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Erythropoietin and Erythropoietin-Like Agents As Pharmacological Countermeasures During Space Exploration Arthur J. Sytkowski, MD Laboratory for Cell and Molecular Biology Department of Medicine Beth Israel Deaconess Medical Center Harvard Medical School Center for Advanced Space Studies, Houston , 28 June 2005
- Page 2 and 3: Outline of the Presentation • Phy
- Page 4 and 5: Peritubular Interstitial Cells Prod
- Page 6 and 7: Cytokine Receptor Superfamily Type
- Page 8 and 9: Structure of the Epo Receptor Phosp
- Page 10 and 11: Principal EpoR Signaling Pathways
- Page 12 and 13: Epo Stimulates Endothelial Cell Gro
- Page 14 and 15: Epo Promotes Wound Healing • Incr
- Page 16 and 17: Affinity Cross-Linking of 125 I-Epo
- Page 18 and 19: EpoR Is Found Within And Around Hum
- Page 20 and 21: Epo Attenuates Brain Injury After B
- Page 22 and 23: Epo Protects Retinal Neurons From A
- Page 24 and 25: Epo and the Gut • Epo found in hu
- Page 26 and 27: So What Does All Of This Have To Do
- Page 28 and 29: We hypothesize that long-acting and
- Page 30 and 31: In addition to these risks for whic
- Page 32 and 33: Hypothetical Action of Epo Dimer Mo
- Page 34 and 35: Chemical Crosslinking of Epo • Pu
- Page 36 and 37: SDS-PAGE of Epo-SH, M-Epo M and Cro
- Page 38 and 39: Pharmacokinetics of Epo Monomer and
- Page 40 and 41: A Second Approach: Epo-Epo Fusion P
- Page 42 and 43: Pharmacokinetics of Epo and Epo-Epo
- Page 44 and 45: New Epo Fusion Proteins Under Study
- Page 46 and 47: For more information about Erythrop
- Page 48 and 49: Greetings from the Laboratory for C
Erythropoiet<strong>in</strong> and Erythropoiet<strong>in</strong>-Like<br />
Agents As Pharmacological<br />
Countermeasures Dur<strong>in</strong>g Space<br />
Exploration<br />
Arthur J. Sytkowski, MD<br />
Laboratory for Cell and Molecular Biology<br />
Department of Medic<strong>in</strong>e<br />
Beth Israel Deaconess Medical Center<br />
Harvard Medical School<br />
Center for Advanced Space Studies, Houston , 28 June 2005
Outl<strong>in</strong>e of the Presentation<br />
• Physiology and cell biology of erythropoiet<strong>in</strong>,<br />
a.k.a. “Epo”.<br />
• Hematopoietic and non-hematopoietic anti-<br />
apoptotic actions of Epo.<br />
• Epo as a pharmacologic countermeasure <strong>in</strong><br />
spaceflight.<br />
• Long-act<strong>in</strong>g Epo-like agents.
Epo Physiology <strong>in</strong> the Adult Human<br />
Ebert & Bunn, 1999<br />
Importantly, Epo also acts on other organs and tissues and is produced<br />
and acts locally (autocr<strong>in</strong>e/paracr<strong>in</strong>e action).
Peritubular Interstitial Cells Produce Epo: mRNA<br />
Detected By In Situ Hybridization and<br />
Autoradiography<br />
Koury et al., 1988
Primary Structure of Human Epo<br />
Oligosaccharides are critical for <strong>in</strong> vivo activity.
Cytok<strong>in</strong>e Receptor Superfamily<br />
Type I,<br />
Homodimer<br />
Watowich, , 1996
EpoR Dimerization and Con<strong>format</strong>ional<br />
Change Upon Epo B<strong>in</strong>d<strong>in</strong>g Initiates Signal<br />
Transduction<br />
There is also evidence of pre-formed EpoR dimers.
Structure of the Epo Receptor<br />
Phosphorylated<br />
tyros<strong>in</strong>es and<br />
other special<br />
doma<strong>in</strong>s are<br />
dock<strong>in</strong>g sites for<br />
signal<br />
transduction<br />
molecules.
