2011

Recombinant DNA Biotechnology

BIOT 503

Fahim Anwar
12-29012
M.Phil Biotechnology

Instructor: Dr Asma Maqbool

Forman Christian College

20-01-2011
 Fahim Anwar

Table of Contents

Introduction: ........................................................................................................................ 3

Recombinant Erythropoietin: .............................................................................................. 3

Novel erythropoiesis stimulating protein: ........................................................................... 4

Future Perspectives: ............................................................................................................ 4

Therapeutic Uses of Erythropoietin: ................................................................................... 4

Literature Review: .............................................................................................................. 5

Bibliography: ...................................................................................................................... 7

BIOT 503 12-29012

 Fahim Anwar

Erythropoietin:
Introduction:
Understanding the function of endogenous hormones and putting them to good use to
treat diseases has been one of the great accomplishments of modern medicine.
Erythropoietin (EPO) is one natural hormone that has turned out to be a blockbuster drug
but not without some controversy.

Erythropoietin is a glycoprotein hormone that controls erythropoiesis, or red blood cell

production. It is a cytokine for erythrocyte (red blood cell) precursors in the bone
marrow. EPO is a glycoprotein (protein-sugar conjugate) that serves as the primary
regulator of red blood cells (erythrocytes) in mammals. It stimulates bone marrow stem
cells to differentiate into red blood cells and controls hemoglobin synthesis and red blood
cell concentration. Human EPO is a 30,400-dalton molecule containing 165 amino acids
and four carbohydrate chains that incorporate sialic acid residues. There are several forms
of EPO, designated by Greek letters that differ only in the carbohydrate content.

In infants, EPO is produced mostly in the liver, but the kidneys become the primary site
of EPO synthesis shortly after birth. EPO production is stimulated by reduced oxygen
content in arterial blood in the kidneys. Circulating EPO binds to receptors on the surface
of erythroid progenitor cells that in turn mature into red blood cells.

Human EPO was first isolated and later purified from urine in the 1970s. Interest in
developing clinical uses for EPO led to the discovery of the gene encoding EPO, and
several groups devised recombinant DNA methods to produce EPO by the mid-1980s.

Recombinant Erythropoietin:
Recombinant EPO quickly made it to market
to treat anemia resulting from a host of
conditions, primarily kidney failure, HIV
infection in patients treated with AZT, and
cancer chemotherapy. Doses of EPO are given
by injection one or more times per week to
maintain a normal hematocrit level, the ratio
of red blood cell volume to total blood
volume. Generally, EPO might be prescribed
for any condition where blood oxygen levels
are depressed and to help eliminate the OXYGEN BOOSTER David S. Goodsell of Scripps
potential need for blood transfusions. Research Institute created this image of
erythropoietin (red) bound to its receptor (blue and
green) from coordinates taken from the Protein
Data Bank.

BIOT 503 12-29012

 Fahim Anwar

Researchers at Amgen and at the Genetics Institute, which became part of Wyeth, were
involved in separate efforts to develop recombinant EPO. Amgen received patent
protection and began selling the form of EPO in 1989. The Genetics Institute also tried to
commercially develop EPO- , and the result was one of the largest patent fights in U.S.
history, according to the Biotechnology Industry Organization. The case was finally
settled in favor of Amgen in 1996, giving the company exclusive U.S. rights.

Novel erythropoiesis stimulating protein:

More recently, a novel erythropoiesis-stimulating protein (NESP) has been
produced. This glycoprotein demonstrates anti-anemic capabilities and has a longer
terminal half-life than erythropoietin. NESP offers chronic renal failure patients a lower
dose of hormones to maintain normal hemoglobin levels. (IC, 2000)

Future Perspectives:
A revolution in the care of patients with acute MI evolving in the mid 1960s followed the
elucidation of the spatial and temporal dynamic evolution and nature of cardiomyocyte
necrosis and its determinants. Among the results were the evolution of pharmacologic
and interventional approaches to prompt and persistent restoration of myocardial
perfusion in patients with ACS that profoundly diminished morbidity and mortality. We
are perhaps on the threshold of another revolution predicated on the understanding of
other mechanisms besides classical necrosis that can result in cell death, including
programmed cell death (apoptosis) and autophagy. The extent to which these phenomena
contribute to overall cardiomyocyte cell death and the extent to which they can be
attenuated have not yet been elucidated. It appears highly likely, however, that
contributions will be substantial, and favorable modification will be possible with the
development of tissue-protective interventions. Although we have focused on Epo in this
manuscript, it appears highly probable that diverse tissue-protective interventions will be
explored and that some of them will indeed confer considerable benefit. Accordingly, it is
likely that tissue protection with pharmacologic agents and physiologic interventions will
constitute a part of the armamentarium in the treatment of patients with ACS 5-10 years
from now. (Ashraf Mikhail, 2008)

Therapeutic Uses of Erythropoietin:

Erythropoietin is available as a therapeutic agent produced by recombinant DNA
technology in mammalian cell culture. It is used in treating anemia resulting from chronic
kidney disease and myelodysplasia, from the treatment of cancer (chemotherapy and
radiation), and from other critical illnesses (heart failure).

