The Physiologic Basis for the Pharmacologic Use of Recombinant
Erythropoietin
Jerry L. Spivak, MD
While many growth factors influence erythroid progenitor cell
proliferation,
the hematopoietic growth factor erythropoietin has the most impact on
the production of erythrocytes.
Introduction
Erythropoiesis, the orderly continuous process by which committed
erythroid progenitor cells differentiate into mature circulating erythrocytes, requires
(1) a normal hematopoietic progenitor cell pool, (2) an adequate supply of essential
nutrients such as iron, folic acid, and vitamin B12, and (3) ongoing exposure to specific
hematopoietic growth factors. While the major function of the red cell is to transport
oxygen from the lungs to the other tissues, the coupling of long-term tissue oxygen
requirements with long-term blood oxygen carrying capacity is the function of the
hematopoietic growth factor erythropoietin.
Endogenous Erythropoietin Production
Among the hematopoietic growth factors, erythropoietin is one of few
that behaves like a hormone. It is unique because its production, under normal
circumstances, is controlled solely at the level of its gene by tissue hypoxia and not by
the absolute number of circulating erythrocytes. Although a variety of growth factors
influence erythroid progenitor cell proliferation, erythropoietin is the most important,
and erythropoiesis cannot continue in its absence.
Erythropoietin is a 30,400 m.w. glycoprotein that is produced
primarily in the kidneys in adults and, to a lesser extent, in the liver. In the kidneys,
erythropoietin is produced in fibroblastoid interstitial cells in the inner renal cortex,1
while in the liver, the hormone is produced by both hepatocytes and interstitial
fibroblastoid cells.2 It is also produced in other tissues such as the testes
and brain in an amount that is negligible in comparison with its production in the kidneys
and liver. Moreover, its function in the testes and brain is unknown.3
Hypoxia is the sole physiologic stimulus for erythropoietin
production. However, transient elevations of serum erythropoietin occur initially with the
use of chemotherapeutic drugs4 and zidovudine5 and have also been
observed with hepatocellular damage.6 More persistent elevation of serum
erythropoietin can occur with certain tumors, primarily those arising in the kidneys,
liver, and cerebellum.7 In the kidney, erythropoietin production occurs on an
all-or-none basis in each cell; normally, there is constitutive production of the hormone
that is commensurate with its role as a survival factor as well as a mitogen.8,9
With increasing tissue hypoxia, more renal tubular interstitial cells are recruited to
produce erythropoietin.10 Erythropoietin production in the liver is also
constitutive, but in contrast to the kidneys, it can be modulated in each hepatocyte
depending on the degree of tissue oxygenation. The behavior of hepatic fibroblastoid cells
with respect to erythropoietin production is still undefined.3 Furthermore, the
threshold for up-regulation of erythropoietin production is much higher in the liver than
in the kidneys. Even though the liver is a larger organ, in the absence of the kidneys,
liver erythropoietin production is unable to maintain an adequate hemoglobin level.
Erythropoietin Interactions With Erythroid Progenitor Cells
Following production in the kidneys and liver, erythropoietin
travels to the bone marrow to interact with specific hematopoietic progenitor cells.
Specificity is provided by the surface of expression of high-affinity receptors for the
hormone,11 and these receptors are primarily expressed on erythroid progenitor
cells. The earliest committed erythroid progenitor cell is called the BFU-E (burst-forming
unit-erythroid) because of the large size of the colonies it forms in vitro. BFU-Es are
characterized by a high proliferative potential, a requirement for other hematopoietic
growth factors, such as IL-3 and stem cell factor, and a low number of erythropoietin
receptors. BFU-Es are largely dormant.12 Because BFU-Es have a low number of
erythropoietin receptors, they require a high concentration of erythropoietin to enter the
cell cycle (Table 1). For these cells, therefore, erythropoietin acts as a mitogen, and
its production must be inducible to reach a level sufficient to trigger these cells into
cycle (Table 2).
Table 1. -- The Biology of Erythropoiesis |
| Erythroid Progenitor Cell |
BFU-E |
CFU-E |
| Proliferative capacity |
High |
Low |
| Cell cycle status |
Dormant |
Active |
| Erythropoietin receptor expression |
Low |
High |
| Erythropoietin function |
Mitogen |
Viability factor |
| Erythropoietin requirement |
High |
Low |
Table 2. -- The Major Functions of Erythropoietin |
| |
Production |
Plasma Level |
| |
| Erythroid cell viability factor |
Constitutive |
Constant |
| Erythroid cell mitogen |
Inducible |
Variable |
As they mature, BFU-Es gradually lose their proliferative capacity
and acquire more erythropoietin receptors. At this stage, the cells are called CFU-E
(colony-forming unit-erythroid). CFU-Es have a reduced capacity for proliferation, form
small colonies in vitro, and are largely in cell cycle. Therefore, CFU-Es require
erythropoietin not to proliferate but rather to maintain their viability as they
differentiate (Table 1).8 It is for this reason that erythropoietin production
is constitutive.
