematocrit change from baseline during the experiment.
It was reasoned that these results might have two possible explanations: the more active isoforms might have a longer serum half-life and/or an increased ability to bind to the EPO receptor. In order to assess the contribution of each of these possibilities, the pharmacokinetics and receptor-binding activity of the individual isoforms was measured.
The isolated EPO isoforms were iodinated and their circulating half-life determined after intravenous injection into rats. At specified intervals after dosing, blood samples were taken and the fraction of iodinated isoform remaining in circulation was measured. The isoforms with the increased sialic acid content had a longer serum half-life than those with the lower sialic acid content (Figure 3). The beta half-life of isoform 14 was 3.2-fold longer than that for isoform 6 (3.97 vs 1.24 hours, respectively). As expected from these results, the serum clearance of the isoforms progressively increased as the sialic acid content decreased. In contrast, the volume of distribution was the same for the individual isoforms and approximately equal to the plasma volume (data not shown). Thus, those isoforms that have a higher sialic acid content have a higher in vivo biological activity, longer serum half-life and slower serum clearance.
Next, the relative affinity of various EPO isoform preparations for the EPO receptor was determined in a radioreceptor assay. This assay measures the quantity of each isoform required to displace 125I-rHuEPO bound to the EPO receptor on the surface of OCIM1 cells. The IC50, the amount of test compound required to compete 50% of the receptor-bound 125I-rHuEPO, was determined for each isoform. As seen in Figure 3, the higher the sialic acid content, the greater the quantity of EPO isoform necessary to compete 125I-rHuEPO binding. Thus, those isoforms having a higher sialic acid content had a lower relative affinity for the EPO receptor. The relative affinity of isoform 6 for the EPO receptor was sevenfold greater than that for isoform 14.
Taken together, these experiments indicate that the carbohydrate moieties of EPO have significant effects on the biological activity of the hormone, modulating both receptor affinity and serum clearance. There is a direct relationship between sialic acid content, in vivo biological activity, and serum half-life, but an inverse relationship with receptor affinity. While conventional wisdom might have predicted that increases in receptor affinity would lead to a more active molecule, clearly this is not the case. In fact, as shown in these experiments, isoform 9, which has a 2.6-fold greater affinity for the EPO receptor than isoform 14, has only approximately one-third of the in vivo activity (Figures 2 and 3). These observations clearly demonstrate that clearance has a far stronger influence on in vivo activity than does receptor-binding affinity. Increases in serum half-life were able to overcome the observed decreases in receptor affinity. Thus, for EPO, serum clearance, not receptor binding affinity, is the primary determinant of in vivo activity.
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