BIOT 503 12-29012

 Fahim Anwar

Literature Review:
Engert stated that anaemia effects up to 90% of cancer patients, with more than 60%
requiring blood transfusion during or after treatment. With the advent of recombinant
human erythropoietins (rHuEPO), an alternative to red blood cell transfusion has become
available. So far, three drugs have been approved for the treatment of anaemia in patients
with malignancies (epoetin alfa, epoetin beta and darbepoetin alfa). New concepts for the
use of erythropoietin in cancer patients include 3- and 4-weekly dosing, as well as
loading-dose concepts. Important factors helping to judge the impact of erythropoietin in
cancer treatment include pharmacoeconomics and better predictive factors. Lately, the
influence of erythropoietin therapy on survival in cancer patients has been discussed very
intensively, because conflicting data have emerged. Studies aimed at correcting anaemia
in cancer patients had indicated a possible survival advantage of those patients receiving
erythropoietin. In contrast, two recent trials aimed at correction of haemoglobin levels
beyond anaemia reported a poorer survival of patients receiving erythropoietin. This
might grossly be attributed to a higher risk of thrombosis in these patients. The largest
systematic review on the use of erythropoietin in cancer patients undergoing treatment
indicates a suggestive but not significant survival advantage of erythropoietin-treated
patients. In addition, very recent results of a Food and Drug Administration meeting on
safety and survival of patients treated with erythropoietin are presented. (Engert, 2005)

Howard, L. Corwin observed that critically ill patients receive an extraordinarily large
number of blood transfusions. Between 40% and 50% of all patients admitted to intensive
care units receive at least 1 red blood cell (RBC) unit during their stay, and the average is
close to 5 RBC units. RBC transfusion is not risk free. There is little evidence that
‘routine’ transfusion of stored allogeneic RBCs is beneficial to critically ill patients. The
efficacy of perioperative recombinant human erythropoietin (rHuEPO) has been
demonstrated in a variety of elective surgical settings. Similarly, in critically ill patients
with multiple organ failure, rHuEPO therapy will also stimulate erythropoiesis. In a
randomized, placebo-controlled trial, therapy with rHuEPO resulted in a significant
reduction in RBC transfusions. Despite receiving fewer RBC transfusions, patients in the
rHuEPO group had a significantly greater increase in hematocrit. Strategies to increase
the production of RBCs are complementary to other approaches to reduce blood loss in
the intensive care unit, and they decrease the transfusion threshold in the management of
all critically ill patients. (Howard, 2004)

Dimitris Georgopoulos et-al stated that the aim of this study was to assess the efficacy of
two dosing schedules of recombinant human erythropoietin (rHuEPO) in increasing
haematocrit (Hct) and haemoglobin (Hb) and reducing exposure to allogeneic red blood
cell (RBC) transfusion in critically ill patients. (Georgopoulos, 2005)

Matthew et-al stated that erythropoietin (Epo) has long been known to be the principal
hematopoietic growth factor that regulates cellular proliferation and differentiation along
the erythroid lineage. Recent studies have shown that Epo is a pleiotropic cytokine that is
proangiogenic and exerts broad tissue-protective effects in diverse non hematopoietic
organs. Recombinant Epo (rEpo) has been widely used in the clinic to prevent or treat

BIOT 503 12-29012

 Fahim Anwar

malignancy-associated anemia. A series of clinical trials have documented the efficacy of

rEpo in reducing RBC transfusion requirements and improving quality of life in cancer
patients, and a recent meta-analysis suggested a positive effect on survival. However, two
randomized trials reported negative outcomes with rEpo, as patients in the rEpo arm fared
worse than their placebo-treated counterparts with respect to progression-free survival.
The expression of Epo receptor (EpoR) in cancer cells has raised the possibility that
exogenous rEpo may exert direct effects on tumor cells associated with the potential for
stimulation of proliferation, inhibition of apoptosis, or modulation of sensitivity to chemo
radiation therapy. The presence of an autocrine-paracrine Epo-EpoR system in tumors
and potential effects of Epo on tumor microenvironment and angiogenesis are consistent
with a complex biology for Epo-EpoR signaling in cancer that requires further research.
This review describes Epo and EpoR biology, focusing on the pleiotropic effects of Epo
on non hematopoietic tissues as well as the expression and function of EpoR in cancer
cells. (Hardee, 2006)

B. Sturm observed that Friedreich’s ataxia (FRDA) is a neurodegenerative disorder

caused by decreased expression of the protein frataxin, recently described to be an iron
chaperone for the assembly of iron-sulphur clusters in the mitochondria, causing iron
accumulation in mitochondria, oxidative stress and cell damage. Searching for
compounds that could possibly influence frataxin expression, we found that the cytokine
recombinant human erythropoietin (rhuEPO) significantly increases frataxin expression
by a still unknown mechanism. (Sturm, 2005)

BIOT 503 12-29012

 Fahim Anwar

Bibliography:
 Ashraf Mikhail, A. C. (2008). Stimulating Erythropoiesis: Future Perspectives.
Kidney Blood Press Res , 234-246.
 Engert, A. (2005). Recombinant human erythropoietin in oncology: current status.
Annals of Oncology , 1584–1595.
 Georgopoulos, D. (2005). Recombinant human erythropoietin therapy in critically
ill. Critical Care , 508-515.
 Hardee, M. E. (2006). Erythropoietin Biology in Cancer. Clin Cancer Res , 332-
339.
 Howard, L. C. (2004). Anemia and blood transfusion in the critically ill patient:
role of. Critical Care , 42-44.
 IC, M. (2000). Novel erythropoiesis stimulating protein. Semin. Nephrol. , 375-
81.
 Sturm, B. (2005). Blackwell Publishing, Ltd. European Journal of Clinical
Investigation , 711-717.

BIOT 503 12-29012