Studies of erythroid progenitor cells overexpressing specific
survival genes indicate that these cells can differentiate in the absence of
erythropoietin. This suggests that the role of erythropoietin in maturing erythroid
progenitor cells with respect to differentiation is permissive (as a survival factor) and
not instructive.13 Thus, with respect to erythroid progenitor cells,
erythropoietin appears to function as a mitogen or a survival factor depending on the
maturation stage of the cell (Table 2). Erythropoietin receptors have been identified in a
variety of other cell types such as endothelial cells14 and neural cells,15
but the role of erythropoietin in the physiology of these cells is unknown.
Regulation of Erythropoietin Production
As mentioned, erythropoietin behaves like a hormone. Tissue hypoxia
stimulates its production, and an excess of oxygen suppresses its production but never
completely. Therefore, if a reliable assay for circulating erythropoietin were available,
it would be possible to assess tissue hypoxia. Fortunately, the immunoassay for serum
erythropoietin using recombinant erythropoietin-derived reagents has proved useful for
this purpose. This is due to several factors: (1) there is only one form of circulating
erythropoietin, (2) there are no preformed stores of erythropoietin,16 (3)
immunologically detectable erythropoietin is equivalent to biologically active
erythropoietin,17 (4) erythropoietin production is regulated at the level of
its gene, (5) hypoxia is the only physiologic stimulus for erythropoietin production, (6)
erythropoietin production is not influenced by its plasma level18 or marrow
cellularity,19 (7) erythropoiesis metabolism is not influenced by its plasma
level,20 (8) neither age nor gender influences the plasma erythropoietin level,21
and (9) plasma erythropoietin is constant in a given individual.22
Given the strong inverse correlation between erythropoietin
production and tissue hypoxia or anemia, there should be an inverse correlation between
plasma erythropoietin and the hemoglobin or hematocrit level. While this is true in
situations such as chronic iron deficiency anemia or chronic hemolytic anemia, it is not
the case in other situations due to a strong central tendency for down-regulation of
erythropoietin production under normal circumstances and with certain diseases (Table 3).
For example, normal men and women differ with respect to red cell mass (and hemoglobin or
hematocrit), but they do not differ with respect to their plasma erythropoietin level
(Table 4). Reduction in testosterone production, however, causes equalization of the
hemoglobin level between men and women.23 Thus, within the normal range of
hemoglobin or hematocrit, factors other than erythropoietin are involved in the regulation
of erythropoiesis. Furthermore, until the hemoglobin level falls below 10.5 g/dL, the
plasma erythropoietin level does not rise outside the normal range.22 This is
not to say that small reductions in hemoglobin will not cause an increase in
erythropoietin production. Rather, the increase is modest and, given the wide range of
normal for plasma erythropoietin (4 to 26 mU/mL), a more significant hemoglobin reduction
is required to raise erythropoietin production to the extent that plasma erythropoietin
increases beyond normal range. This has important clinical implications since it suggests
that a substantial degree of anemia or tissue hypoxia must occur before a significant
increase in erythropoietin production occurs. For this reason, the amount of blood that
can be donated at any one time or over a short duration is limited, since the increase in
erythropoietin production caused by phlebotomy is not marked.24
Table 3. -- Factors
Impairing Erythropoietin Production |
| Renal dysfunction |
| Hyperviscosity |
| Inflammation |
| Infection |
| Neoplasia |
| Cancer chemotherapy |
| Bone marrow transplantation |
| Surgery |
| Prematurity |
| Pregnancy |
Table 4. -- Serum Immunoreactive Erythropoietin in Normal and Castrate Individuals
|
| |
Normal Men |
Normal Women |
Castrate Men |
| Hemoglobin (g/dL) |
15.1 ± 0.1 |
13.4 ± 0.1 |
13.7 ± 0.2 |
| Erythropoietin (mU/mL) |
12.0 ± 0.8 |
11.0 ± 0.7 |
9.1 ± 2.1 |
| |
| *Mean ± SEM |
Impairment of Erythropoietin Production
In addition to the normal central tendency for down-regulation of
erythropoietin production, a number of disease states impact negatively on erythropoietin
production (Table 3). Because erythropoietin is produced primarily in the kidneys, its
production and therefore its plasma level are sensitive to impairment of renal function.
Thus, once the serum creatinine rises outside the normal range, the expected correlation
between plasma erythropoietin and the hemoglobin level is lost.22
Hyperviscosity also impairs erythropoietin production by an unknown mechanism.25,26
This probably serves as a protective mechanism against exacerbation of hyperviscosity by a
rising red cell mass in situations such as hypoxic erythrocytosis or a plasma protein
dyscrasia.
