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Erythropoietic Agents as Neurotherapeutic Agents: What Barriers Exist?

Erythropoietic Agents as Neurotherapeutic Agents: What Barriers Exist?

ABSTRACT: Erythropoietin is the primary physiological regulator of erythropoiesis, and it exerts its effect by binding to cell surface receptors. It has recently been shown that both erythropoietin and its receptor are found in the human cerebral cortex, and that, in vitro, the cytokine is synthesized by astrocytes and neurons, has neuroprotective activity, and is up-regulated following hypoxic stimuli. In animal models, exogenous recombinant human erythropoietin has been reported to be beneficial in treating experimental global and focal cerebral ischemia and reducing nervous system inflammation. These findings suggest that exogenous administration of erythropoietic agents (darbepoetin alfa [Aranesp], epoetin alfa [Epogen, Procrit], and epoetin beta [NeoRecormon]) may be a potential therapeutic tool for central nervous system injury. However, transport of protein therapeutics to the brain’s extracellular environment via systemic blood supply generally does not occur due to the negligible permeability of the brain capillary endothelial wall. Therefore, in order to pharmacologically exploit and fully realize the therapeutic benefits of exogenous erythropoietic agents in CNS dysfunction, mechanisms of action and the potential impact of biodistribution barriers need to be elucidated. [ONCOLOGY 16(Suppl 10):91-107, 2002]

This report is structured
to serve
two purposes. The first is to
summarize current understanding of the biology of erythropoietin in the central
nervous system (CNS) and to highlight missing pieces of the puzzle. Some of the
basic questions to be addressed include: What is the role of erythropoietin in
the CNS? Does it differ from its function in systemic erythropoiesis? Is
erythropoietin an instructive or survival/trophic factor during CNS development
and in the adult brain? Is there a link between CNS and peripheral
erythropoietin regulation? Do exogenously administered erythropoietic agents
cross the blood-brain barrier/blood-cerebrospinal fluid barrier? Do
erythropoietic agents need to cross the blood-brain barrier in order to elicit
central therapeutic effects?

The second purpose of this report is to discuss ongoing
research addressing these questions. Suggestions of how this information may be
integrated to fully realize the potential benefits of intervention with
erythropoietic agents to prevent or reduce pathological neuronal loss in CNS
disease or injury are proposed where appropriate.

As it is beyond the scope of this report to provide a
comprehensive review of erythropoietin literature, the reader is referred to
available reviews.[1-7]

Erythropoietin: Gene Structure and Activity

The human endogenous erythropoietin (EPO) gene, a single-copy
gene located on chromosome 7, consists of 5 exons and 4 introns. Erythropoietin
is initially formed as a 197-amino-acid glycoprotein. The leader sequence and
terminal amino acid are cleaved prior to secretion of the mature 165-amino-acid
peptide with a molecular weight of 34,000 Da.[8] Endogenous erythropoietin has
three N-linked glycosylation sites and one O-linked glycosylation site.
Glycosylation of these sites results in four complex carbohydrate chains
containing sialic acid,[9-11] and the duration of erythropoietin circulation in
blood appears to be governed by these moieties.

For biological activity, erythropoietin amino acids 150-160
interact with the erythropoietin receptor.[12] Studies have shown that there is
a direct relationship between sialic acid-containing carbohydrate content,
serum half-life, and in vivo biological activity, and an inverse relationship
with its receptor binding affinity.[13]

Erythropoietic stimulation was believed to be the sole
physiological function of erythropoietin. However, evidence now exists that
suggests that erythropoietin also acts as an instructive signal during fetal
development and an antiapoptotic agent promoting survival and differentiation in
the brain, heart, and uterus. The main erythropoietin production site is the
kidney in the adult and the liver in the fetus.

