Erythropoietin

Erythropoietin

Erythropoietin (Epo) is a hormone produced by the kidneys in response to hypoxia and is also a prescription drug used in treatment of anemia. It is essential for normal development and maturation of red blood cells (RBC), and abnormally high levels of either the endogenous or drug form can lead to dangerously high hematocrit values.

History

Carnot and DeFlandre (ref) made initial observations in rabbits that suggested the existence of a factor in peripheral blood that could stimulate production of reticulocytes. Their experiment involved bleeding a rabbit to induce accelerated RBC production, then transfer of some of this animal's plasma to a recipient animal. The key observation of increased reticulocytes in the recipient animal prompted the search for a substance, which they named hemopoietin, that regulated the rate of RBC production.

A major breakthrough came In 1977 when small amounts of erythropoietin were purified from the urine of patients with aplastic anemia (Miyake). Amino acid sequence data from this protein were used in subsequent efforts to clone the gene for erythropoietin in 1983 (lin ref). The gene was then inserted into a suitable mammalian cell line, Chinese hamster ovary cells, allowing large-scale manufactureof the protein as a commercial product. It was approved for use in 1991. About $10B was spent worldwide in 2006 for treatment of patients with rHuEpo, with about $2B for the cost of treating Medicare patients on dialysis (Smith).

Nomenclature

Erythropoietin exists in several forms and goes by several names. The endogenous form is also referred to as epoetin alfa and sometimes spelled as erythropoetin; it can be abbreviated to EPO, Epo, or EP. Various synthetic forms of recombinant (r) human (h) Epo are available, collectively referred to as rHuEpo or rhEpo. These include the following:

• Procrit is the trade name of epoetin alfa marketed in the US by Ortho Biotech Products, L.P., a member of the Johnson & Johnson Family of Companies. It is approved for treatment of chemotherapy-related anemia in patients with most types of cancer; for the treatment of anemia in chronic kidney disease patients who are not on dialysis; for treatment of anemia related to zidovudine treatment in HIV patients; and for reducing the need for transfusions in patients undergoing some types surgery who are anemic or at significant risk for blood loss.
• Eprex is the trade name of epoetin alfa marketed outside the US by Ortho Biotech Products, L.P., a member of the Johnson & Johnson Family of Companies.
• Epogen is the trade name of epoetin alfa made and marketed by Amgen in the US for treatment of anemia in patients with chronic renal failure on dialysis.
• NeoRecormon is the trade name of epoetin beta marketed by Roche in Europe for treatment of anemia in patients with chronic renal failure and for treatment of anemia in people with solid tumours who are receiving platinum-based chemotherapy. Even though epoetin alfa and epoetin beta are both synthesized in Chinese hamster ovary cells, they differ in their erythropoietin isoform compositions and biological properties (Storring et al., BJH 100:79)
• Aranesp is the trade name of darbepoetin alfa, a hyperglycosylated mutant form of Epo produced and marketed by Amgen or the treatment of anemia associated with chronic renal failure (CRF), including patients on dialysis and patients not on dialysis, and for the treatment of anemia in patients with nonmyeloid malignancies where anemia is due to the effect of concomitantly administered chemotherapy.

Structure

Epo is a glycoprotein with a molecular mass of 30.4 kD. Its strucure includes a 165-amino acid backbone with three N-linked carbohydrates attached to asparagines at amino acid positions 24, 38, and 83 and one O-linked carbohydrate attached to Ser126 (CSHQB ref). The carbohydrate residues allow for many possible isoforms and contribute to stability of the hormone in vivo. Darbepoetin (see above) was created through site-directed mutation of two amino acid residues, allowing for two additional N-linked carbohydrate chains.

Production

Epo is produced by peritubular cells in the adult kidney, and in hepatocytes in the fetus. In adults, a small amount of Epo is produced by the liver. The rate of Epo synthesis and secretion depends on local oxygen concentrations; hypoxia is the main stimulus for Epo production. The serum concentration of Epo in adults is normally 4-27 mU/mL. In adults with non-renal anemias, the serum concentration of Epo tends to increase with the severity of the anemia.

Actions

Epo's activities depend on successful interaction with its receptor, which is prominent on the surface of developing RBC in the bone marrow. Epo signaling acts to prevent or retard apoptosis, i.e., it acts as a survival factor for developing cells. The increase in RBC mass brought about by Epo stimulation of the bone marrow completes a self-regulating feedback loop, since (other things being equal), the increased RBC mass would lessen the hypoxia experienced by the kidney and thus, lessen Epo production.

Pharmaceutical Epo

EPO as a blood doping agent

Pharmaceutical Epo is sometimes used by nonanemic athletes to increase their body's oxygen-carrying capacity and thus gain an unfair advantage in competition. Besides the risk of disqualification for cheating, athletes who participate in this illicit use of Epo risk the complications of abnormally high RBC concentrations, which include abnormal blood clotting. Detection of illicit Epo use is challenging because endogenous and exogenous (pharmaceutical) Epo are almost identical. Several tests rely on altered patterns of glycosylation of Epo molecules shed in the urine. Other detection methods rely on altered parameters of RBC production such as hematocrit, reticulocyte hematocrit, the proportion of abnormally large RBC, the serum Epo level, and the soluble transferrin receptor concentration. (Parisotto 2001).

Potential future applications

Epo's activity in the bone marrow to increase RBC production hinges on its ability to inhibit apoptosis. Experimental treatment of diseases in which apoptosis is prominent have yielded promising initial results. For example, Epo has been proposed as being both safe and beneficial in acute stroke (ref).

References

Browne JK, Cohen AM, Egrie JC, Lai PH, Lin FK, Strickland T, Watson E,Stebbing N. Erythropoietin: gene cloning, protein structure, and biological properties. Cold Spring Harb Symp Quant Biol. 51:693-702, 1986.

Carnot P, DeFlandre C. Sur l’activit´e h´ematopoi´etique des diff´erents organes au cours de la r´eg´en´eration du sang. C. R. Acad. Sci. Paris 143:432–435, 1906

Ehrenreich H, Hasselblatt M, Dembowski C, Cepek L, Lewczuk P, Stiefel M, Rustenbeck HH, Breiter N, Jacob S, Knerlich F, Bohn M, Poser W, Ruther E, Kochen M, Gefeller O, Gleiter C, Wessel TC, De Ryck M, Itri L, Prange H, Cerami A, Brines M, Siren AL. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med 8:495-505, 2002

Storring PL, Tiplady RJ, Gaines Das RE, Stenning BE, Lamikanra A, Rafferty B, Lee J. Epoetin alfa and beta differ in their erythropoietin isoform compositions and biological properties. Br J Haematol 100:79-89, 1998.

Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, Chen KK, Fox GM, Martin FM, Stabinsky Z, Badrawi SM, Lai PH, Goldwasser E. Cloning and expression of the human erythropoietin gene. PNAS 82: 7580-7584, 1985.

Miyake T, Kung CK, Goldwasser E. Purification of human erythropoietin. J Biol Chem 252:5558-5564, 1977

Parisotto R, Wu M, Ashenden MJ, Emslie KR, Gore CJ, Howe C, Kazlauskas R, Sharpe K, Trout GJ, Xie M. Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis. Haematologica 86:128-137, 2001

Smith M. Aggressive anemia treatment increases mortality. MedPage Today, February 2, 2007, http://www.medpagetoday.com/Nephrology/GeneralNephrology/tb/4987.