Most disease-related causes of impaired erythropoietin production
(Table 3) probably occur by a common mechanism involving the production of inflammatory
cytokines that not only suppress the production of erythropoietin but also interfere with
the proliferation of erythroid progenitor cells.27 Thus, patients with chronic
inflammatory or infectious disorders or neoplasia, while capable of increasing their
erythropoietin production, produce less erythropoietin for any degree of anemia than do
patients with uncomplicated iron deficiency anemia or chronic hemolytic anemia.5,28,29
Furthermore, the quantity of erythropoietin produced is also insufficient to overcome the
suppressive effects of inflammatory cytokines on the proliferative response of erythroid
progenitor cells to erythropoietin.
In addition to the disorders that impair erythropoietin production,
an inappropriate increase in erythropoietin production is caused by a number of
conditions, including liver disease or the use of zidovudine and chemotherapeutic agents.4-6
With respect to liver disease, the increase in erythropoietin production may represent
hepatocyte regeneration with a recapitulation of ontogeny when the liver is the primary
site of erythropoietin production. The mechanism by which certain drugs cause elevation of
plasma erythropoietin remains unknown.
Measurement of the plasma erythropoietin level can provide a useful
gauge of erythropoietin production if the guidelines listed in Table 5 are followed.
Generally, a low level of plasma erythropoietin in an anemic patient suggests a
hormone-deficient state that can be corrected with recombinant erythropoietin therapy if
the bone marrow is responsive. In an anemic patient, a high level of plasma erythropoietin
(particularly over 1000 mU/mL) in the absence of zidovudine, liver disease, or
chemotherapeutic agents suggests a bone marrow that is unable to respond to the hormone.
Before considering the use of recombinant erythropoietin, it is important to exclude
correctable causes of anemia such as nutritional deficiency states or lesions that cause
bleeding, as well as underlying conditions that predispose to anemia (ie, hypothyroidism).
Table 5. -- Caveats
Regarding the Clinical Use of the Immunoassay for Plasma Erythropoietin |
| The best indicator of erythropoietin deficiency in nonrenal anemias |
| Insensitive if the hemoglobin level is more than 10.5 g/dL |
| Not useful if the serum creatinine is more than 1.5 g/dL |
| Not useful if the serum bilirubin is greater than 2.0 g/dL |
| Plasma erythropoietin is not useful with recent chemotherapy |
| Plasma erythropoietin is not useful if correctable causes of anemia
have not been excluded |
| Plasma erythropoietin levels greater than 1000 mU/mL suggest that
erythropoietin may not work |
References
1. Koury ST, Bondurant MC, Koury MJ. Localization of erythropoietin synthesizing cells
in murine kidneys by in situ hybridization. Blood. 1988;71:524-527.
2. Koury ST, Bondurant MC, Koury MJ, et al. Localization of cells producing
erythropoietin in murine liver by in situ hybridization. Blood. 1991;77:2497-2503.
3. Tan CC, Eckardt KU, Firth JD, et al. Feedback modulation of renal and hepatic
erythropoietin mRNA in response to graded anemia and hypoxia. Am J Physiol.
1992;263:F474-F481.
4. Piroso E, Erslev AJ, Caro J. Inappropriate increase in erythropoietin titres during
chemotherapy. Am J Hematol. 1989;32:248- 254.
5. Spivak JL, Barnes DC, Fuchs E, et al. Serum immunoreactive erythropoietin in
HIV-infected patients. J Am Med Assoc. 1989;261: 3104-3107.
6. Klassen DK, Spivak JL. Hepatitis-related hepatic erythropoietin production. Am J
Med. 1990;89:684-686.
7. Thorling EB. Paraneoplastic erythrocytosis and inappropriate erythropoietin
production. Scand J Haemat. 1976;17(suppl):1-16.
8. Koury MJ, Bondurant MC. A survival model of erythropoietin action. Science.
1990;248:378-381.
9. Spivak JL, Pham TH, Isaacs MA, et al. Erythropoietin is both a mitogen and a
survival factor. Blood. 1991;77:1228-1233.
10. Koury ST, Koury MJ, Bondurant MC, et al. Quantitation of erythropoietin-producing
cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal
erythropoietin mRNA and serum erythropoietin concentration. Blood. 1989;74:645-651.
11. D’Andrea AD, Zon LI. Erythropoietin receptor. J Clin Invest.
1990;86:681-687.
12. Sawada K, Krantz SB, Dai CH, et al. Purification of human blood
burst-forming-units-erythroid and demonstration of the evolution of erythropoietin
receptors. J Cell Physiol. 1990;142:219-230.
13. Silva M, Grillot D, Benito A, et al. Erythropoietin can promote erythroid
progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2. Blood.
1996;88:1576-1582.