Erythropoietin Receptors

The action of erythropoietin is mediated by binding to the
specific erythropoietin receptor, which belongs to the family of cytokine
receptors that do not have a tyrosine kinase domain. This family includes
receptors for growth hormone, granulocyte-colony stimulating factor (G-CSF [Neupogen]), and thrombopoietin, among others.[14] The erythropoietin
receptor is a 507-amino-acid (55,000-68,000 Da) polypeptide with a single
domain,[15] which has an unusual tryptophan-serine-X (any amino acid)-tryptophan-serine
extracellular motif.[16] The extracellular N-terminal region contains the
erythropoietin-binding domain and the C-terminal intracellular region
participates in signal transduction.[17]

Once erythropoietin binds to the erythropoietin receptor (EPO-R),
rapid tyrosine phosphorylation is induced by receptor dimerization. The
phosphorylated tyrosines act as docking sites for intracellular proteins, which
are subsequently phosphorylated, leading to activation and downstream signal
transduction. Signal termination occurs via dephosphorylation and inactivation
of the kinase, which down-regulates the signaling cascade.[18]

In mammalian embryos, EPO-Rs are initially found in the yolk
sac blood islands, where ‘primitive’ erythropoiesis occurs and then later
shifts to the fetal liver. At term, liver erythropoiesis is largely attenuated,
supposedly by the increase of glucocorticoid levels.[19] The EPO-Rs are
initially manifested in burst-forming unit-erythroid (BFU-e) cells and reach
maximum expression in the colony-forming unit-erythroid (CFU-e) cells, following
which receptor numbers decline sharply (see Figure
). In small animals such as
rats and mice, "definitive" erythropoiesis occurs in the bone marrow
and spleen, where erythropoietin receptors are localized. In adult humans,
erythropoiesis largely occurs in the bone marrow. The erythropoietin requirement
is absolute for "definitive" erythropoiesis in the developing fetus
and human adult erythropoiesis.[20-22]

Erythropoietic Agents

The erythropoietic agents (darbepoetin alfa [Aranesp],
epoetin alfa [Epogen, Procrit], and epoetin beta [NeoRecormon]) have the
biologic activity of endogenous erythropoietin, which is mediated via
erythropoietin receptor. Recombinant human erythropoietin (rHuEPO) has an amino
acid sequence identical to that of human erythropoietin. Endogenous
erythropoietin and rHuEPO have microheterogeneity in their structures and are
comprised of several isoforms, including some with charge differences.[23]
Charge differentiation by isoelectric focusing shows that rHuEPO-alpha and
rHuEPO-beta patterns are similar (isoelectric point [pI] 4.4-5.1), but
distinguishable from purified urinary erythropoietin, which is more acidic (pI
3.92-4.42). Such differences permit the differentiation of exogenously
administered erythropoietin from endogenous protein.[23-25]

Darbepoetin alfa is a new erythropoietic agent generated by
site-directed mutagenesis of the erythropoietin gene, resulting in an increased
number of glycosylation sites and greater carbohydrate content.[13]
Consequently, darbepoetin alfa has an increased serum residence time and greater
potency in vivo.[26] In clinical trials of patients with renal failure,
darbepoetin alfa was shown to have a threefold longer terminal half-life than
epoetin alfa (25.3 h vs 8.5 h) and to be as efficacious as epoetin alfa despite
reductions in dosing frequency to weekly and once-every-2-week regimens.[26,27]
In cancer patients receiving chemotherapy, previous studies have shown that
darbepoetin alfa effectively increases hemoglobin concentrations when
administered once weekly, once every 2 weeks, or once every 3 weeks. [28-30]

The quest to identify small-molecule agonists of the
erythropoietin receptor led to the discovery of both peptide[31-33] and
nonpeptide[34] erythropoietin mimetics. These molecules mimic EPO-R binding and
activate the signal transduction pathways used by erythropoietin. While these
molecules have yet to be exploited clinically, they have proven to be valuable
tools for investigating the biological and functional structure of EPO-R, which
will be useful for continued exploration of erythropoietin mimetic agents.


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