Signal<strong>in</strong>g Prote<strong>in</strong>s That<br />
Associate With the EpoR<br />
• Jak2, STAT5, , Cis1.<br />
• PTKs: : Lyn, Syk, , Tec<br />
• PTPs: : SHP1 & SHP2<br />
• Phospholipid modify<strong>in</strong>g<br />
enzymes: : PI3-K, PLCγ<br />
& SHIP<br />
• Adaptor prote<strong>in</strong>s:<br />
GRB2, Shc, Cbl,<br />
CrkL, APS, IRS-2,<br />
Gab1 & Gab2.<br />
• Nucleotide exchange<br />
factors: : Vav, C3G &<br />
mSOS
Pr<strong>in</strong>cipal EpoR Signal<strong>in</strong>g Pathways<br />
• JAK2/STAT5…Phospho<br />
…Phospho-STAT 5b is a hallmark<br />
of Epo’s anti-apoptotic apoptotic signal.<br />
• PI3-K<strong>in</strong>ase<br />
K<strong>in</strong>ase…regulates c-mycc<br />
transcription and<br />
phosphorylates Akt, another anti-apoptotic apoptotic signal.<br />
• Raf/MEK/MAP k<strong>in</strong>ase …also regulates c-mycc<br />
transcription by a different mechanism.<br />
• PKC…the<br />
epsilon isoform is especially important.
EpoR Is Found on Other Tissues: Epo<br />
Also Has Non-Hematopoietic Actions<br />
• Endothelium: : angiogenesis and wound heal<strong>in</strong>g.<br />
• Central nervous system: : neuroprotection.<br />
• Heart: : tissue protection.<br />
• Gastro<strong>in</strong>test<strong>in</strong>al tract: : tissue protection.<br />
• Other cell types: : actions unknown.
Epo Stimulates Endothelial Cell Growth<br />
and B<strong>in</strong>ds to Cell Surface Receptors<br />
Anagnostou et al. 1990
Epo and Endothelial Cells<br />
• Increases angiogenesis <strong>in</strong> rat aortic r<strong>in</strong>g. Carl<strong>in</strong>i et al.<br />
1995.<br />
• Up-regulates immediate/early genes (growth).<br />
Fod<strong>in</strong>ger et al. 2000.<br />
• Induces phosphorylation of JAK2 and STAT5 anti-<br />
apoptosis. Fuste et al. 2002.<br />
• Protects aga<strong>in</strong>st hypoxia-<strong>in</strong>duced <strong>in</strong>duced apoptosis by<br />
activation of AKT-1. Chong et al. 2002.
Epo Promotes Wound Heal<strong>in</strong>g<br />
• Increases granulation tissue an <strong>in</strong> vivo wound-<br />
heal<strong>in</strong>g assay. Haroon 2003.<br />
• Stimulates angiogenesis and heal<strong>in</strong>g of<br />
experimental ischemic sk<strong>in</strong> wounds. Buemi 2004.<br />
• Stimulates angiogenesis and wound heal<strong>in</strong>g <strong>in</strong> the<br />
genetically diabetic mouse. Galeano 2004.
Epo and EpoR <strong>in</strong> the CNS<br />
• Hypoxia <strong>in</strong>duces appearance of endogenous Epo mRNA <strong>in</strong> rat<br />
bra<strong>in</strong>. Tan et al. 1992.<br />
• 125 I-Epo b<strong>in</strong>ds to mouse bra<strong>in</strong> slices. Endogenous Epo and<br />
EpoR mRNA detected by RT-PCR.<br />
Digicaylioglu et al. 1995.<br />
• Epo and EpoR detected <strong>in</strong> monkey and human bra<strong>in</strong> by RT-<br />
PCR. Marti et al. . 1996.<br />
• Epo and EpoR detected <strong>in</strong> develop<strong>in</strong>g human bra<strong>in</strong> by<br />
immunohistochemistry. Juul et al. 1999.
Aff<strong>in</strong>ity Cross-L<strong>in</strong>k<strong>in</strong>g of 125 I-Epo to<br />
EpoR of Erythroid and Neuronal Cells<br />
Difference <strong>in</strong> size of cross-l<strong>in</strong>ked complexes implies<br />
fundamental difference between EpoRs of the two cell types.