14. Anagnostou A, Lee ES, Kessimian N, et al. Erythropoietin has a mitogenic and
positive chemotactic effect on endothelial cells. Proc Natl Acad Sci U S A.
1990;87:5978-5982.
15. Digicaylioglu M, Bichet S, Marti HH, et al. Localization of specific erythropoietin
binding sites in defined areas of the mouse brain. Proc Natl Acad Sci U S A.
1995;92:3717-3720.
16. Schooley JC, Mahlmann LJ. Evidence for the de novo synthesis of erythropoietin in
hypoxic rats. Blood. 1972;40:662-670.
17. Egrie JC, Cotes PM, Lane J, et al. Development of radioimmunoassays for human
erythropoietin using recombinant erythropoietin as tracer and immunogen. J Immunol Meth.
1987;99:235- 241.
18. Fried W, Barone-Varelas J. Regulation of the plasma erythropoietin level in hypoxic
rats. Exp Hematol. 1984;12:706-711.
19. Piroso E, Erslev AJ, Flaharty K, et al. Erythropoietin life span in rats with
hypoplastic and hyperplastic bone marrows. Am J Hematol. 1991;36:105-110.
20. Spivak JL, Hogans BB. The in vivo metabolism of recombinant human erythropoietin in
the rat. Blood. 1989;73:90-99.
21. Powers JS, Krantz SB, Collins JC, et al. Erythropoietin response to anemia as a
function of age. J Am Geriatr Soc. 1991;39:30-32.
22. Spivak JL, Hogans BB. Clinical evaluation of a radioimmunoassay for serum
erythropoietin using reagents derived from recombinant erythropoietin. Blood.
1987;70:143a.
23. Weber JP, Walsh PC, Peters CA, et al. Effect of reversible androgen deprivation on
hemoglobin and serum immunoreactive erythropoietin in men. Am J Hematol.
1991;30:190-194.
24. Kicker TS, Spivak JL. Effect of repeated whole blood donations on serum
immunoreactive erythropoietin levels in autologous donors. J Am Med Assoc.
1988;260:65-67.
25. Kilbridge TM, Fried W, Heller P. The mechanism by which plethora suppresses
erythropoiesis. Blood. 1969;33:104-113.
26. Singh A, Eckardt KU, Zimmerman A, et al. Increased plasma viscosity as a reason for
inappropriate erythropoietin formation. J Clin Invest. 1993;91:251-256.
27. Means RT Jr, Krantz SB. Progress in understanding the pathogenesis of the anemia of
chronic disease. Blood. 1992;80:1639-1647.
28. Hochberg MC, Arnold CM, Hogans BB, et al. Serum immunoreactive erythropoietin in
rheumatoid arthritis: impaired response to anemia. Arthrit Rheum.
1988;31:1318-1321.
29. Miller CB, Jones RJ, Piantadosi S, et al. Decreased erythropoietin response in
patients with the anemia of cancer. N Engl J Med. 1990;322:1689-1692.
DR BALDUCCI
One of our fellows completed a cost-effectiveness study of
erythropoietin and blood transfusions in cancer patients and found that costs are
comparable. Regarding the prophylactic use of erythropoietin, I believe the study reported
by the group in Genoa shows that if erythropoietin is used before the patient becomes
anemic, anemia can be prevented in many patients. What endpoint should we be looking for
to consider erythropoietin to be cost effective?
DR SPIVAK
I think it is true that there may almost be a tie between the cost
of a transfusion and the cost of erythropoietin therapy, depending on how many units of
blood are given. But you would always rather give a pharmacologic agent that is close to
what the body produces. On that score, erythropoietin comes out ahead in every way.
DR BALDUCCI
Could continuous infusion of erythropoietin reduce substantially the
cost of the drugs, such as the case with insulin?
DR SPIVAK
I don’t know the answer to that, but when you
make an animal hypoxic, it gets a spike in erythropoietin production. If you go up to the
top of a mountain, you would get a spike in your erythropoietin production. And then, even
though you stayed hypoxic, it would down-regulate to within the normal range because other
compensatory mechanisms take over. So rather than think of a constant infusion of EPO, you
might think of administering one large dose at the beginning of the week and see what
happens. That’s never been tested. If I were going to use a continuous infusion, I
would give a bolus and then take the level to just above what it needed. Subcutaneous
administration does this. So maybe the way to use erythropoietin is to give a bolus dose
and then give a subcutaneous dose, and make sure that the level stays constant but higher
than normal.
From The Johns Hopkins University School of Medicine, Baltimore, Md.
Address reprint requests to Jerry L. Spivak, MD, at the Division of
Hematology, Department of Medicine, The Johns Hopkins University School of Medicine,
Baltimore, MD 21205.
Back to Cancer Control Journal Supplement Volume 5 Number 2