Neuroprotective activity of a new erythropoietin formulation with increased penetration in the central nervous system.

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MEETING ABSTRACTOpen Access
Neuroprotective activity of a new erythropoietin
formulation with increased penetration in the
central nervous system
Marina Etcheverrigaray1*, Natalia Ceaglio1, Mónica Mattio1, Marcos Oggero1, Ignacio Amadeo1,2,
Guillermina Forno1,2, Norma Perotti1, Ricardo Kratje1
From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies
Vienna, Austria. 15-18 May 2011
Background
Apart from its hematopoietic effect, erythropoietin
(EPO) is a molecule with high neuroprotective potential.
However, its prolonged application may cause serious
adverse effects due to the erythropoiesis stimulation.
Therefore, an EPO derivative with neuroprotective prop-
erties but low hematopoietic activity, designated as neu-
ropoietin (rhNEPO), was developed in our lab using an
alternative purification process of the recombinant
human erythropoietic counterpart (rhEPO) produced in
CHO cells [1]. The in vitro cytoprotective activity of
rhNEPO on neural phenotype cells and its brain uptake
from blood are herein analyzed.
Results
In vitro citoprotective activity of rhNEPO was analyzed
on rat pheochromocytoma cells (PC-12) differentiated to
neural phenotype with neural growth factor (NGF).
Apoptosis was triggered by NGF and serum withdrawal
from cell cultures. Thus, nuclear DNA fragmentation
was analyzed by colorimetric TUNEL detection. One-
way analysis of variance was carried out followed by
Dunnett´s multiple comparison test. Probabilities lower
than 0.05 were considered significant (p<0.05).
As shown in Figure 1, serum and NGF withdrawal sig-
nificantly increased the number of apoptotic cells to
17.4 ± 1.5% (apoptosis control) compared to 3.7± 4.3%
from growth control (p<0.05). rhEPO slightly protected
differentiated PC-12 cells from death since apoptotic
cells decreased to 12.1 ± 2.8% compared to the apopto-
sis control. Interestingly, rhNEPO and desialylated
rhEPO (an EPO derivative where sialic acid were enzi-
matically removed) completely protected cells from
apoptosis as both analogues showed 7.4 ± 1.3% and 7.1
± 1.0% of apoptotic cells respectively (p<0.05). Besides,
none of the derivatives exhibited significant differences
with the growth control (p>0.05), confirming their prop-
erties to protect PC-12 cells from apoptosis. Therefore,
this novel combination of erythropoietin glycoforms
(rhNEPO) with lower sialic acid content and antennarity
than rhEPO [1] preserved its binding receptor capacity
exerting an in vitro neuroprotective activity even better
than the mentioned counterpart. Also, rhAEPO showed
an in vitro activity that is similar to that of rhNEPO,
having both derivatives the lowest content of carbohy-
drates. It is well known that the affinity of EPO analo-
gues for EPO receptor is inversely related to the
sialylation of their attached carbohydrate [2] and that
removal of sialic acid turns it into a molecule with a
very short half-life with almost no erythropoietic activ-
ity. This is the case of rhAEPO that explains its rapid
hepatic clearance from blood [3]. For that reason,
rhNEPO emerges as a neuroprotective candidate dis-
playing higher in vitro activity than rhEPO.
Taking into account the cytoprotective activity of
rhNEPO on neural phenotype cells, cerebrospinal fluid
(CSF) and blood pharmacokinetics of rhEPO and
rhNEPO were evaluated in rats following intravenous
administration of a single dose of each protein, aiming
to evaluate their CSF uptake from plasma.
The distribution and the elimination half-lives of
rhNEPO in blood were significantly shorter than the
corresponding ones for rhEPO. Differences in the sialic
* Correspondence: marina@fbcb.unl.edu.ar
1Cell Culture Laboratory, School of Biochemistry and Biological Sciences,
Universidad Nacional del Litoral. Ciudad Universitaria – C.C.242 – (S3000ZAA)
Santa Fe, Provincia de Santa Fe, Argentina
Full list of author information is available at the end of the article
Etcheverrigaray et al. BMC Proceedings 2011, 5(Suppl 8):P3
http://www.biomedcentral.com/1753-6561/5/S8/P3
© 2011 Etcheverrigaray et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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acid content and oligosaccharide anntenarity status [1]
may describe the faster elimination rate of rhNEPO.
Thus, this derivative, which is a less sialylated and less
branched molecule, was rapidly cleared from blood but
reached the CSF in a shorter time (5 min vs 30 min of
rhEPO) at concentrations high enough to bind to the
EPO receptors. Consequently, the faster transport
through the blood-brain-barrier (BBB) might accelerate
rhNEPO distribution into the nervous system, causing a
faster appearance into the action site. Moreover, the
rapid clearance of rhNEPO from plasma represents an
advantage in the treatment of neurological diseases,
because the continuous presence of EPO in blood is the
stimulus that triggers the production of sanguineous
cells with the resulting appearance of adverse side-
effects.
Conclusions
The in vitro anti-apoptotic effect of rhNEPO becomes
a remarkable fact to predict its neuroprotective
action in the nervous system. Furthermore, despite
its short plasma half-life, rhNEPO appears promptly
within the CSF, being also a further and a significant
fact that encourage the study of neuroepoetin as a
potential drug for the treatment of neurological dis-
eases, in which, a highly neuroprotective activity with
low side effects and a fast blood-to-brain influx are
desirables.
Figure 1 Protective effect of rhEPO derivatives on differentiated PC-12 cells upon induction of apoptosis by NGF and serum withdrawal. rhEPO,
rhNEPO and desialylated rhEPO (rhAEPO) were added to the culture 24 h prior to induction of cell death. Apoptosis was measured by TUNEL
technique.
Table 1 Pharmacokinetics of rhEPO and rhNEPO in plasma and CSF after intravenous administration of a single dose
of 500 µg of each protein in rats. The quantification of EPO derivatives were carried out by sandwich ELISA [1].
PharmacokineticsrhEPO
Plasma1
t1/2a(h)a
1.7±
t1/2b(h)b
12.3±
CSF2
tCSF(min)c
30
rhNEPO
0.1
1.0
0.4
8.9
±
±
5
0.1
0.4
at1/2a, distribution half-life;bt1/2b, elimination half-life;ctime to reach CSF after administration of EPO derivatives.1Distribution and elimination half-lives and
their corresponding standard deviations were obtained using a bi-compartmental pharmacokinetic model.2The tCSFrefers to the first time point in which EPO
derivatives were detected in CSF, and for this reason standard deviations were not considered.
Etcheverrigaray et al. BMC Proceedings 2011, 5(Suppl 8):P3
http://www.biomedcentral.com/1753-6561/5/S8/P3
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Author details
1Cell Culture Laboratory, School of Biochemistry and Biological Sciences,
Universidad Nacional del Litoral. Ciudad Universitaria – C.C.242 – (S3000ZAA)
Santa Fe, Provincia de Santa Fe, Argentina.2Zelltek S.A. Ruta Nacional 168 –
PTLC – (3000) Santa Fe, Provincia de Santa Fe, Argentina.
Published: 22 November 2011
References
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16(Suppl 3):3-13.
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doi:10.1186/1753-6561-5-S8-P3
Cite this article as: Etcheverrigaray et al.: Neuroprotective activity of a
new erythropoietin formulation with increased penetration in the
central nervous system. BMC Proceedings 2011 5(Suppl 8):P3.
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