Some Effects of Epo on Neuronal Cells<br />
• Increases <strong>in</strong>tracellular free calcium. Assandri<br />
1999.<br />
• Increases membrane polarization, dopam<strong>in</strong>e<br />
release and tyros<strong>in</strong>e hydroxylase activity.<br />
Koshimura 1999.<br />
• Promotes differentiation of oligodendrocytes<br />
and growth of astrocytes. Sugawa 2002.
EpoR Is Found With<strong>in</strong> And Around<br />
Human Bra<strong>in</strong> Capillaries.<br />
Br<strong>in</strong>es 2000<br />
(A)) EpoR <strong>in</strong> capillaries (arrow). (B)(<br />
) High-power view (C)(<br />
) EpoR at capillary (c)<br />
identified as an astrocytic process (a). Astrocytes conta<strong>in</strong> EpoR. . (D)(<br />
) EM shows<br />
EpoR with<strong>in</strong> astrocytic foot processes (*),(<br />
and ECs (arrows).
Epo Crosses the Blood-Bra<strong>in</strong> Bra<strong>in</strong> Barrier<br />
(A)) Biot<strong>in</strong>ylated Epo (bEPO) seen around capillaries 5 h after i.p. <strong>in</strong>jection.<br />
(B) bEPO surrounds the lumen of capillaries (arrow). (C)(<br />
bEpo not observed if<br />
<strong>in</strong>jected along with 100 times excess of unlabeled Epo (bEPO(<br />
+ EPO).
Epo Attenuates Bra<strong>in</strong> Injury<br />
After Blunt Trauma<br />
Injury without<br />
Epo pretreatment<br />
Injury with<br />
Epo pretreatment<br />
Br<strong>in</strong>es et al. PNAS 2000
Anti-Inflammatory Effect on the CNS:<br />
Exptl Autoimmune Encephalomyelitis<br />
H & E sta<strong>in</strong><br />
Anti-glial<br />
fibrillary acidic<br />
prote<strong>in</strong><br />
(GFAP) sta<strong>in</strong><br />
Anti-CD11b sta<strong>in</strong><br />
Rat lumbar sp<strong>in</strong>al cord sections from unimmunized<br />
(control), EAE rat (immunized with myel<strong>in</strong> basic<br />
prote<strong>in</strong>), and EAE/Epo rat.<br />
Agnello et al., , 2002.
Epo Protects Ret<strong>in</strong>al Neurons From<br />
Acute Ischemia-Reperfusion Injury<br />
Non-ischemic<br />
Ischemic Ischemic +<br />
Epo Pre-Rx<br />
Ischemic +<br />
Epo Post-Rx<br />
Histological appearances of the non-ischemic (control) and<br />
ischemic (vehicle and Epo-treated) ret<strong>in</strong>as at 7 days of<br />
reperfusion after ischemia. A, , control; B, , ischemic (45-m<strong>in</strong>)<br />
vehicle-treated;<br />
C, , ischemic (45-m<strong>in</strong>) and Epo-pretreated;<br />
pretreated;<br />
D, , ischemic (45-m<strong>in</strong>), Epo-posttreated<br />
posttreated. . Epo-treated animals have<br />
significantly less ret<strong>in</strong>al th<strong>in</strong>n<strong>in</strong>g compared with vehicle-treated<br />
controls.<br />
Junk et al, , 2002
Epo and the Heart<br />
• Epo and ret<strong>in</strong>oic acid are secreted from<br />
epicardium dur<strong>in</strong>g embryogenesis.<br />
• Block<strong>in</strong>g of endogenous Epo <strong>in</strong>hibits<br />
proliferation and survival of cardiomyocytes.<br />
• Epo (both endogenous and adm<strong>in</strong>istered)<br />
protects aga<strong>in</strong>st ischemia/reperfusion <strong>in</strong>jury.
Epo and the Gut<br />
• Epo found <strong>in</strong> human milk.<br />
• EpoR detected <strong>in</strong> small bowel of human fetuses.<br />
• Enterocytes of post-natal rats migrate faster if exposed<br />
to Epo.<br />
• Reduced <strong>in</strong>cidence of necrotiz<strong>in</strong>g enterocolitis <strong>in</strong> low<br />
birth weight <strong>in</strong>fants receiv<strong>in</strong>g rhEpo.
Epo and Other Cell Types<br />
• EpoR <strong>in</strong> kidneys.<br />
• Epo <strong>in</strong> myoblasts.<br />
• EpoR on pancreatic islets.
So What Does All Of This Have To<br />
Do With Spaceflight?
• Long-term human spaceflight presents unique<br />
problems <strong>in</strong> autonomous medical care as well as<br />
identified risks to health for which countermeasures,<br />
<strong>in</strong>clud<strong>in</strong>g novel pharmaceuticals, , especially long-<br />
act<strong>in</strong>g pharmaceuticals, will be needed.<br />
• Among the risks identified <strong>in</strong> the Bioastronautics<br />
Roadmap of special relevance to Epo are<br />
• hemorrhage/anemia /blood replacement,<br />
• major trauma and<br />
• pharmacology of space medic<strong>in</strong>e delivery.
We hypothesize that long-act<strong>in</strong>g and ultra-long<br />
long-<br />
act<strong>in</strong>g forms of Epo will prove to be valuable<br />
therapeutic agents and pharmacological<br />
countermeasures <strong>in</strong> support of the<br />
treatment/prevention of trauma and of<br />
hemorrhage/anemia/blood replacement.
Importantly, Epo has several crucial non-hematopoietic<br />
actions <strong>in</strong>clud<strong>in</strong>g<br />
• <strong>in</strong>creased wound heal<strong>in</strong>g<br />
• neuroprotection and<br />
• cardioprotection.<br />
Thus, Epo can serve as a new countermeasure for these<br />
and, potentially, other diverse risks of human<br />
spaceflight.<br />
Long-act<strong>in</strong>g forms of Epo, , such as those that we are<br />
develop<strong>in</strong>g, should have even greater impact.
In addition to these risks for which Epo and<br />
derivatives may serve as valuable<br />
countermeasures/treatments, additional<br />
health risks may be counteracted by other<br />
long-act<strong>in</strong>g recomb<strong>in</strong>ant prote<strong>in</strong> therapeutic<br />
agents, , <strong>in</strong>clud<strong>in</strong>g, but not limited to, muscle<br />
wast<strong>in</strong>g and bone loss.
Problems With Conventional<br />
Recomb<strong>in</strong>ant Epo<br />
• Must be glycosylated- produced <strong>in</strong> mammalian cells.<br />
• Relatively short <strong>in</strong> vivo half-life: life: 6-126<br />
hr.<br />
• Must be <strong>in</strong>jected.<br />
• Delay <strong>in</strong> physiological response: 2-42<br />
4 weeks.<br />
There are several approaches to address these problems,<br />
<strong>in</strong>clud<strong>in</strong>g darbepoet<strong>in</strong>, , an Epo mutant with two additional<br />
glycosylation sites, which <strong>in</strong>crease its half-life..<br />
life..<br />
Our strategy is the production of Epo dimers by<br />
chemical crossl<strong>in</strong>k<strong>in</strong>g or as a fusion prote<strong>in</strong>.
Hypothetical Action of Epo Dimer<br />
Monomer<br />
Epo Dimer<br />
L<strong>in</strong>ker length is critical<br />
Increased aff<strong>in</strong>ity and<br />
EpoR cluster<strong>in</strong>g<br />
amplifies signal<strong>in</strong>g.
• Also, the <strong>in</strong>creased size and glycosylation<br />
of the Epo-Epo dimer should result <strong>in</strong> a<br />
markedly prolonged plasma half-life.<br />
life.<br />
• So we predict a double effect of Epo<br />
dimerization, <strong>in</strong>creased action on the cell<br />
and <strong>in</strong>creased survival <strong>in</strong> the circulation.
Chemical Crossl<strong>in</strong>k<strong>in</strong>g of Epo<br />
• Purpose<br />
• Hypothesize that T 1/2 is a function of size<br />
• Create a “large Epo” or oligomer with<br />
<strong>in</strong>crease number of EpoR b<strong>in</strong>d<strong>in</strong>g sites<br />
• Approach<br />
• Control degree of crossl<strong>in</strong>k<strong>in</strong>g and size<br />
• Use two chemical modify<strong>in</strong>g agents to<br />
prepare two pools of modified Epo
First Approach: Chemical<br />
Crossl<strong>in</strong>k<strong>in</strong>g of Epo<br />
LC-SPDP<br />
SMCC<br />
Epo A<br />
SH<br />
M<br />
Epo B<br />
LC-SPDP<br />
= succ<strong>in</strong>imidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate<br />
SMCC = succ<strong>in</strong>imidyl 4-(N-maleimidomethyl<br />
M = maleimido group
SDS-PAGE of Epo-SH, M-Epo M<br />
and<br />
Crossl<strong>in</strong>ked Epo Dimer and Trimer<br />
Trimer<br />
Dimer<br />
Monomer<br />
SH M Epo-SH<br />
+<br />
M-Epo
Activity of Epo Oligomers<br />
Separated By SE HPLC<br />
IU/µg<br />
Monomer 160<br />
Dimer 210<br />
Trimer 100
Pharmacok<strong>in</strong>etics of Epo<br />
Monomer and Dimer <strong>in</strong> Rabbits
Epo Dimer ( ) Activity Is<br />
Superior to Monomer ( ) In Vivo<br />
A: 300 IU/kg 3X<br />
B: 300 IU/kg 1X<br />
C: 30 IU/kg 3X<br />
D: 30 IU/kg 1X<br />
Groups of 6 mice were <strong>in</strong>jected subcutaneously.
A Second Approach:<br />
Epo-Epo Fusion Prote<strong>in</strong> cDNA<br />
Encodes A “Recomb<strong>in</strong>ant Dimer”<br />
5’ UTR Epo<br />
Epo<br />
3’ UTR<br />
Leader<br />
L<strong>in</strong>ker<br />
Peptide<br />
Stop
SDS-PAGE and Western Blot of<br />
Epo and Epo-Epo Fusion Prote<strong>in</strong><br />
Specific Activity,<br />
U/µg U/pmol<br />
Epo 350 13<br />
Epo-Epo<br />
1000 76
Pharmacok<strong>in</strong>etics of Epo and Epo-Epo<br />
<strong>in</strong> Mice<br />
Intravenous <strong>in</strong>jection. Each curve represent 1 mouse.<br />
Epo<br />
Epo-Epo<br />
Note much longer half-life life of Epo-Epo
Epo-Epo is Active In Vivo And is<br />
Superior To Epo<br />
S<strong>in</strong>gle subcutaneous dose of 300 U/kg. Post-Hct done after 7 days.<br />
Each l<strong>in</strong>e represent 1 mouse.
New Epo Fusion Prote<strong>in</strong>s Under<br />
Study In Our Laboratory<br />
M.W.<br />
Tetramer<br />
Trimer<br />
Hexamer<br />
Pentamer<br />
SDS PAGE<br />
and<br />
Western Blot<br />
Dimer = Epo-Epo
Summary<br />
• Long-term human spaceflight is associated with<br />
novel risks that require countermeasures,<br />
<strong>in</strong>clud<strong>in</strong>g new pharmacological agents, and with<br />
unique problems <strong>in</strong> autonomous medical care.<br />
• Long-act<strong>in</strong>g and ultra-long<br />
long-act<strong>in</strong>g prote<strong>in</strong><br />
therapeutic agents can be designed to address<br />
some of these risks.<br />
• The Epo dimer and fusion prote<strong>in</strong> concept can<br />
serve as a paradigm for the development of these<br />
new agents.
For more <strong>in</strong><strong>format</strong>ion<br />
about<br />
Erythropoiet<strong>in</strong><br />
www.wiley.com
Acknowledgment<br />
• Julia Sue<br />
• James Fisher<br />
• Laurie Feldman<br />
• Yijuang Chern<br />
• Jennifer Grodberg<br />
• Elizabeth Lunn<br />
• Mary Ris<strong>in</strong>ger<br />
• Kelly Donahue<br />
• Kerry Wellenste<strong>in</strong><br />
• Rudy Spangler<br />
• Stephen Bailey<br />
• Hiren Patel<br />
• Yuqui Li<br />
• Changm<strong>in</strong> Chen<br />
Fund<strong>in</strong>g from NIH, DOD and NASA
Greet<strong>in</strong>gs from the Laboratory for<br />
Cell and Molecular Biology
Thank you for your k<strong>in</strong>d attention<br />
Arthur J. Sytkowski, MD 617 632 9980<br />
asytkows@bidmc.harvard.edu